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 Group I (claims 1-14) in the reply filed on Jul 31, 2026 is acknowledged. Claims 15-21 withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected Group II and III, there being no allowable generic or linking claim.
Applicant’s election without traverse of (i) nucleic acid as the single elected cargo, and (ii) CRISPR-based nuclease as the single elected cargo protein in the reply filed on Jul 31, 2026 is acknowledged. Claim 10 is directed to a cargo that is (i) a protein or (ii) a nucleic acid encoding a protein. Its dependent claim 13 is directed to a positively supercharged protein and the protein cargo as a fusion protein, which further limits claim 10’s cargo that is (i) a protein. Accordingly, claim 13 is directed to nonelected species of the cargo. Thus, claim 13 withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected species, there being no allowable generic or linking claim.
Claims 1-12, and 14 are currently pending and under examination.
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).
The disclosure of the prior-filed application, Application No. 63/178,444, fails to provide adequate support or enablement in the manner provided by 35 U.S.C. 112(a) or pre-AIA 35 U.S.C. 112, first paragraph for one or more claims of this application. Regarding instant claim 7, ’444 discloses SEQ ID NO: 4 that is identical to instantly claimed SEQ ID NO: 4, but ‘444 identifies this sequence as a supercharged mCerulean3 instead of “+scGFP”. Regarding instant claim 8, ’44 fails to disclose wherein the cargo comprises a small molecule. Regarding instant claim 12, ‘444 fails to disclose wherein the genome editing or epigenome modulating protein is or comprises a zinc finer or a TALE, optionally fused to an epigenome modulator. Regarding instant claim 14, ‘444 discloses “scsgRNAViruses” encoding for guide RNA were exogenously loaded with Cas9 conjugated scProteins or plasmids expressing Cas9 together with scProtein (lines 684-685), which does not provide support for a cargo further comprising a guide RNA complexed with a positively supercharged protein required in instant claim 14. On the contrary, support for the following claims can be found in ‘444: claim 1 (lines 559-561), claim 2 (lines 555-556; lines 710-711), claim 3 (lines 707-710), claim 4 (line 360), claim 5 (lines 360-361), claim 6 (lines 889-894), claim 9 (lines 668-670), and claims 10-11 (lines 684-685). Accordingly, claims 1-6, 9-11, and 14 have an effective filling date of Apr 22, 2021.
The disclosure of the prior-filed application, Application No. 63/180,489, fails to provide adequate support or enablement in the manner provided by 35 U.S.C. 112(a) or pre-AIA 35 U.S.C. 112, first paragraph for one or more claims of this application. ‘489 fails to disclose instantly recited limitations of claims 7, 8, and 12.
Applicant’s claim for the benefit of the International Patent Application PCT/US2022/071873 filed on Apr 22, 2022 is acknowledged. Support for instant claims 7-8, and 12 can be found in claims 7-8, and 12. Accordingly, claims 7-8, and 12 have an effective filling date of Apr 22, 2022.
Information Disclosure Statement
The listing of references in the specification (e.g., pg. 25, lines 31-33) is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
Drawings
The drawings are objected to because 37 CFR 1.84 (u)(1) states “Partial views intended to form one complete view, on one or several sheets, must be identified by the same number followed by a capital letter.” In the current case, the view numbers for the partial views for Figures 9G, 9H, and 17 that appear on several sheets are followed by "Continued." instead of a capital letter such as FIG. 9G.
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.
Nucleotide and/or Amino Acid Sequence Disclosures
REQUIREMENTS FOR PATENT APPLICATIONS CONTAINING NUCLEOTIDE AND/OR AMINO ACID SEQUENCE DISCLOSURES
Items 1) and 2) provide general guidance related to requirements for sequence disclosures.
