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 .
Election/Restrictions
Applicant’s election without traverse of the following invention
Group II (claims 42-67 and 91) drawn to a method for genetically modifying a cell in the reply filed on 16th Sept, 2025 was previously acknowledged.
Applicants have amended claim 91 to require a limitation of withdrawn group I: a method for preparing ssDNA. Therefore, as previously conveyed, claim 91 will be examined to the extent that does not require limitations from a withdrawn group.
Claims 1-18, 19-27, 28-32, 33-41, 68-83, and 84-90, remain withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention and newly amended claim 91 is being added to the group of withdrawn claims.
Priority
This application is filed under 35 U.S.C. 371 as a national stage of international application PCT/US2020/054526, filed on Oct 07, 2020, which claims domestic priority under 35 U.S.C. 119(e) to U.S. provisional application no. 62/912,115, filed on Oct 8, 2019.
Amendments and Status of the Claims
This action is in response to papers filed 15 May 2026 in which claims 42-47, 50-67 and 91 were amended, no claims were canceled, and no new claims were added. All of the amendments have been thoroughly reviewed and entered.
Any rejection or objection not reiterated herein has been overcome by amendment.
Applicant's remarks have been fully considered and are persuasive. As a result, i. the priority date has been corrected; ii. the amendments have overcome previous objections, §112a and §102 rejections. However, in view of amendments, and claims previously not examined on account of objection, are being examined in this office action. Accordingly, new rejections have been presented in this Office Action. This Office Action is Final.
Status of Claims
Claims 42-67 and 91 are under consideration.
Withdrawn Claim Objections
Applicants have amended claims 50-67 and 91 to remove their dependency on another multiple dependent claim. Accordingly, the objection to claims 50-67 and 91 has been withdrawn.
Withdrawn Claim Rejections - 35 USC § 112a
Scope of Enablement
Applicants have amended claims 42-49 by changing in vivo to in vitro. Accordingly, the § 112a rejection of claims 42-49 has been withdrawn.
Withdrawn Claim Rejections - 35 USC § 102
Applicants have amended claims 42-49. Accordingly, the § 102 rejection of claims 42-49 is withdrawn.
Applicants have persuasively argued that in view of amendments, Cemák no longer anticipates instant claims.
Therefore, and as stated above, the § 102 rejection of claims 42-49 is withdrawn.
New Claim Rejections on account of Amendment follows.
Claim Rejections - 35 USC § 112b
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claim 55 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 55 recites the limitation "the DNA binding protein" in first line of the claim. But claim 55 depends from claim 54, which recites “the non-specific DNA binding protein”. There is insufficient antecedent basis for the limitation "the DNA binding protein" in claim 55.
Claim Rejections - 35 USC § 112a
Written Description
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 55-56 and 91 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.
The purpose of the written description requirement is to ‘ensure that the scope of the right to exclude, as set forth in the claims, does not overreach the scope of the inventor’s contribution to the field of art as described in the patent specification.” Ariad Pharm., Inc. v. Eli Lilly & Co., 598 F.3d 1336, 1353-54 (Fed. Cir. 2010) (en banc) (quoting Univ. of Rochester v. G.D. Searle & Co., 358 F.3d 916, 920 (Fed. Cir. 2004)). To satisfy the written description requirement, the specification must describe the claimed invention in sufficient detail that one skilled in the art can reasonably conclude that the inventor had possession of the claimed invention. Vas-Cath, Inc. v. Mahurkar, 935 F.2d 1555, 1562-63, 19 USPQ2d 1111 (Fed. Cir. 1991). See also MPEP 2163.04.
For a claim to a genus, a generic statement that defines a genus of substances by only their functional activity does not provide an adequate written description of the genus. Reagents of the University of California v. Eli Lilly, 43 USPQ2d 1398 (CAFC 1997).
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 a disclosure of a representative number of species to describe the complete structure of the claimed genus and/or disclosure of a complete or partial structure, physical and/or chemical properties, functional characteristics, structure/function correlation, and any combination thereof.
