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 .
Drawings
The drawings filed on 5/22/24 are objected to for the following reasons:
37 C.F.R. 1.84 states “Character of lines, numbers, and letters. All drawings must be made by a process which will give them satisfactory reproduction characteristics. Every line, number, and letter must be durable, clean, black (except for color drawings), sufficiently dense and dark, and uniformly thick and well-defined.”
In the current case, the words in Figure 9 are illegible.
Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
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 1-3, 7-10, 12, 16-18, 20-22, 26-29, 69, and 124 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 is directed to a system for altering a host cell DNA comprising any DNA endonuclease and any gRNA comprising a spacer sequence complementary to any host cell locus and any donor template comprising a nucleic acid encoding a synthetic FVIII protein, wherein the protein comprises a B-domain substitute from 1-9 N-linked glycosylation sites and from 3-40 amino acids in length.
The specification does not adequately describe the structure required for the function of the DNA endonuclease to function as claimed. DNA endonucleases as a class do not inherently operate through gRNAs. CRISPR-Cas nucleases are RNA-guided, unlike other DNA nucleases which recognize their targets through protein-DNA interaction, as taught by Zhang et al. (Molecular Therapy Vol. 27 No 4 April 2019, 735-746).
The species of the specification are not representative of the entire claimed genus of any DNA endonuclease. The specification does not adequately describe the structure required for any given DNA endonuclease to have the required function.
Additionally, the claims are directed to a system comprising a gRNA that has a spacer that is complementary to any host cell locus and would therefore guide the DNA endonuclease and insert the FVIII donor template into the host cell locus. The specification describes insertion of a specific FVIII donor template into an Albumin host cell locus, which is not representative of insertion of any donor template comprising a nucleic acid encoding a synthetic FVIII protein, wherein the protein comprises a B-domain substitute from 1-9 N-linked glycosylation sites and from 3-40 amino acids in length into any host cell locus with a resultant functional system for altering host cell DNA sequence.
The MPEP states that for a generic claim, the genus can be adequately described if the disclosure presents a sufficient number of representative species that encompass the genus. See MPEP § 2163. If the genus has a substantial variance, the disclosure must describe a sufficient variety of species to reflect the variation within that genus. See MPEP § 2163. Although the MPEP does not define what constitute a sufficient number of representative species, the courts have indicated what do not constitute a representative number of species to adequately describe a broad genus. In Gostelli, the courts determined that the disclosure of two chemical compounds within a subgenus did not describe that subgenus. In re Gostelli, 872, F.2d at 1012, 10 USPQ2d at 1618. Additionally, in Carnegie Mellon University v. Hoffman-La Roche Inc., Nos. 07-1266, -1267 (Fed. Cir. Sept. 8, 2008), the Federal Circuit affirmed that a claim to a genus described in functional terms was not supported by the specification’s disclosure of species that were not representative of the entire genus. Furthermore, for a broad generic claim, the specification must provide adequate written description to identify the genus of the claim. In Regents of the University of California v. Eli Lilly & Co. the court stated:
"A written description of an invention involving a chemical genus, like a description of a chemical species, 'requires a precise definition, such as by structure, formula, [or] chemical name,' of the claimed subject matter sufficient to distinguish it from other materials." Fiers, 984 F.2d at 1171, 25 USPQ2d 1601; In re Smythe, 480 F.2d 1376, 1383, 178 USPQ 279, 284985 (CCPA 1973) ("In other cases, particularly but not necessarily, chemical cases, where there is unpredictability in performance of certain species or subcombinations other than those specifically enumerated, one skilled in the art may be found not to have been placed in possession of a genus ...") Regents of the University of California v. Eli Lilly & Co., 43 USPQ2d 1398.
The Guidelines for Examination of Patent Applications under the 35 USC § 112, first paragraph, “Written Description” Requirement”, published at Federal Register, Vol. 66, No. 4, pp. 1099-1111 outline the method of analysis of claims to determine whether adequate written description is present. The first step is to determine what the claim as a whole covers, i.e., discussion of the full scope of the claim. Second, the application should be fully reviewed to understand how applicant provides support for the claimed invention including each element and/or step, i.e., compare the scope of the claim with the scope of the description. Third, determine whether the applicant was in possession of the claimed invention as a whole at the time of filing.
