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 .
Application Status
Applicant’s response filed October 22, 2025 is acknowledged. Claims 1-2, 4, 6-11, 13-14, 16-17, 19-25, 27, 29, 31-34, 36, 38-48, and 50-51 are pending.
Restriction/Election
Applicant elected Group I (claims 1-2, 4, 6-11, 13-14, 16-17, 19-25, 27, 29, 31-34, 36, and 38-41) in the response filed October 22, 2025. Applicant did not indicate whether the election was made with or without traverse, or distinctly and specifically point out the supposed errors in the restriction requirement. The election has been treated as an election without traverse, accordingly. See MPEP § 818.01(a). Claims 42-48, and 50-51 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention. Claims 1-2, 4, 6-11, 13-14, 16-17, 19-25, 27, 29, 31-34, 36, and 38-41 are under examination hereinafter.
Priority
Applicant's claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged. Applicant has not complied with one or more conditions for receiving the benefit of an earlier filing date under 35 U.S.C. 119(e) as follows:
The later-filed application must be an application for a patent for an invention which is also disclosed in the prior application (the parent or original nonprovisional application or provisional application). The disclosure of the invention in the parent application and in the later-filed application must be sufficient to comply with the requirements of 35 U.S.C. 112(a) or the first paragraph of pre-AIA 35 U.S.C. 112, except for the best mode requirement. See Transco Products, Inc. v. Performance Contracting, Inc., 38 F.3d 551, 32 USPQ2d 1077 (Fed. Cir. 1994).
A review of Application No. 62/952,981 failed to uncover support for an “IscB system or component thereof” as recited in instant claim 4. The first disclosure of an “IscB system or component thereof” is in PCT/US2020/066949 (see at least [0131]). Accordingly, the effective filing date of claims 4, 6-11, and 13-14 is December 23, 2020. The remaining claims under examination find support in Application No. 62/952,981, and therefore, have an effective filing date of December 23, 2019.
Specification
The specification is objected to because of the following informalities:
The Brief Description of Drawings makes reference to color drawings (“red arrow,” [0075]). 37 CFR 1.84(a)(2) states “the Office will accept color drawings in utility patent applications only after granting a petition filed under this paragraph explaining why the color drawings are necessary.” There is no granted petition to accept color drawings on file. Applicants may either comply with all requirements of 37 CFR 1.84(a)(2)(i)-(iii), or submit replacement sheets in black and white, and remove the references to color drawings throughout the specification. The Examiner takes no position on whether color drawings are necessary as the only practical medium by which to disclose the subject matter sought to be patented in this utility patent application.
The disclosure is objected to because it contains an embedded hyperlinks and/or other form of browser-executable code. The embedded hyperlinks and/or other form of browser-executable code are in at least paragraphs [0260], [0474], [0480], [0581], [0673], [0821], [0935], [0952], [01088], [01093]. Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01.
The use of terms which are trade names or marks used in commerce, has been noted in this application, e.g., “Lipofectin” ([0564]), and Lipofectamine 2000” ([0539]). 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. The terms should be accompanied by the generic terminology; furthermore, the terms should be capitalized wherever they appear or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the terms.
Appropriate correction is required.
Claim Objections
Claims 1-2 are objected to because of the following informalities:
Claim 1 recites “one or more DNA-nucleases” which should be amended to recite “DNA programmable DNA
Claim 2 recites “one or more programmable DNA nuclease polypeptides.” It would be preferable to amend the claim to recite “one or more programmable DNA nucleases.
Appropriate correction is required.
Claim Rejections - 35 USC § 112(b)
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 4, 6-11, 13-14, 19, 21, and 24 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.
Claim 4 recites that the one or more programmable DNA nucleases are “one or more RNA-guided DNA nucleases.” The claim then recites that the one or more RNA-guided DNA nucleases are optionally, “one or more CRISPR-Cas systems or a component thereof,” “an IscB system or component thereof,” a Zinc Finger Nuclease or system thereof,” “a TALE nuclease or system thereof,” or a “meganuclease or a system thereof.” With the exception of “CRISPR-Cas systems or a component thereof” and “an IscB system or component thereof,” none of the remaining options would be considered “RNA-guided” by the ordinarily skilled artisan. Indeed, Zhang (Zhang et al., 4 April 2019, Molecular Therapy, Vol. 27, No. 4, pg. 735-746) teaches that each of the remaining options are guided by the interaction between DNA and protein, not RNA (Table 1). The specification also discusses the use of catalytically dead Cas proteins (at least [0181]; [0231]; [01187]). This renders the scope of the term “RNA-guided DNA nuclease” unclear, because it appears, based on the claim and specification, to encompass DNA nucleases which would not generally be considered “RNA-guided,” or even have functional nuclease capability.