Regarding claims 55-56 and 91, while Applicant’s disclosure provides support for
SSB as the ssDNA binding protein,
the full breadth of ssDNA binding proteins, or a variant thereof, as recited, are not supported by Applicant’s disclosure for the reasons stated below.
Scope of the Claimed Invention
In the instant case, the genera are:
ssDNA binding protein (claim 91) and variants of (claims 55-56).
The disclosure defines the genera of ssDNA binding proteins to encompass all proteins known to interact with ssDNA [0093]. Within this genera is the sub genera of single stranded DNA binding proteins that are non-specific, in that it does not bind to a specific DNA sequence [0093], which is a large genus. If variants or homologs of ssDNA binding proteins are included, this genera becomes even larger. Thus, instant claims encompass all possible proteins of all possible species, sizes, or even fragments of known DNA binding proteins, which is an unfathomable number of proteins.
Also, given a broadest reasonable interpretation, the recited “variant thereof” is interpreted as being unlimited with respect to nucleotide sequence.
Disclosure of Structure and Disclosure of Species
ssDNA binding protein
Regarding the ssDNA binding proteins, Applicant discloses the following ssDNA binding protein: SSB (para. [0144] onwards) encompassing SSB from different species. E.coli SSB, SEQ ID NOs:1 and 4; Citrobacter genus SSB, SEQ ID NOs:2 and 3 and consensus is SEQ ID NO 19; genus Thermus SSB, SEQ ID NOs:5 – 7 and consensus is SEQ ID NO 20.
Sequences of some non-SSB ssDNA binding proteins listed are: SEQ ID NOs: 12-18: (Table 3).
The working examples solely utilize E.coli SSB.
Structure/Function Correlation
The instant specification fails to disclose any details of any of the components required for the method as claimed other than simply listing them in the background.
ssDNA binding protein
Regarding the ssDNA binding proteins, the specification only describes SSB proteins from 3 species of bacteria which is not representative of such a diverse genus of ssDNA binding proteins.
Since the ssDNA binding proteins include non-specific ssDNA binding proteins, without any disclosure on how to identify such proteins, the disclosed sequences do not represent the large number of possible structurally distinct proteins with functional consequences of binding to ssDNA in order to enable integration of the insert in a particular loci that are within the scope of instant claims.
Because the breadth of the claims encompasses an unfathomably large genus of ssDNA binding proteins, and the specification has not provided sufficient structural-functional relationship for the claimed genera, a person of ordinary skill in the art cannot envision carrying out the method of genome editing with any generic SSB or variant thereof from the instant disclosure. Furthermore, a person of ordinary skill in the art would not know how to tailor any of the components to achieve the method at a predetermined locus in the genome.
Guidance from the Prior Art and the Art
ssDNA binding protein
All ssDNA binding proteins are characterized by the presence of OB-fold (reviewed by Guo and Malik, Biomolecules . 2022 Aug 26;12(9):1187). The OB fold results in non-specific binding of the proteins with its binding partners, a consequence of a lack of sequence conservation among OB folds. Guo and Malik recite on 2nd to last para on pg. 2:
While the sequence diversity of the OB fold provides a variety of interaction surface for recognition of different ssDNA sequences, especially for the sequence-specific OB-fold SSBs, it presents challenges in identification of new SSBs based on sequence information alone.
Regarding the genera of SSB, the ssDNA binding proteins from bacteria, Applicant is directed to the review by Oakley (PROTEIN SCIENCE 2019 | VOL 28:990–1004) which evidences that the SSB, which is narrower than instant claims, varies between bacterial species, and modes of binding to ssDNA depends upon ion concentration.