To achieve the desired function, it appears that the structure is required to be a Cas9 endonuclease in combination with a specific gRNA that has been determined to target a precise site necessary within a host cell locus (not any host cell locus) to result in endogenous FVIII expression. The locus, orientation, promoter/enhancer, splice configuration, reading frame, and donor design can determine if FVIII is actually expressed from a given DNA endonuclease/gRNA system. Expression depends on whether the inserted sequence is placed in a transcriptionally competent context and is configured so it can be transcribed and translated, as evidenced by Chen et al. (Scientific Reports, 2019, 9:16838, 1-15).
Thus, having analyzed the claims with regard to the Written Description guidelines, it is clear that the specification does not disclose a representative number of species within the instant genuses that function as claimed. Thus, one skilled in the art would be led to conclude that Applicant was not in possession of the claimed invention at the time the application was filed.
Claims 29, 69, and 124 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for the instant methods via delivery of a specific construct (LNP encapsulating mALbT1 gRNA and spCas9 mRNA) of the specification that resulted in FVIII coding sequence integration into intron 1 of albumin (page 129) , does not reasonably provide enablement for a method of editing a genome of host cell, treating hemophilia A, and increasing the amount of FVIII in a subject via delivering any DNA endonuclease, any guide RNA comprising a spacer sequence complementary to any host locus, and the donor template as claimed. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention commensurate in scope with these claims.
Factors to be considered in a determination of lack of enablement include, but are not limited to:
(A) The breadth of the claims;
(B) The nature of the invention;
(C) The state of the prior art;
(D) The level of one of ordinary skill;
(E) The level of predictability in the art;
(F) The amount of direction provided by the inventor;
(G) The existence of working examples; and
(H) The quantity of experimentation needed to make or use the invention based on the content of the disclosure.
In re Wands, 858 F.2d 731, 737, 8 USPQ2d 1400, 1404 (Fed. Cir. 1988)
The instant claims are directed to a method of editing a genome of host cell, treating hemophilia A, and increasing the amount of FVIII in a subject via delivering any DNA endonuclease, any guide RNA comprising a spacer sequence complementary to any host locus, and the donor template as claimed.
Certainly delivery of any possible DNA endonuclease won’t predictably function in a CRISPR system. DNA endonucleases as a class do not inherently operate through gRNAs. CRISPR-Cas nucleases are RNA-guided, unlike other DNA nucleases which recognize their targets through protein-DNA interaction, as taught by Zhang et al. (Molecular Therapy Vol. 27 No 4 April 2019, 735-746).
For example, EcoR1 is a DNA endonuclease that does not function as a nuclease component of a CRISPR system, but rather recognizes its own fixed DNA sequence.
Chen et al. (Scientific Reports, 2019, 9:16838, 1-15) is evidence that not any guide RNA comprising a spacer sequence complementary to any host locus will function in a predictable manner. The experimental evidence of Chen et al. demonstrates that merely having a gRNA complementary to the intended host locus does not mean that every such gRNA will perform in the same way or that its activity can be assumed from the presence of complementarity to a target sequence. Chen et al. identified five different gRNA candidates, Alb-sg1-Alb-sg5, all directed to the mouse albumin intron 13 region and experimentally tested each one. The results were substantially different for each. Chen et al. teaches different mutation/cleavage rates ranging from 6% to 40%, with Alb-sg1 producing the highest activity (40% in mouse embryonic fibroblasts and 20% in Heap1-6 liver cells). Therefore, Chen et al. demonstrates that sequence complementarity to a given host locus by itself does not establish that a gRNA will provide the desired CRISPR/Cas9 activity. Even with gRNAs targeted to the same intron of albumin, the activities were not the same. Selection of intron 13 was crucial to keep Albumin protein and expression intact after BDD-FVIII insertion. The specific biological architecture needed was present in this specific region of a specific target. Chen et al. determined the precise site necessary at the Alb intron 13/exon 14 junction to result in endogenous albumin-driven BDD-FVIII expression in order for the sequence to be placed under control of the endogenous albumin expression machinery. Insertion in the opposite direction did not express BDD-F8.