The phrase “or a component thereof” adds further confusion to the scope of the term. The phrase “component thereof” is not explicitly defined by the specification, but based on its usage, appears to refer to any element which could be considered part of the claimed system (“a CRISPR-Cas system component (e.g., an effector protein, including but not limited to a Cas protein,” [0087]; “where a programmable DNA nuclease system or component thereof is described below (such as a guide molecule, Cas protein, IscB protein, or other component) that such a system or component is referring to one that can include or associate with a programmable DNA nuclease associated ligase,” [0096]). It is not clear what minimum structural requirements are implied by the term “RNA-guided DNA nuclease,” and therefore, it is not clear which of the many, undefined “components thereof” of the CRISPR-Cas and IscB “systems” would be encompassed by this term.
Claims 6-11, and 13-14 are rejected for depending from claim 4 and failing to remedy the indefiniteness. Claims 6-11 are included in this rejection, because while the claims further limit “the one or more CRISPR-Cas systems or components thereof” to “one or more Cas polypeptides,” they do not resolve the issues described above related to the scope of the term “RNA-guided DNA nucleases.”
In the interest of compact prosecution, the scope of “RNA-guided DNA nucleases” is interpreted as encompassing any DNA nuclease, which may or may not have functional nuclease capability. The phrase “components thereof” of the systems are interpreted the same, i.e., as encompassing any DNA nuclease, which may or may not have functional nuclease capability.
Claim 14 recites “the donor sequence,” but no such donor sequence is explicitly required, implied by, or inherent to claims 1, 4, or 13, from which the claim depends. This element lacks sufficient antecedent basis in the claim. In the interest of compact prosecution, the claim is interpreted as having sufficient antecedent basis for this element.
Claim 19 recites “the donor sequence is protected from degradation.” It is not clear what, if anything, is required of the claim to achieve “protect[ion] from degradation.” The specification describes chemical modifications to the donor sequence, which the skilled artisan would know could result in protection from degradation ([0244]), but the claim does not explicitly require any chemical modifications to the donor sequence. It is not clear whether the donor sequence is inherently “protected from degradation,” or whether there are additional elements required of the claim, and/or structural modifications required of the donor sequence, to achieve the recited “protect[ion] from degradation.” Because the implications of the phrase “protected from degradation” are unclear, the claim is rendered indefinite. Hereinafter, “protect[ion] from degradation” is interpreted as an inherent feature of the donor sequence.
Claim 21 recites “the first and the optional second guide molecules,” but no such first or optional second guide molecules are explicitly required, implied by, or inherent to claims 1 or 16 from which the claim depends. These elements lack sufficient antecedent basis in the claim. It is also noted that the use of the phrase “when present,” appears to make optional each preceding limitation, such that even should the elements have sufficient antecedent basis, it is not clear whether the claim even requires any of its limitations. This claim will not be addressed further, due to uncertainty about the basis for its limitations, and what limitations are actually required.
Claim 24 also recites “the first, or optional second guide molecule,” but no such first or optional second guide molecule are explicitly required, implied by, or inherent to claim 1 from which the claim depends. These elements lack sufficient antecedent basis in the claim. Hereinafter, the claim is interpreted as having sufficient antecedent basis for this element.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claims Directed to or Encompassing a Human Organism
Section 33(a) of the America Invents Act reads as follows:
Notwithstanding any other provision of law, no patent may issue on a claim directed to or encompassing a human organism.
Claims 39-41 are rejected under 35 U.S.C. 101 and section 33(a) of the America Invents Act as being directed to or encompassing a human organism. See also Animals - Patentability, 1077 Off. Gaz. Pat. Office 24 (April 21, 1987) (indicating that human organisms are excluded from the scope of patentable subject matter under 35 U.S.C. 101).
Claim 39 is drawn to “a cell comprising: the engineered composition of claim 1….” Claim 40 recites that the cell is “a human cell.” The specification discusses delivery of the engineered compositions to cells within a human (see at least [0927]-[0928]; [0937]; [0947]-[0950]), and therefore, claims 39-40 could reasonably be interpreted as encompassing cells within a human organism, which is non-statutory subject matter.
Claim 41 recites “A tissue, organ, or organism comprising: the cell of claim 39.” The cells encompass human cells (see claim 40, discussed above), and therefore, claim 41 could also reasonably be interpreted as encompassing tissues or organs within a human organism, or a human organism itself. This is non-statutory subject matter.
Claims Directed to a Judicial Exception
Claims 1-2, 16-17, 19-20, 22-25, 27, 29, 31-34, 36, and 38-41 are rejected under 35 U.S.C. 101 because the claimed invention is directed to a judicial exception (i.e., a law of nature, natural phenomenon, or an abstract idea) without significantly more. The claims recite “An engineered composition for modifying polynucleotides, the composition comprising: one or more programmable DNA nucleases; and one or more ligases, wherein each ligase is connected to or otherwise capable of forming a complex with one or more of the one or more DNA-nucleases.” This judicial exception is not integrated into a practical application and does/do not include additional elements that are sufficient to amount to significantly more than the judicial exception for the reasons that follow.