Interactions between components in genome editing
Church et al. (International Publication No. WO 2017/184227 A2) discloses a method of altering a target nucleic acid sequence within a cell by providing to the cell a donor nucleic acid, a single strand annealing protein, and a single strand DNA binding protein (see, e.g., abstract). Church discloses the lambda Red operon, which comprises the SSAP Redbeta and the 5’-3’ exonuclease Redgamma and further discloses that lambda beta improves recombination of ssDNA into the E. coli chromosome (see page 2, second paragraph). Church discloses SSBs protect ssDNA and inhibit complementary annealing and that lambda beta interacts with SSB to facilitate recombination (see page 33). Church discloses species tropism between SSAPs and SSBs and evaluates foreign SSAPs for a gain of function when they are co-expressed with a phylogenetically matched SSB homologs (see Example V on page 35). Church discloses that oligo recombination via SSAPs is enhanced by expressing its corresponding SSB (see page 37, first paragraph; Fig. 6A-6B depict that co-expression of species-matched SSAP-SSB pairs enable gain of recombinase function; Example 5). Given the species tropism disclosed by Church, and without information about porting components across species, there is uncertainty regarding the combination of components required for instant method.
Written Description - Conclusion
In summary, the claims embrace targeted, enzymatic integration of a gene (of any nucleotide sequence) into any cell type, exemplified by rescue of GFP gene in an animal cell line using CRISPR complex. The evidence of record suggests that the art of forming such integrations is unpredictable. The specification as filed provides no guidance that would allow one of skill in the art to predict which SSB and variants thereof, will support genomic integration. The disclosure of one SSB that supports integration does not provide the basis for predicting which of the practically infinite number of ssDNA binding proteins embraced by the claims will have the requisite activity of improving gene integration. The disclosure of only one SSB with two nucleotide sequences as donor DNAs, in an unpredictable art would not convey to one of skill that Applicant was in possession of the breadth of the genus that is claimed. Thus, in the face of an unpredictable art, one of ordinary skill in the art could not envision the requisite structural elements and the elements that could be modified from the disclosures of the application or art at the time of filing.
Examiner Suggestion: i) Amend the claims to recite the species of donor DNA, ssDNA binding protein and the cutting entity in independent claims for which there is support in the disclosure; ii) delete the phrase “or variant thereof” in claims 55-56.
For e.g., A method for …, comprising introducing into the cell a ssDNA and ssDNA binding protein of SEQ ID NO:… in the presence of a nucleic acid cutting entity or a nucleic acid encoding the nucleic acid cutting entity may be considered.
Scope of Enablement
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claim 91 is 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:
A method for genetically modifying an animal cell in vitro, the method comprising introducing into the cell a donor DNA, at least one ssDNA binding protein or nucleic acid encoding the at least one single stranded DNA binding protein, and at least one nucleic acid cutting entity or a nucleic acid encoding the nucleic acid cutting entity
does not reasonably provide enablement for methods of modifying an animal cell in vivo (including all administration types, vector types, dosage types, and subject types)
The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to practice the method of the invention commensurate in scope with these claims.
Wands Factors
The factors to be considered in determining whether undue experimentation is required are summarized In re Wands 858 F.2d 731, 8 USPQ2nd 1400 (Fed. Cir, 1988). The Court in Wands states: “Enablement is not precluded by the necessity for some 'experimentation.'” Clearly, enablement of a claimed invention cannot be predicated on the basis of quantity of experimentation required to make or use the invention. “Whether undue experimentation is needed is not a single simple factual determination, but rather is a conclusion reached by weighing many factual considerations.” (Wands, 8 USPQ2d 1404). The factors to be considered in determining whether undue experimentation is required include: (1) the quantity of experimentation necessary, (2) the amount or direction or guidance presented, (3) the presence or absence of working examples, (4) the nature of the invention, (5) the state of the prior art, (6) the relative skill of those in the art, (7) the predictability or unpredictability of the art, and (8) the breadth of the claims. While all of these factors are considered, a sufficient amount for a prima facie case is discussed below.
Breadth of the Claims
Instant claims encompass:
“modifying”
This includes all types of genetic modification including all possible editing types such as but not necessarily limited to CRISPR, TALEN, ZFN or Mega nucleases.
“a cell”
This encompasses all possible types of cells in vitro from all possible species types or in vivo in all possible subject types
“introducing … in to the cell”
This encompasses all types of delivery in vitro or in vivo
Regarding in vivo delivery, this includes all possible routes of delivery.
“a single stranded DNA”
Regarding both in vitro and in vivo delivery, this encompasses all possible single stranded DNA delivery vehicles including but not necessarily limited to viral (such as but not necessarily limited to adenoviruses, lentivirus, or AAVS) and non-viral vector types (such as but not necessarily limited to LNPs or naked DNA).