The locus, orientation, promoter/enhancer, splice configuration, reading frame, and donor design can determine if FVIII is actually expressed from a given DNA endonuclease/gRNA system. Expression depends on whether the inserted sequence is placed in a transcriptionally competent context and is configured so it can be transcribed and translated, as evidenced by Chen et al.
The instant claims are directed to the delivery of the combination of any DNA endonuclease and any gRNA comprising a spacer complementary to any host cell locus in combination with the instantly recited template, which clearly would not result in each of the instantly recited outcomes in a predictable manner without undue experimentation to determine which specific DNA endonucleases in combination with which specific gRNAs would result in insertion and expression of the recited donor template. Additionally, the instant claims encompass insertion into a poorly expressed or silenced gene.
As outlined above, it is well known that there is a high level of unpredictability in the CRISPR art for therapeutic in vivo applications and design. The scope of the claims in view of the specification as filed together do not reconcile the unpredictability in the art to enable one of skill in the art to make and/or use the claimed invention, namely a broad method of achieving each of the instantly recited outcomes via delivery via any means of a combination of any DNA endonuclease, any guide RNA comprising a spacer sequence complementary to any host locus, and the donor template as claimed encompassing in vivo effects.
MPEP 2164.01
Any analysis of whether a particular claim is supported by the disclosure in an application requires a determination of whether that disclosure, when filed, contained sufficient information regarding the subject matter of the claims as to enable one skilled in the pertinent art to make and use the claimed invention.
Also, MPEP 2164.01(a)
A conclusion of lack of enablement means that, based on the evidence regarding each of the above factors, the specification, at the time the application was filed, would not have taught one skilled in the art how to make and/or use the full scope of the claimed invention without undue experimentation. In re Wright, 999 F.2d 1557,1562, 27 USPQ2d 1510, 1513 (Fed. Cir. 1993).
Given the teachings of the specification as discussed above, one skilled in the art could not predict a priori whether introduction of any combination of the instantly recited agents of the instant genuses in vivo by the broadly disclosed methodologies of the instantly claimed invention, would result in editing a genome in a host cell, treating hemophilia A, or increasing the amount of FVIII in a subject. To practice the claimed invention, one of skill in the art would have to de novo determine; the stability of the molecule in vivo, delivery of the molecule to the whole organism, specificity to the target tissue in vivo, dosage and toxicity in vivo, and entry of the molecule into the cell in vivo and the effective action therein. Without further guidance, one of skill in the art would have to practice a substantial amount of trial and error experimentation, an amount considered undue and not routine, to practice the instantly claimed invention.
A conclusion of lack of enablement means that, based on the evidence regarding each of the above factors, the specification, at the time the application was filed, would not have taught one skilled in the art how to make and/or use the full scope of the claimed invention without undue experimentation (see MPEP 2164.01(a)).
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.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claim(s) 1-3, 7-10, 12, 16-18, 20-22, 26-29, 69, and 124 is/are rejected under 35 U.S.C. 103 as being unpatentable over Miller et al. (US 2015/0166618 A1), in view of McIntosh et al. (Blood, 2013, 121, 17, 3335-3344), Anguela et al. (WO 2017/075619 A1), Zhang et al. (Genome Biology, 2017, 18:35, 1-18), and Yu et al. (Biotechnol Lett (2016) 38:919–929).
Miller et al. teach methods and compositions for insertion of transgene sequences encoding proteins involved in clotting into the genome of a cell for treating conditions including hemophilias (abstract).
Miller et al. teach: [0006] Gene therapy for patients with Hemophilia A or B, involving the introduction of plasmid and other vectors (e.g., AAV) encoding a functional Factor VIII or Factor IX proteins have been described. However, in these protocols, the formation of inhibitory anti-factor VIII or IX (anti-F8 or anti-F.IX) antibodies and antibodies against the delivery vehicle remains a major complication of Factor VIII and Factor IX replacement-based treatment for hemophilia.