Subject Matter Eligibility Test for Products and Processes – Claim 1
Step 1 – Is the Claim to a Process, Machine, Manufacture, or Composition of Matter? YES
The claim is directed to an engineered composition, which is a composition of matter. The claim is directed to a statutory category.
Step 2A, Prong One – Does the Claim Recite an Abstract Idea, Law of Nature, or Natural Phenomenon? YES
Laws of nature and natural phenomena, as identified by the courts, include naturally occurring principles/relations and nature-based products that are naturally occurring or that do not have markedly different characteristics compared to what occurs in nature. MPEP 2106.04(c) outlines the markedly different characteristics analysis.
Claim 1 recites “An engineered composition for modifying polynucleotides.” The term “engineered composition” is interpreted as mixture of two or more elements which is designed, rather than naturally occurring. However, “a synthetic, artificial, or non-naturally occurring product… is not automatically eligible because it was created by human ingenuity or intervention.” “Instead, the key to eligibility of all non-naturally occurring products is whether they possess markedly different characteristics from any naturally occurring counterpart.” See MPEP 2106.04(b)(II). The phrase “for modifying polynucleotides” is interpreted as a reciting an intended use for the composition which does not structurally limit the claim, because the body of the claim recites a structurally complete invention.
The engineered composition comprises “one or more programmable DNA nucleases,” which is interpreted as encompassing agents with DNA cleaving function (e.g., “a protein or a small molecule,” or non-functional nucleases derived therefrom (e.g., dead Cas proteins). See at least paragraphs [0097] and [0181]. The term “programmable” is not explicitly defined by the specification, and is interpreted as requiring that the nuclease be amenable to modification to achieve a desired result, e.g., through mutagenesis. This encompasses essentially any known protein, and therefore, does not appear to further structurally limit the nucleases. The composition also comprises “one or more ligases” each “connected to or otherwise capable of forming a complex with” the one or more of the programmable DNA nucleases. A “ligase” is interpreted as an enzyme which joins breaks between adjacent nucleotides in a nucleic acid ([0131]). The phrase “connected to or otherwise capable of forming a complex” is interpreted as encompassing modes of direct physical connection (e.g., through a linker), and indirect connection within an “effective proximity” to achieve a desired effect, e.g., nuclease or ligase-activity ([0107]-[0110]).
The closest naturally occurring counterparts to the claimed engineered composition are I) the nucleoplasm of a mammalian cell comprising a DNA ligase IV-Artemis complex taught by De Ioannes (De Ioannes et al., 27 December 2012, Cell Reports, pg. 1505-1512), II) the cytoplasm of an M. tuberculosis cell comprising the multidomain multifunctional ATP-dependent DNA ligase taught by Pitcher (Pitcher et al., 2005, J. Mol. Biol. 351, pg. 531-544), and III) the nucleoplasm of a human cell comprising a MRN-DNA ligase IIIα/XRCC1 complex taught by Della-Maria (Della Maria et al., 2011, Journal of Biological Chemistry, Vol. 286, No. 39, pg. 33845-33853).
De Ioannes teaches that DNA ligase IV and Artemis, which possesses DNA nuclease activity, form a complex involved in the process of non-homologous end joining (NHEJ) in mammals (“LigIV is unique among other mammalian ligases in its ability to perform nuclear DNA DSB repair… Artemis… has a diverse array of nuclease activities and is a necessary factor for the processing of DBSs during NHEJ… Association-binding experiments indicate that the C-terminal region (C-ter) of Artemis directly interacts with the DNA binding domain (DBD) of LigIV,” pg. 1505, right col.). NHEJ takes place in the nucleus of a mammalian cell, in which the DNA that DNA ligase IV and Artemis act upon is suspended in the nucleoplasm. Thus, based on De Ioannes, the nucleoplasm of a mammalian cell is a naturally occurring counterpart to the instantly claimed composition.
Pitcher teaches that M. tuberculosis comprises a multidomain multifunctional ATP-dependent DNA ligase (Mt LigD) which “exhibits polymerase and nuclease activity in addition to DNA ligation activity” (pg. 531, right col.; Fig. 1). Based on Pitcher, the cytoplasm of a M. tuberculosis cell is a naturally occurring counterpart to the instantly claimed composition.
Della-Maria teaches that the MRN complex, comprising the nuclease Mre11, physically interacts with DNA ligase IIIα/XRCC1, in an alternative nonhomologous end joining (alt-NHEJ) pathway in human cells (“we demonstrate that DNA ligase IIIα/XRCC1 and MRN are constitutively associated in undamaged wild-type cells… we show that DNA ligase IIIα/XRCC1 and MRN physically and functionally interact to join DNA ends in a reaction that mimics alt-NHEJ,” pg. 33845-33846; “Cell Culture,” pg. 33846, left col.). This pathway takes place in the nucleus, and thus, based on Della-Maria, the nucleoplasm of a human cell is a naturally occurring counterpart to the instantly claimed composition.