Direction or Guidance Presented
While contemplating methods in vivo in subjects (para. [0105, 0140 - 0141, and 0151] see for example para. [0140] Some embodiments include compositions and methods designed to result in high efficiency of genome editing in cells (e.g., eukaryotic cells such as plant cells and animal cells, such as insect cells and mammalian cells, including mouse, rat, hamster, rabbit and human cells). Applicant provides no guidance on in vivo methods.
Present Working Examples
Applicant discloses Examples 1-3 and 6, which appear to be drawn to in vitro gene editing of cells of a human cell line in vitro.
A description of Examples is discussed in the Written Description section.
in vitro Cell Types:
293FT cells (Examples 1-2, and 6) U2OS cells (Example 3) are described.
Nuclease Types:
CRISPR/Cas9 RNP (Examples 1 - 3) and variants of CRISPR/Cas9 RNP (Example 6) were tested. Nucleotides were introduced in to cells with a transfection reagent.
Electroporation of different single stranded DNA binding proteins on delivery of Cas9 ribonucleoprotein (RNP) into EmGFP cell line (having a disrupted EmGFP gene) with a single stranded donor DNA (approximately 100 nucleotides) was also performed and depicted in Fig. 1.
Absent Working Examples
Since the cells recited in claims are unlimited, the scope of the claims reads on cells within a subject. Importantly, no working examples are provided that are drawn to in vivo gene editing of cells, which is encompassed by instant claims.
State of the Art and Unpredictability of the Art
Providing to a cell and in vitro vs. in vivo
As stated above, while Applicant’s broad claim encompasses in vivo embodiments Applicant only provides working examples of gene editing in vitro.
Regarding translation of in vitro methods to in vivo methods, Applicant is directed to the post-filing art of Mattes (Current Opinion in Toxicology 2020, 23-24:114–118). Matts evidences that around the time of filing, and post-filing, translating in vitro data to in vivo data is unpredictable. Specifically, Mattes evidences “the challenge of validating a system’s performance and extrapolating it’s responses to those of an animal or human remains” (abstract) and “the question of relevance to the in vivo setting remains an issue” (Summary; pg. 116).
Accordingly, because translation of in vitro methods to in vivo methods is still unpredictable, Applicant cannot be enabled for instant claims, which encompass in vivo methods.
Applicant is also directed to the post-filing art of Zhang (J Cell Physiol. 2021 Apr;236(4):2459-2481.). Zhang teaches significant challenges also remain before CRISPR/Cas technology can be routinely employed in the clinic for treating different genetic diseases, which include toxicity and immune response of treated cells to CRISPR/Cas component, highly throughput delivery method, and potential off‐target impact (abstract). Zhang teaches the off‐target effect is one of the major concerns for CRISPR/Cas9 gene therapy, more research should be focused on limiting this impact by designing high specific gRNAs and using high specificity of Cas enzymes. Modifying the CRISPR/Cas9 delivery method not only targets a specific tissue/cell but also potentially limits the off‐target impact (abstract).
Thus the art before the effective filing date of the claimed invention teaches methods of administering gene editing components to a cell were highly unpredictable, with challenges of a lack a specificity, adverse immune responses with the nucleases and cleavage agents, as well as questionable translatability of rat in vivo results to other mammal for specific results, such as therapeutic effects or providing a predictable amount of such a composition to get the intended result. Further, the post-filing arts of Zhang and Mattes described above demonstrate that these unpredictability and hurdles to in vivo embodiment persist from the prior time of effective filing and post-filing.
Unpredictability of the Art and the Quantity of Experimentation
As the arts of Mattes and Zhang demonstrate, the obstacles that hinder the use of the claimed method in in vivo embodiments are not easy tasks to be done or solely routine experimentation to enabled particular embodiments of the claimed method. The type of experimentation would require new methodologies. This level of experimentation goes beyond what would be routine optimization know at the time of filing. As such, the amount of experimentation would be undue.