Miller et al. teach: [0007] Various methods and compositions for targeted cleavage of genomic DNA have been described. Such targeted cleavage events can be used, for example, to induce targeted mutagenesis, induce targeted deletions of cellular DNA sequences, and facilitate targeted recombination at a predetermined chromosomal locus. These methods often involve the use of engineered cleavage systems to induce a double strand break (DSB) or a nick in a target DNA sequence such that repair of the break by an error born process such as non-homologous end joining (NHEJ) or repair using a repair template (homology directed repair or HDR) can result in the knock out of a gene or the insertion of a sequence of interest (targeted integration). This technique can also be used to introduce site specific changes in the genome sequence through use of a donor oligonucleotide, including the introduction of specific deletions of genomic regions, or of specific point mutations or localized alterations (also known as gene correction). Cleavage can occur through the use of specific nucleases such as engineered zinc finger nucleases (ZFN), transcription-activator like effector nucleases (TALENs), or using the CRISPR/Cas system with an engineered crRNA/tracr RNA (`single guide RNA`) to guide specific cleavage. Further, targeted nucleases are being developed based on the Argonaute system, which also may have the potential for use in genome editing and gene therapy.
Miller et al. teach: [0008] This nuclease-mediated targeted transgene insertion approach offers the prospect of improved transgene expression, increased safety and expressional durability, as compared to classic integration approaches, since it allows exact transgene positioning for a minimal risk of gene silencing or activation of nearby oncogenes.
Miller et al. teach: [0009] Targeted integration of a transgene may be into its cognate locus, for example, insertion of a wild type transgene into the endogenous locus to correct a mutant gene. Alternatively, the transgene may be inserted into a non-cognate locus, for example a "safe harbor" locus. Several safe harbor loci have been described, including CCR5, HPRT, AAVS1, Rosa and albumin (instant claim 10).
Miller et al. teach: [0011] Disclosed herein are methods and compositions for targeted integration of a sequence encoding a protein, such as a functional clotting factor protein (Factor VIII). Expression of a functional Factor VIII ("F8") can result, for example, in the treatment and/or prevention of hemophilia A (Factor VIII) (instant claim 69). Nucleases, for example engineered meganucleases, zinc finger nucleases (ZFNs), TALE-nucleases (TALENs including fusions of TALE effectors domains with nuclease domains from restriction endonucleases and/or from meganucleases (such as mega TALEs and compact TALENs)), Ttago system and/or CRISPR/Cas nuclease systems are used to cleave DNA at a `safe harbor` gene locus (e.g. CCR5, AAVS1, HPRT, Rosa or albumin) (instant claim 10) in the cell into which the gene is inserted. Targeted insertion of a donor transgene may be via homology directed repair (HDR) or non-homology repair mechanisms (e.g., NHEJ donor capture). The nuclease can induce a double-stranded (DSB) or single-stranded break (nick) in the target DNA. In some embodiments, two nickases are used to create a DSB by introducing two nicks. In some cases, the nickase is a ZFN, while in others, the nickase is a TALEN or a CRISPR/Cas nickase. In some embodiments, the methods and compositions involve at least one protein that binds to an albumin gene in a cell (instant claims 8 and 9).
Miller et al. teach: [0014] In another aspect, described herein is a CRISPR/Cas system that binds to target site in a region of interest (e.g., an albumin gene) in a genome, wherein the CRISPR/Cas system comprises one or more engineered single guide RNA or a functional equivalent, as well as a Cas9 nuclease (instant claims 8, 9, and 12). It is noted that albumin is expressed in the liver (instant claim 7).
Miller et al. teach: [0016] In another aspect, described herein is a polynucleotide encoding one or more nucleases (e.g., ZFNs, CRISPR/Cas systems, Ttago and/or TALENs described herein) or other proteins. The polynucleotide may be, for example, mRNA. (instant claim 16).
Miller et al. teach: [0145] Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids encoding nucleases and donor constructs in cells (e.g., mammalian cells) and target tissues. Non-viral vector delivery systems include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell (instant claim 26).
Miller et al. teach: [0023] Thus, an isolated cell may be introduced into the subject (ex vivo cell therapy) or a cell may be modified when it is part of the subject (in vivo). Also provided is the use of the donors and/or nucleases described herein for the treatment of a hemophilia (e.g., hemophilia A with Factor VIII donor), for example, in the preparation of medicament for treatment of hemophilia. In certain embodiments, the Factor VIII protein comprises a B-domain deletion. In certain embodiments, the Factor VIII encoding sequence is delivered using a viral vector, a non-viral vector (e.g., plasmid) and/or combinations thereof.