In view of the foregoing, the engineered composition of claim 1 is not markedly different from the naturally occurring counterparts, and recites a product of nature judicial exception.
Step 2A, Prong Two – Does the Claim Recite Additional Elements that Integrate the Judicial Exception into a Practical Application? NO
The Supreme Court has long distinguished between principles themselves, which are not patent eligible, and the integration of those principles into practical applications, which are patent eligible. The phrase "integration into a practical application" requires an additional element or a combination of additional elements in the claim to apply, rely on, or use the judicial exception in a manner that imposes a meaningful limit on the judicial exception, such that it is more than a drafting effort designed to monopolize the exception.
As described above, the limitations of claim 1 are present in each of the natural counterparts. Claim 1 does not recite any additional elements that would integrate the natural product into a practical application.
Step 2B – Does the Claim Recite Additional Elements that Amount to Significantly More than the Judicial Exception? NO
The Supreme Court has identified a number of considerations for determining whether a claim with additional elements amounts to "significantly more" than the judicial exception(s) itself. The claim as a whole is evaluated as to whether it amounts to significantly more than the recited exception, i.e., whether any additional element, or combination of additional elements, adds an inventive concept to the claim (MPEP 2106.05).
Each limitation of claim 1 is present in the natural counterparts to the claimed invention. Claim 1 does not recite any additional elements which would amount to significantly more than the judicial exception.
Dependent Claims
The following claims are not markedly different from the naturally occurring counterpart(s), and do not recite any additional elements which would integrate the judicial exception into a practical application or amount to significantly more than the judicial exception.
Claim 2 recites that the one or more programmable DNA nucleases are “nickases,” which is interpreted as a nuclease which is capable of “nicking” a single DNA strand ([0181]). Garcia (Garcia et al., 2011, Nature, 479, pg. 241-244) teaches that Mre11 is a nickase (“Mre11 nicks the strand to be resected,” Abstract).
Claims 16-17 recite that the composition comprises a “donor molecule comprising a donor sequence configured for insertion into a target polynucleotide,” wherein the donor sequence is “a double-stranded oligonucleotide or polynucleotide.” The term “donor” is interpreted as referring to an intended use for the molecule and sequence. The claims are interpreted as requiring a nucleotide sequence, or a double-stranded nucleotide sequence, which may be inserted by any means into a target polynucleotide. The claims do not appear to limit the sequence of the donor molecule or donor sequence, because the target polynucleotide is unlimited in structure. The nucleoplasm of the mammalian and human cells taught by De Ioannes and Della-Maria possess double-stranded nucleotide sequences, i.e., the double-stranded DNA sequences of chromosomes, which would be “configured for insertion into a target polynucleotide,” e.g., through the process of homologous recombination taught by Kumari (Kumari et al., 2025, Current Opinion in Pharmacology, 80:102496), or through any non-naturally occurring means, e.g., CRISPR-Cas genome editing.
Claim 19 recites that the “donor sequence is protected from degradation,” which is interpreted as an inherent feature of the donor sequence due to the indefiniteness described above. Claim 19 is rejected for the reasons described immediately above.
Claim 20 recites that the “donor sequence is covalently or non-covalently attached” to one of the programmable DNA nucleases. The nucleases taught by De Ioannes and Della-Maria bind to the double-stranded DNA sequences of chromosomes in mammalian and human cells, and therefore, would be considered non-covalently attached thereto.
Claim 22 requires a “splint oligonucleotide” comprising a “region capable of hybridizing to a cleaved strand of the target polynucleotide and a region capable of hybridizing the donor molecule.” The term “splint” is interpreted as referring to an intended use for the oligonucleotide. Neither the target polynucleotide or donor molecule are limited to a specific sequence. The oligonucleotide is, therefore, not limited to any specific sequence, and is essentially any additional oligonucleotide which could be used as a “splint.” The nucleoplasm of the mammalian and human cells taught by De Ioannes and Della-Maria possess nucleotide sequences, i.e., portions of the double-stranded DNA sequences of chromosomes, which would comprise a region capable of hybridizing to a cleaved strand of a target polynucleotide and a region capable of hybridizing to a donor molecule.” For example, consider a donor molecule intended to replace a portion of a gene on a DNA strand (i.e., a target polynucleotide). The naturally occurring complementary DNA strand to the target polynucleotide would comprise regions capable of hybridizing to “a cleaved strand of the target polynucleotide” and “the donor molecule.”
Claim 23 requires that the donor sequence is configured to “introduce one or more mutations to the target polynucleotide.” As stated above, given that the target polynucleotide is unlimited in structure, the claim does not appear to further limit the sequence or structure of the donor molecule or donor sequence. The donor sequence remains essentially any nucleotide sequence. Claim 23 is rejected for the reasons described above for at least claims 16-17.