The physiological art is recognized as unpredictable (MPEP 2164.03). As set forth in In re Fisher, 166 USPQ 18 (CCPA 1970), compliance with 35 USC 112(a) requires: “That scope of claims must bear a reasonable correlation to scope of enablement provided by specification to persons of ordinary skill in the art; in cases involving predictable factors, such as mechanical or electrical elements, a single embodiment provides broad enablement in the sense that, once imagined, other embodiments can be made without difficulty and their performance characteristics predicted by resort to known scientific laws; in cases involving unpredictable factors, such as most chemical reactions and physiological activity, scope of enablement varies inversely with degree of unpredictability of factors involved.” Moreover, the courts have also stated that reasonable correlation must exist between scope of exclusive right to patent application and scope of enablement set forth in the patent application (27 USPQ2d 1662 Ex parte Maize!.). In view of the foregoing, due to the lack of sufficient guidance provided by the specification regarding the issues set forth above, the state of the relevant art, and the breadth of the claims, it would have required undue experimentation for one skilled in the art to practice the instant broadly claimed invention.
Scope of Enablement - Conclusion
In conclusion, the breadth of the claims lacks enablement because the specification provides limited working examples in vitro and the specification provides no guidance and no working examples of the claimed method in vivo. The art at the time of effective filing fail to provide specific guidance that supplement to shortcomings of the specification and further teaches that the breadth of claims cannot predictably be performed. Further, a great deal of new methodology would need to be developed to enable the full breadth of the claims and this level of experimentation is undue.
Claim Interpretation
Claims 42 - 47 recite, method for genetically modifying a cell, method for improving targeting efficiency of a nucleic acid cutting entity for genetic modification of a cell, method for reducing off-target integration of a donor DNA during genetic modification of a cell, method for enhancing delivery of a donor DNA to a cell for genetic modification of the cell, method for reducing degradation of a donor DNA for genetic modification of a cell, method for genetically modifying a cell, and method for genetically modifying a cell, respectively.
These recitations are preamble recitations. MPEP 2111.02 (II) states:
If the body of a claim fully and intrinsically sets forth all of the limitations of the claimed invention, and the preamble merely states, for example, the purpose or intended use of the invention, rather than any distinct definition of any of the claimed invention’s limitations, then the preamble is not considered a limitation and is of no significance to claim construction. Pitney Bowes, Inc. v. Hewlett-Packard Co., 182 F.3d 1298, 1305, 51 USPQ2d 1161, 1165 (Fed. Cir. 1999). See also Rowe v. Dror, 112 F.3d 473, 478, 42 USPQ2d 1550, 1553 (Fed. Cir. 1997) ("where a patentee defines a structurally complete invention in the claim body and uses the preamble only to state a purpose or intended use for the invention, the preamble is not a claim limitation"); Kropa v. Robie, 187 F.2d at 152, 88 USPQ2d at 480-81 (preamble is not a limitation where claim is directed to a product and the preamble merely recites a property inherent in an old product defined by the remainder of the claim); STX LLC. v. Brine, 211 F.3d 588, 591, 54 USPQ2d 1347, 1350 (Fed. Cir. 2000) (holding that the preamble phrase "which provides improved playing and handling characteristics" in a claim drawn to a head for a lacrosse stick was not a claim limitation).
In the instant case, the body of the claims recites active steps such as, forming a composition … and introducing into the cell … .
Therefore, the stated recitations of the preamble of these claims are not considered a limitation and are of no significance to claim construction.
Response to Arguments
Applicant’s arguments, see Pgs. 13 - 14, filed 15th May2026, with respect to amended claim interpretation have been fully considered and are persuasive. The recitations of A. in vitro and B. under conditions that allow for genetically modifying the cell at a predetermined locus, are limitations of significance in determining patentability and will be considered.
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) 42-59 and 61-67 are rejected under 35 U.S.C. 103 as being unpatentable over Cemák (US 2020/0407754 A1 published Dec. 31, 2020, supported by its provisional application 62/866,317, filed on Jun 25, 2019) in view of Church et al. (International Publication No. WO 2017/184227 A2). Claim 53 is evidenced by Wikipedia (at wikipedia.org [retrieved on 2026-01-12], retrieved from the Internet on 12th Jan 2026 from <URL: https://www.wikipedia.org/>, 2 pgs.) and Claim 58 is evidenced by Park (US 11293019 B2).