Miller et al. teach: [0024] In any of the compositions and methods described, the nuclease(s) and/or transgene(s) may be carried on an AAV vector, including but not limited to AAV1, AAV3, AAV4, AAV5, AAV6, AAV8, AAV9 and AAVrh10 or pseudotyped AAV such as AAV2/8, AAV8.2, AAV2/5 and AAV2/6 and the like. In some embodiments, the AAV vector is an AAV2/6 vector. In certain embodiments, the nucleases and transgene donors are delivered using the same AAV vector types. In other embodiments, the nucleases and transgene donors are delivered using different AAV vector types. The nucleases and transgenes may be delivered using one or more vectors, for example, one vector carries both the transgene and nuclease(s); two vectors where one carries the nuclease(s) (e.g., left and right ZFNs of a ZFN pair, for example with a 2A peptide) and one carries the transgene; or three vectors where one vector carries one nuclease of a nuclease pair (e.g., left ZFN), a separate vector carries the other nuclease of a nuclease pair (e.g., right ZFN) and a third separate vector carries the transgene (instant claim 20).
Miller et al. teach: [0164] The compositions are administered to a human patient in an amount effective to obtain the desired concentration of the therapeutic Factor VII, Factor VIII, Factor IX or Factor X polypeptide in the serum, the liver or the target cells (instant claim 124)/
Therefore, Miller et al. teach a system for altering a host cell DNA sequence comprising a Cas9 endonuclease or a nucleic acid encoding the Cas9 endonuclease, a guide RNA comprising a spacer that is complementary to the host cell locus [0014][0023]; and a method of treating hemophilia A via delivery of the system [0023] (instant claims 29, 69, and 124). It is noted that the methods of claims 29, 69, and 124 recite identical method steps of delivering the same components and therefore the method of Miller et al. would result in each of the outcomes.
Miller et al. teach that in certain embodiments, the Factor VIII protein comprises a B-domain deletion [0023] and teaches that in some embodiments, the donor includes additional modifications, including but not limited to codon optimization, addition of glycosylation sites, truncations and the like [0141] (instant claim 17). Miller et al. cites McIntosh et al. (2013) Blood (17):3335-44 with regards to these modifications.
McIntosh et al. teach: Recombinant adeno-associated virus (rAAV) vectors encoding human factor VIII (hFVIII) were systematically evaluated for hemophilia A (HA) gene therapy. A 5.7-kb rAAV-expression cassette (rAAV-HLP-codop-hFVIII-N6) containing a codon-optimized hFVIII cDNA in which a 226 amino acid (aa) B-domain spacer replaced the entire B domain and a hybrid liver-specific promoter (HLP) mediated 10-fold higher hFVIII levels in mice compared with non–codon-optimized variants. A further twofold improvement in potency was achieved by replacing the 226-aa N6 spacer with a novel 17-aa peptide (V3) in which 6 glycosylation triplets from the B domain were juxtaposed. The resulting 5.2-kb rAAV-HLP-codop-hFVIII-V3 cassette was more efficiently packaged within AAV virions and mediated supraphysiologic hFVIII expression (732 ± 162% of normal) in HA knock-out mice following administration of 2 × 1012 vector genomes/kg, a vector dose shown to be safe in subjects with hemophilia B. Therefore, rAAV-HLP-codop-hFVIII-V3 substantially improves the prospects of effective HA gene therapy (abstract).
McIntosh et al. teach: This is in part due to improved secretion of hFVIII, which is thought to be facilitated by interactions of 6 N-linked glycosylation triplets with the mannose-binding lectin, LMAN1, within this region
McIntosh et al. teach: In this report, we describe a novel 5.2-kb AAV expression cassette (rAAV-HLP-codop-hFVIII-V3) in which HLP is a hybrid liver-specific promoter and the N6 226-aa B-domain spacer has been replaced with a 17-aa peptide that contains the 6 N-linked glycosylation signals required for efficient and safe expression of hFVIII.