Claim 24 recites that “the one or more ligases are each covalently or non-covalently attached to at least one of the programmable DNA nucleases.” The naturally occurring counterparts taught by De Ioannes and Della-Maria comprise a ligase which forms a complex with the DNA nuclease, and therefore would be considered “non-covalently attached.” The naturally occurring counterpart taught by Pitcher comprises a ligase covalently attached (i.e., through peptide bonds) to the DNA nuclease.
Claims 25 and 27 recite the one or more ligases is/are capable of ligating a single-strand break, or a double-strand break, respectively. The ligases in the naturally occurring counterparts catalyze the formation of a covalent bond between a 5’ phosphate and 3’ hydroxyl group, i.e., they are capable of ligating a single-strand break. As illustrated by Kumari, the action of the ligases of De Ioannes and Della-Maria in NHEJ and alt-NHEJ results in the ligation of a double-strand break (see Fig. 1, middle and right panels).
Claim 29 recites that the one or more ligases is/are fused to a C-terminus or N-terminus, or both, of the programmable DNA nucleases. As shown in Fig. 1 of Pitcher, Mt-LigD comprises a ligase fused to the C-terminus of the DNA nuclease.
Claim 31 requires that the one or more programmable DNA nucleases comprise “one or more nuclear localization signals.” A “nuclear localization signal” is interpreted as a sequence which promotes localization of a nuclease in the nucleus. Transport of proteins into the nucleus from the cytoplasm (e.g., post-synthesis) is an active process mediated by the interactions of the cargo protein (i.e., its constituent amino acids), and the nuclear pore complex (NPC). The nucleases in the naturally occurring counterparts of De Ioannes and Della-Maria must, necessarily, comprise one or more nuclear localization signals, given that they are synthesized in the cytoplasm of eukaryotic cells, and function in nuclear processes (i.e., NHEJ or alt-NHEJ) as evidenced by De Ioannes and Della-Maria.
Claims 32-33 are directed to a vector composition comprising one or more vectors, or a single vector, comprising nucleic acid sequences encoding one or more components of the engineered composition of claim 1. The term “vector” is interpreted as referring to an intended use, which does not structurally limit the composition. The “one or more vectors” are interpreted as essentially any one or more nucleic acid sequences which encode one or more components of the engineered composition of claim 1. The closest naturally occurring counterparts to the claimed composition are the nucleoplasm and cytoplasm comprising the genomes, or portions thereof (e.g., chromosomes, single genes, or single-strand of a gene), encoding the components of the DNA nuclease-ligase complexes taught by De Ioannes, Pitcher, and Della-Maria.
Claim 34 requires that the one or more vectors “comprise viral vectors.” The claimed invention is essentially a composition comprising viral nucleic acid sequences encoding one or more components of the engineered composition of claim 1, i.e., one or more DNA nucleases and/or one or more ligases. Yamada (Yamada et al., 1999, Journal of Bioscience and Bioengineering, Vol. 88, Issue 4, pg. 353-361) teaches that the Chlorella virus (Phycodnaviridae) genome encodes DNA nucleases and DNA ligases (“DNA methyltransferase and DNA site-specific endonuclease,” pg. 357; “DNA restriction endonuclease,” Table 2; “DNA ligase. Chlorella viruses also encode a DNA ligase gene,” pg. 357-358). Thus, the closest naturally occurring counterpart to the claimed composition is the cytoplasm of a Chlorella cell infected by Chlorella virus, which would thereby comprise the aforementioned sequences. The claim does not recite any additional elements to the natural counterpart.
Claim 36 is directed to a “delivery composition,” comprising “a vector composition comprising one or more vectors comprising nucleic acid sequences encoding one or more components of the engineered composition of claim 1,” and a “delivery vehicle.” The term “delivery” is interpreted as referring to an intended use of the composition. The term “delivery vehicle” is also interpreted as referring to an element which is intended to be used for delivery. The specification does not explicitly define the term “delivery vehicle,” but states that “[e]xamples of delivery vehicles include vectors, viruses (e.g., virus particles), non-viral vehicles, and other delivery reagents described herein” ([0438]). The specification describes various carriers, e.g., “water, salt solutions… vegetable oils… carbohydrates such as lactose,” which the skilled artisan would understand to meet the scope of “other delivery reagents described herein” ([0617]). The closest naturally occurring counterparts to the claimed composition are the nucleoplasm and cytoplasm comprising the genomes, or portions thereof (e.g., chromosomes, single genes, or single-strand of a gene), encoding the components of the DNA nuclease-ligase complexes taught by De Ioannes, Pitcher, and Della-Maria. The nucleoplasm and cytoplasm comprise the recited “vectors,” as well as water, salt solutions, “lipids,” “proteins,” etc., encompassed by the term “delivery vehicle.”