Cemák discloses methods for genome editing and improving CRISPR/Cas-mediated genome editing and teach that to do so, the cell must be contacted with a composition comprising an HDR-enhancer (see, e.g., title and abstract). HDR-enhancer is also an HDR promoting agent, and comprises: an SSAP, an exonuclease, and an SSB. Cemák teach these components are required for methods of modification of the target editing site of the eukaryotic cell genome and increasing efficiency of HDR-mediated genome editing (claim 1, table B), increasing HDR precision (reducing off-target integration) ([0284] and Table 4A), transient expression of HDR promoting agents (enhancing delivery of a donor DNA) ([0081] onwards), and improving functionality of polynucleotides by including additional nucleotide sequences into polynucleotides to provide useful functionality by complexing with a non-nucleic acid element (reducing degradation) ([0083]); where the method comprises the step of providing genome editing molecules comprising (i) at least one sequence-specific endonuclease which cleaves a DNA sequence in the target editing site; and (ii) a donor template DNA molecule having homology to the target editing site; template nucleic acids for correcting mutation having homology arms, and the above mentioned HDR promoting agents such as SSB [0006].
Regarding claims 42 – 47, Cemák discloses a method for genetically modifying a cell and improving targeting efficiency, the method comprising introducing into the cell: (i) a donor template (donor DNA molecule);(ii) SSB (a single stranded DNA binding protein) or nucleic acid encoding SSB; and (iii) at least one sequence-specific endonuclease which cleaves a DNA sequence in the target editing site or at least one polynucleotide encoding the sequence-specific endonuclease (at least one nucleic acid cutting entity or nucleic acid encoding the at least one nucleic acid cutting entity) (see, e.g., [0245]; nucleic acid that encodes… SSB [0169]).
Regarding claims 48 and 49, Cemák discloses many examples of nucleases and specifically, Cas nuclease (nucleic acid cutting entity) (e.g. [0100]; see Fig. 1, [0017], and Examples starting on pg. 48 for Cas).
Regarding claims 50 - 52, Cemák teaches the construction of vectors encoding the various polynucleotides required for the method of modifying a cell, followed by transfection of the cells to be modified (polynucleotides encoding donor templates, Cas polynucleotides (Cas9 and gRNA), and Lambda RED (SSB and other proteins)) (Example 1 – 2, starting on pg. 48).
With respect to the order of steps recited in claims 50-52, it is noted that the courts have held that any order of performing process steps is prima facie obvious in the absence of new or unexpected results (In re Gibson, 39 F.2d 975, 5 USPQ 230 (CCPA 1930); Ex parte Rubin, 128 USPQ 440 (Bd. App. 1959)). See MPEP §2144.04 IV C.
In the instant case, the claimed order of steps is an obvious variant of the steps of the cited prior art.
Regarding claim 53, Cemák teaches SSB, as discussed above. As evidenced by pg. 1 of Wikipedia, SSB has an OG-fold.
Regarding claim 54-56, Cemák discloses that the SSB is an E. coli SSB (see, e.g., [0061, 0263]; SEQ ID NOs. in Table 3).
Regarding claims 57 and 61, Cemák teaches the SSB comprises a NLS (A DNA sequence encoding a tobacco c2 nuclear localization signal (NLS) of SEQ ID NO:15 was operably linked to the DNA sequences encoding the … and the E. coli SSB [0264]).
Regarding claim 62, Cemák teaches polynucleotides of their invention were introduced into the cell by PEG-mediated transfection essentially according to published procedures (citing Cermak et al. 2017, [0267]).
Regarding claim 63, Cemák teaches HDR promoting agents of their invention of their invention were introduced into the cell by electroporation [0085].
Regarding claims 65-66, Cemák teaches donor template DNA was added either as a linear double stranded DNA molecule, or as part of a circular vector flanked by specific nuclease recognition sequences [0111, 0283].