Therefore, it was known to incorporate the B-domain substitute, N-linked glycosylation sites and codon-optimization of McIntosh et al. (17 aa, 6 N-linked glycosylation sites), as cited by Miller et al. IT would have been obvious to incorporate these elements into the system of Miller et al. with the motivation and expectation of improved secretion of hFVIII, as well as efficient and safe expression of hFVIII, as taught by McIntosh et al. (instant claims 1-3).
There was clear motivation from Miller et al. to utilize a CRISPR (Cas9/gRNA) system with a donor template encoding FVIII, with Miller et al. citing McIntosh et al. for the benefits of incorporation of N-linked glycosylation sites and B domain substitution in the instant amino acid size range.
Miller et al. does not teach that the donor template comprises a reduced content of CpG di-nucleotides as compared to wild type sequence. However, it would have been obvious for the donor template to comprise a reduced content of CpG di-nucleotides as compared to wild type sequence because Anguela et al. teach that CpG reduced nucleic acid variants encoding FVIII are expressed more efficiently by cells, are secreted at increased levels by cells over wild-type Factor VIII proteins, exhibit enhanced expression and/or activity over wild-type Factor VIII proteins or are packaged more efficiently into viral vectors (abstract).
Since Anguela et al. teach reducing the content of CpG di-nucleotides in the same sequence, FVIII, and teach the benefits of doing such, it would have been obvious to design the donor template to comprise a reduced content of CpG di-nucleotides as compared to wild type sequence with the motivation and reasonable expectation of achieving the benefits taught by Anguela et al. (instant claim 18).
Miller et al. do not teach that the donor cassette is flanked on both sides by a gRNA target site. However, Zhang et al. teach: we show that a double cut HDR donor, which is flanked by single guide RNA (sgRNA)-PAM sequences and is released after CRISPR/Cas9 cleavage, increases HDR efficiency by twofold to fivefold relative to circular plasmid donors at one genomic locus in 293 T cells and two distinct genomic loci in iPSCs. We find that a 600 bp homology in both arms leads to high-level genome knockin, with 97–100% of the donor insertion events being mediated by HDR.
Zhang et al. teach that the sgRNA specifically recognizes the chromosomal locus of interest with a 17-20 nucleotide sequence. This is an obvious design element of the gRNA of Miller et al. as gRNAs are known to have a spacer sequence that is complementary to the target locus.
It would have been obvious for the donor template to be in a cassette comprising the nucleic acid sequence encoding the protein, wherein the cassette is flanked on both sides by a gRNA target site because Zhang et al. teach that flanking the donor with single guide RNA (sgRNA)-PAM sequence results in increased HDR efficiency, a benefit that one would want in the system of Miller et al. Additionally, Zhang et al. teach: ribonucleoproteins (RNPs), the Cas9 protein in complex with in vitro transcribed guide RNA, have been used together with a single-stranded oligonucleotide HDR template for nucleotide replacement or insertion. We believe that the double cut donor vector can also be delivered together with Cas9 protein/RNA and sgRNA transcripts, which may even be preferable in applications such as zygote injection for creating knockin animals (page 13 (instant claim 28).
Zhang et al. teach that the same sgRNA can target both the genomic locus and the donor plasmid.
One would reasonably expect that incorporating the elements of the system of Zhang et al. into the system of Miller et al. would result in increased HDR efficiency of inserting the FVIII template sequence (instant claims 21 and 22).
It would have been obvious to incorporate both the gRNA and Cas9 in the liposome or lipid nanoparticle because Miller et al. teaches that Cas9/gRNA CRISPR system can be used, Miller et al. teaches delivery of Cas9 or a sequence encoding Cas9 from a lipoma, and Miller et al. teaches delivery of the donor construct from the same liposome. Yu et al. teach delivery of Cas9 and gRNA RNPs from lipid nanoparticles together (abstract). Therefore, it would have been obvious to incorporate both the gRNA and Cas9 of Miller et al. in the same lipid nanoparticle for ease of delivery with a reasonable expectation and motivation of successful co-delivery, as taught by Yu et al. (instant claim 27).
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Amy R Hudson whose telephone number is (571)272-0755. The examiner can normally be reached M-F 8:00am-6:00pm.
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/AMY ROSE HUDSON/Primary Examiner, Art Unit 1636
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