Claim 38 states that “the delivery vehicle comprises ribonucleoproteins.” The claim essentially encompasses a composition comprising one or more vectors comprising nucleic acid sequences encoding one or more components of the engineered composition of claim 1, and a delivery vehicle comprising ribonucleoproteins. The closest naturally occurring counterpart to the claimed composition is the nucleoplasm comprising the genomes, or portions thereof (e.g., chromosomes, single genes, or single-strand of a gene), encoding the components of the DNA nuclease-ligase complexes taught by De Ioannes and Della-Maria. Fox (Fox and Stanek, 2017, Current Opinion in Cell Biology, 46:94-101) teaches the nucleoplasm comprises various nuclear bodies comprising ribonucleoproteins (“we focus on findings describing the role of nuclear bodies in the biogenesis of specific ribonucleoprotein complexes,” Abstract; “Nuclear bodies (NBs) are non-membrane bound structures in the nucleoplasm that… concentrate specific nuclear factors, namely proteins or RNAs,” pg. 94, left col.). Thus, in its natural state, the nucleoplasm of the cells taught by De Ioannes and Della-Maria comprise the recited “vectors” and “delivery vehicle,” i.e., water, salt solutions, etc., comprising “ribonucleoproteins.”
Claims 39-41 encompass a “cell comprising: the engineered composition of claim 1” wherein the cell may be a “eukaryotic cell,” and an “organism” comprising the cell. The closest naturally occurring counterparts to the claimed inventions are the mammalian and human cells taught by De Ioannes and Della-Maria, and M. tuberculosis taught by Pitcher.
Claim Rejections - 35 USC § 102 - Zymergen
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 1-2, 4, 6-11, 13-14, 16-17, 19, 23-25, 27, 29, 31-34, 36, and 38-41 are rejected under 35 U.S.C. 102(a)(1) and 102(a)(2) as being anticipated by Zymergen (DeLoache et al., WO 2018/013990 A1, published 18 January 2018; of record).
Regarding claim 1, Zymergen teaches an engineered composition for modifying polynucleotides, the composition comprising: one or more programmable DNA nucleases (e.g., "Cpf1," "Cas9"); and one or more ligases, wherein each ligase is connected to or otherwise capable of forming a complex with one or more of the one or more DNA nucleases (“the present disclosure teaches fusing a Cpf1 or other CRISPR polypeptide with a polypeptide with ligase activity,” "In some embodiments, a linker is used to genetically fuse an enzymatic ligase to a Cpf1 or other Targetable Enzyme gene to create an engineered, non-naturally occurring protein," "Fusion of protein subunits of a complex has been performed on other systems and can be accomplished ... by one skilled in the art with knowledge of the nucleic acid sequences to be fused to the Cas9 or Cpf1," [0123]-[0130]; "enzymatic ligases, include, but are not limited to, bacteriophage T4 ligase, T7 ligase ... Afu ligase," [120]; “the present disclosure teaches methods and compositions… methods of gene editing using any targetable DNA nuclease (e.g., Cpf1, Cas9…), [0102]; “Persons skilled in the art will immediately recognize that the aforementioned references to vectors encoding Cpf1 endonucleases are equally applicable to other CRISPR endonucleases or Targetable Enzymes,” [0180]).
Regarding claims 4, and 6-10, Zymergen teaches the one or more programmable DNA nucleases are one or more Cas polypeptides, wherein the one or more Cas polypeptides are a Cas9 polypeptide or Cas12 polypeptide, which is referred to as “Cpf1” throughout Zymergen (“the present disclosure teaches fusing a Cpf1 or other CRISPR polypeptide with a polypeptide with ligase activity,” "In some embodiments, a linker is used to genetically fuse an enzymatic ligase to a Cpf1 or other Targetable Enzyme gene to create an engineered, non-naturally occurring protein," "Fusion of protein subunits of a complex has been performed on other systems and can be accomplished ... by one skilled in the art with knowledge of the nucleic acid sequences to be fused to the Cas9 or Cpf1," [0123]-[0130]; “methods of gene editing using any targetable DNA nuclease (e.g., Cpf1, Cas9…), [0102]; “Persons skilled in the art will immediately recognize that the aforementioned references to vectors encoding Cpf1 endonucleases are equally applicable to other CRISPR endonucleases or Targetable Enzymes,” [0180]).
Regarding claims 2 and 11, Zymergen teaches the one or more Cas polypeptides is a nickase (“the systems and methods disclosed herein can be used with… Cas9 mutants that act as single stranded nickases,” [0112]).
Regarding claim 13, Zymergen teaches the engineered composition further comprises a first guide molecule (“the present disclosure teaches methods and compositions of vectors, constructs, and nucleic acid sequences encoding the gene editing complexes of the present disclosure,” [0167]; “the plasmids and vectors of the present disclosure will encode for the Cpf1 protein(s) and also encode the crRNA and/or donor insert sequences,” [0169]; Fig. 1). Zymergen teaches the guide molecule forms a complex with an RNA-guided DNA nuclease and comprises a guide sequence capable of directing site-specific binding to a first target sequence in a target polynucleotide (Fig. 1).