Regarding claim 67, Cemák teaches an example wherein a 42-bp heterologous sequence flanked on both sides by homology arms about 600-800 bp in length, are introduced into the cell [0265]. This example encompasses instant recitation of between 35 and 10,000 nucleotides long.
Cemák does not explicitly disclose a method or a system where the donor DNA molecule is contacted with HDR promoting agent comprising SSB forming a composition prior to the method step of introducing the composition into the cell, as recited in claims 42-47, regarding claim 64, Cemák does not disclose that the donor DNA is a single stranded DNA, and regarding claims 58-59, Cemák does not teach advantages of the SSB protein with respect to protection from degradation or improving targeting efficiency.
Church teaches methods of in vitro recombination-mediated genetic engineering including providing in a suitable in vitro environment a target nucleic acid sequence, a target cell's translational machinery (i.e., those proteins and other components responsible for translation within the cell), a recombinase and a single strand binding protein and a donor nucleic acid (i.e., a single stranded nucleic acid, a single stranded DNA, a double stranded nucleic acid or a double stranded DNA), wherein either one or both of the recombinase and a single strand binding protein are foreign to the cell. The pair of the recombinase and the single strand binding protein, in combination with the host cell's translational machinery, is sufficient to insert a ssDNA sequence into a target nucleic acid sequence (see para bridging pgs. 8-9).
Church further teaches an assay, Fluorescent oligo quenching assay, see pgs.6-7, and Fig. 5, wherein the significance of SSB in protecting ssDNA is seen (see page 2, second paragraph). Pertinent to this rejection is Church recitation from top of pg. 7:
If the oligos are coated with SSB prior mixing, they will be prevented from annealing unless additional factors are able to remove the inhibition.
Church further teaches the C-terminal 8-amino acid tail of SSB is required for its interaction with a HDR enzyme (lambda beta, middle of pg. 7). Thus, Church discloses SSBs protect ssDNA and inhibit complementary annealing (of donor DNA) (see page 33).
Church teaches advantages of the SSB protein with respect to protection from degradation or improving targeting efficiency. See recitation from pg. 34:
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Church teaches the amounts of SSB protein that can bind until saturation. See Fig. 4D.
As evidenced by Park, SSB proteins such as E. coli. SSB proteins stimulate RecA protein-promoted pairing, trap ssDNA produced by DNA helicase activity, and protects ssDNA from nucleolytic degradation in the process of genetic recombination (220).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to use a ssDNA or dsDNA as the donor template and to additionally contact the ssDNA to the SSB, prior to introduction in to the cell, as taught by Church for recombination genome editing. It would have amounted to a simple combination of elements by known means to yield predictable results. One would have been motivated to do so because Church teaches that there is an advantage to making such contact with respect to protecting the ssDNA and inhibiting complementary annealing. One would have a reasonable expectation that donor DNA such as ssDNA contacted to the SSB could be recognized and accessed by the homologous recombination machinery because Church teaches SSB, and ssDNA, along with other components result in efficient recombination and Cemák too teaches the same components in HDR are effective in modifying the genome. See MPEP 2143 I A and 2144.05 II.
Thus, Cemák in view of Church make obvious instant claims 42-59 and 61-67.
Claim 60 is rejected under 35 U.S.C. 103 as being unpatentable over Cemák (US 2020/0407754 A1 published Dec. 31, 2020, supported by its provisional application 62/866,317, filed on Jun 25, 2019) in view of Church et al. (International Publication No. WO 2017/184227 A2) Retrieved from the Internet on 12th Jan 2026 from <URL: https://www.wikipedia.org/>, 2 pgs.) as applied to claims 42-59, 61–65, and 67 above, where claim 53 is evidenced by Wikipedia (at wikipedia.org [retrieved on 2026-01-12], 2 pages) and claim 58 is evidenced by Park (US 11293019 B2), and further in view of Zanta (Zanta et al., Proc. Natl. Acad. Sci. (1999), 96: 91-96).
Regarding claim 60, Cemák does not disclose that the donor DNA comprises a nuclear localization signal (NLS).
Zanta teaches efficient delivery of double stranded DNA to the nucleus by attaching the SV40 NLS to the DNA (Abstract). Zanta also teaches "[t]ranslocation of exogenous DNA through the nuclear membrane is a major concern of gene delivery technologies." (Abstract). Zanta teaches covalently linking a hairpin oligonucleotide with an NLS (Figure 2). Zanta teaches attaching an NLS to DNA greatly increases expression of a gene encoded by the DNA (Figure 3). Zanta teaches that DNA with a covalently attached NLS is functional in a variety of cell types (Table 2).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to additionally attach an NLS to the DNA donor as taught in Zanta for genome editing via Cas9 with two or more bipartite NLSs. It would have amounted to a simple combination of elements by known means to yield predictable results. One would have a reasonable expectation that donor DNA covalently attached to an NLS could be recognized and accessed by the homologous recombination machinery because Zanta teaches that DNA-NLS fusions can be accessed by the transcriptional machinery. One would have been motivated to do so because Zanta teaches attaching an NLS greatly increases import into the nucleus into a variety of cells.
Thus, Cemák in view of Church and further in view of Zanta make obvious instant claim 60.
Claim 91 is rejected under 35 U.S.C. 103 as being unpatentable over Cemák (US 2020/0407754 A1 published Dec. 31, 2020, supported by its provisional application 62/866,317, filed on Jun 25, 2019).
See pertinent recitation from Cemák:
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Thus, Cemák makes obvious instant claim 91.
Given the teachings of the prior art and the level of the ordinary skilled artisan before the effective filing date of the applicant’s invention, it must be considered, absent evidence to the contrary, that said skilled artisan would have had a reasonable and predictable expectation of success in practicing the claimed invention.
Relevant Prior Art Not relied Upon
The following art is made note of and not currently relied on, but is relevant to applicants invention. Cotta-Ramusino (US Patent Application Publication 2018/0298392 A1, published on October 18, 2018, filed on November 9, 2015) discloses methods for improving CRISPR/Cas-mediated genome editing and that to increase the likelihood that the break is repaired by HDR, the cell can be contacted with an HDR-enhancer (see, e.g., title and abstract). Cotta-Ramusino discloses a method for increasing HDR-mediated genome editing where the method comprises the step of providing genome editing molecules comprising (i) at least one sequence-specific endonuclease which cleaves a DNA sequence in the target editing site (contacting the cell with a gRNA that targets a target position and a Cas9 molecule, see, e.g., [1231]); and (ii) a donor template DNA molecule having homology to the target editing site (a template nucleic acid, see, e.g., [1231]; template nucleic acids for correcting mutation… homology arms, see e.g. [1216]). Cotta-Ramusino further discloses providing HDR promoting agents (an HDR-enhancer, see, e.g., [1231]) to a eukaryotic cell (see [0089]) to a eukaryotic cell (see e.g. [1474]). Cotta-Ramusino discloses HDR-enhancer molecules that are up-regulators of SSA and include the protein RAD52 (see, e.g., [0045]). Cotta-Ramusino discloses exonucleases which can at least partially convert a double stranded DNA substrate to a single stranded DNA product (EXO1, see, e.g., [1334]). Cotta-Ramusino discloses single stranded DNA binding proteins (RPA1, RPA2, and RPA3, see, e.g., [1334]) and that RPA proteins prevent repeat sequences from inappropriate annealing (see [1321]). Cotta-Ramusino discloses methods involving stimulating or overexpressing one or more components of an HDR pathway (see, e.g., [1334]). Cotta-Ramusino discloses that HDR enhancing molecules, e.g. an HDR-enhancing gRNA, enhances HDR as compared to what would occur in absence of the HDR-enhancing molecule (see, e.g., [0292-0293]).
Cotta-Ramusino does not disclose SSB as an efficient single stranded DNA binding protein in the process of genome editing.
The closest prior art disclosing preferred embodiments is applied above.
Conclusion
No claim is allowable.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Correspondence
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/SHABANA S MEYERING/Examiner, Art Unit 1635
/CATHERINE KONOPKA/Primary Examiner, Art Unit 1635
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