Regarding claim 14, Zymergen teaches a first target sequence on a first strand of a double stranded target polynucleotide, and a second target sequence on a second strand of the double stranded target polynucleotide (Figs. 2, 4). As shown in Fig. 2 and 4, the first and second target sequences define an intervening target region for insertion of a donor sequence. Zymergen also teaches methods with such a first and second target sequence (Fig. 7).
Regarding claims 16-17, Zymergen teaches the engineered composition further comprises a donor molecule comprising a donor sequence configured for insertion into a target polynucleotide (“the plasmids and vectors of the present disclosure will… also encode the crRNA and/or donor insert sequences of the present disclosure,” [0169]). Zymergen teaches donor sequences which are double-stranded DNA (see Fig. 2 and 4, which illustrate a donor insert sequence with two strands).
Regarding claim 19, the claim recites that the “donor sequence is protected from degradation,” which is interpreted as an inherent feature of the donor sequence due to the indefiniteness described above. Claim 19 is rejected for the reasons described immediately above for claims 16-17.
Regarding claim 23, Zymergen teaches the donor sequence is configured to insert a gene or gene fragment at one or multiple copies of the target polynucleotide (“GOI,” Fig. 2; “repair fragment,” “Reconstituted gene (e.g., marker/GFP/etc),” Fig. 4).
Regarding claim 24, Zymergen teaches the one or more ligases are each covalently or non-covalently attached to at least one of the programmable DNA nucleases (“the present disclosure teaches fusing a Cpf1 or other CRISPR polypeptide with a polypeptide with ligase activity,” [0128]; “In some embodiments, a linker is used to genetically fuse an enzymatic ligase to a Cpf1 or other Targetable Enzyme gene,” [0129]).
Regarding claims 25 and 27, Zymergen teaches the ligase “is an enzymatic ligation reagent or catalyst that, under appropriate conditions, forms phosphodiester bonds between the 3’-OH and the 5’-phosphate of adjacent nucleotides in DNA molecules, RNA molecules, or hybrids” ([0123]). Thus, Zymergen teaches one or more ligases which are capable of ligating a single-strand break. Zymergen also teaches that the ligase is used to “sew” DNA back together ([0121]). See also Zymergen’s examples comprising ligation of double-strand breaks (Fig. 2, 4, and 7). Thus, Zymergen also teaches one or more ligases which are capable of ligating a double-strand break.
Regarding claim 29, Zymergen teaches that the sequence encoding a ligase is linked at the 3’ end or 5’ end of a sequence encoding a programmable DNA nuclease ([0130]). The skilled artisan would understand that the 3’ end and 5’ ends of the sequences referred to by Zymergen correspond to N- and C-termini of the encoded protein. Thus, Zymergen teaches that the one or more ligases are fused to a C- or N-terminus of a programmable DNA nuclease.
Regarding claim 31, Zymergen teaches the one or more programmable DNA nucleases comprises one or more nuclear localization signals ([0136]-[0138]).
Regarding claims 32-33, Zymergen teaches a vector composition comprising one or more vectors comprising nucleic acid sequences encoding one or more components of the engineered composition ([0087]; [0167]-[00180]; [0187]-[0190]). Zymergen teaches “single vector” compositions, which is interpreted as one vector, e.g., a plasmid, encoding one or more components of the engineered composition (“introducing into the cell, one or more vectors encoding for…,” “introducing into the cell a CRISPR complex encoded in one or more vectors,” see at least embodiments 12-18, and 22-26.1 on pgs. 20-21).
Regarding claim 34, Zymergen teaches viral vectors encoding one or more components of the engineered composition (“Vectors can be… viruses,” [0087]; “persons having skill in the art will recognize that viral vectors or plasmids for gene expression can be used to deliver the complexes herein,” [0179]).
Regarding claims 36 and 38, Zymergen teaches delivery compositions comprising one or more components of the engineered composition of claim 1, and a delivery vehicle, wherein the delivery vehicle comprises ribonucleoproteins (“Virus-like particles (VLP) can be used to encapsulate ribonucleoprotein complexes or recombinant expression, and purified ribonucleoprotein complexes disclosed herein can be purified and delivered to cells via electroporation or injection,” [0179]).
Regarding claim 39, Zymergen teaches a cell comprising the engineered composition of claim 1, or a vector composition comprising one or more vectors encoding one or more components of the engineered composition of claim 1 (“the present disclosure teaches methods for getting exogenous protein (Cpf1 and DNA ligase), RNA (crRNA), and DNA (target DNA to be ligated into the genome”) into the cell… Various methods for achieving this have been described previously including direct transfection of protein/RNA/DNA or DNA transformation followed by intracellular expression of RNA and protein,” [0176]; [0179]-[0180]).
Regarding claim 40, Zymergen teaches eukaryotic cells, including human cells (“In some embodiments, the vectors of the present disclosure may be introduced into the host cells using any of a variety of techniques,” [0174]; “host cell (e.g., bacteria, yeast cell, fungal cell, CHO, human cell, etc.,” [0071]; “In some embodiments, viable genome-editing tools must be delivered to the nucleus of eukaryotic cells,” [0137]).
Regarding claim 41, Zymergen teaches organisms (“In yet other embodiments, the genome-editing tools of the present disclosure are used in organisms without nuclei,” [0137]).
Notice to Joint Inventors
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 Rejections - 35 USC § 103 – Zymergen in view of Kennedy
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.
Claims 20 and 22 are rejected under 35 U.S.C. 103 as being unpatentable over Zymergen (DeLoache et al., WO 2018/013990 A1, published 18 January 2018; of record) as applied to claims 1-2, 4, 6-11, 13-14, 16-17, 19, 23-25, 27, 29, 31-34, 36, and 38-41 above, and further in view of Kennedy (Kennedy et al., 2 March 2017, US 2017/0058298 A1).
The teachings of Zymergen are described above and applied as to claims 1-2, 4, 6-11, 13-14, 16-17, 19, 23-25, 27, 29, 31-34, 36, and 38-41 therein.
Regarding claim 22, the term “splint” is interpreted as referring to an intended use for the oligonucleotide. Neither the target polynucleotide or donor molecule are limited to a specific sequence. The oligonucleotide is, therefore, not limited to any specific sequence, and is essentially any additional oligonucleotide which could be used as a “splint” between the target polynucleotide and donor molecule, i.e., an oligonucleotide which “comprises a region capable of hybridizing to a cleaved strand of the target polynucleotide and a region capable of hybridizing to the donor molecule.”
Zymergen does not teach that the engineered composition further comprises a splint oligonucleotide as interpreted above (claim 22), or that the donor sequence of the donor molecule is covalently or non-covalently attached to the one or more programmable DNA nucleases (claim 20).
However, Kennedy teaches splint oligonucleotides (“adaptor segment”) designed to “tether[] [] the guide RNA to polynucleotide sequences that can act as donors for homologous recombination (donor polynucleotides)” ([0038]-[0066]). Kennedy teaches that the splint oligonucleotides comprise a region capable of hybridizing to a donor sequence in a donor molecule, and a region capable of hybridizing to a target sequence (“the adaptor segment of the guide RNA comprises ssDNA or ssRNA adapted for hybridizing to a polynucleotide (e.g., a donor polynucleotide…) through Watson Crick base-pairing,” [0044]; Fig. 3-4; “the crRNA segment comprises a guide sequence that is capable of hybridizing to a target sequence,” [0068]-[0078]). Kennedy teaches that the splint oligonucleotides result in non-covalent attachment of the DNA nuclease (i.e., Cas protein) to the donor molecule comprising the donor sequence (“When loaded with the guide RNA:donor polynucleotide complex, the Cas protein will bring the donor polynucleotide to a target cleavage site,” [0039]). See also Fig. 4-6 which illustrate a Cas protein complexed with a gRNA comprising an adaptor segment, wherein the adaptor segment is attached to a donor molecule. These teachings meet the limitations of claims 20 and 22.
Kennedy teaches use of the splint oligonucleotides in CRISPR-Cas genome editing strategies similar to that of Zymergen (see at least Fig. 4-6; [0017]; [0129]-[0138]). Kennedy teaches that “[h]omologous gene targeting approaches have been used… for gene correction, and in theory, for the correction of mutations linked with monogenic diseases. However, this application is difficult, due to the low efficiency of the process” ([0007]; [0005]). Kennedy teaches that splint oligonucleotides “increase[] the local concentration of donor polynucleotide around the Cas cleavage site and increases the likelihood that a homologous recombination (HR) event will occur” ([0039]).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have included a splint oligonucleotide taught by Kennedy in an engineered composition of Zymergen. It would have amounted to a simple combination of a known element usable in methods of CRISPR-Cas genome editing, with a known composition for CRISPR-Cas genome editing, by known means to yield predictable results. The design and synthesis of splint oligonucleotides, and their combination with compositions comprising DNA nucleases, was known in the art as evidenced by Kennedy. The skilled artisan would have expected Kennedy’s splint oligonucleotide and Zymergen’s engineered composition to have the same functions described by the respective references when combined; the splint oligonucleotide would tether the donor molecule to the gRNA, and thereby, increase the local concentration of the donor molecule and efficiency of its integration at the target polynucleotide by the elements in the composition of Zymergen. The skilled artisan would have been motivated to arrive at the claimed engineered composition because Zymergen and Kennedy are both concerned with site-specific insertion of donor sequences via CRISPR-Cas strategies, and Kennedy teaches an element which is likely to improve the efficiency of these methods.
Conclusion
No claims are allowed.
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/JENNA L PERSONS/Examiner, Art Unit 1637
/Soren Harward/Primary Examiner, TC 1600