37 CFR 1.821(c) requires that patent applications which contain disclosures of nucleotide and/or amino acid sequences that fall within the definitions of 37 CFR 1.821(a) must contain a "Sequence Listing," as a separate part of the disclosure, which presents the nucleotide and/or amino acid sequences and associated information using the symbols and format in accordance with the requirements of 37 CFR 1.821 - 1.825. This "Sequence Listing" part of the disclosure may be submitted:
In accordance with 37 CFR 1.821(c)(1) via the USPTO patent electronic filing system (see Section I.1 of the Legal Framework for Patent Electronic System (https://www.uspto.gov/PatentLegalFramework), hereinafter "Legal Framework") as an ASCII text file, together with an incorporation-by-reference of the material in the ASCII text file in a separate paragraph of the specification as required by 37 CFR 1.823(b)(1) identifying:
the name of the ASCII text file;
ii) the date of creation; and
iii) the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(1) on read-only optical disc(s) as permitted by 37 CFR 1.52(e)(1)(ii), labeled according to 37 CFR 1.52(e)(5), with an incorporation-by-reference of the material in the ASCII text file according to 37 CFR 1.52(e)(8) and 37 CFR 1.823(b)(1) in a separate paragraph of the specification identifying:
the name of the ASCII text file;
the date of creation; and
the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(2) via the USPTO patent electronic filing system as a PDF file (not recommended); or
In accordance with 37 CFR 1.821(c)(3) on physical sheets of paper (not recommended).
When a “Sequence Listing” has been submitted as a PDF file as in 1(c) above (37 CFR 1.821(c)(2)) or on physical sheets of paper as in 1(d) above (37 CFR 1.821(c)(3)), 37 CFR 1.821(e)(1) requires a computer readable form (CRF) of the “Sequence Listing” in accordance with the requirements of 37 CFR 1.824.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed via the USPTO patent electronic filing system as a PDF, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the PDF copy and the CRF copy (the ASCII text file copy) are identical.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed on paper or read-only optical disc, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the paper or read-only optical disc copy and the CRF are identical.
Specific deficiencies and the required response to this Office Action are as follows:
Specific deficiency - This application fails to comply with the requirements of 37 CFR 1.821 - 1.825 because the application does not contain a statement that the CRF is identical to the "Sequence Listing" part of the disclosure, as described above in item 1), as required by 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii).
Required response - Applicant must provide such statement.
Claim Objections
Claim 5 is objected to because of the following informalities: an article is missing preceding “supercharged eGFP”. Appropriate correction is required.
Claim 5 is objected to because of the following informalities: the acronyms “+scmCerulean3” and “+scGFP” are recited but not defined in the claim. An acronym should be defined the first time it appears in an independent claim or in the group of claims under an independent claim. Appropriate correction is required.
Claim Rejections - 35 USC § 112
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 3, 5, 7, 11-12, and 14 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.
In claim 3, the phrase “preferably” reads as an exemplary term. MPEP 2173.05(d) states "Description of examples or preferences is properly set forth in the specification rather than the claims. If stated in the claims, examples and preferences may lead to confusion over the intended scope of a claim. In those instances where it is not clear whether the claimed narrower range is a limitation. It is unclear whether claim 3 requires that (i) the extracellular vesicles (EVs) be obtained from a biofluid or tissue of any living animal, or (ii) the EVs be obtained from a biofluid or tissue of a human.
In claim 5, the phrase “preferably reads as an exemplary term (see MPEP 2173.05(d) discussed above). It is unclear whether claim 5 requires the positively supercharged protein to comprise (i) a supercharged eGFP, (ii) a variant thereof, (iii) a +scmCerulean3, or (iv) a +scGFP. It is unclear whether claim 5 requires the positively supercharged protein to comprise only (i) or (ii).
Claim 5 also recites “wherein the positively supercharged protein comprises [a] supercharged eGFP or a variant thereof” (emphasis added), which renders the claim indefinite. There is ambiguity in the antecedent basis of “thereof”, and the specification provides support for two different scopes: (i) a variant of eGFP (i.e., SEQ ID NO: 1 disclosed on pg. 27) that is then supercharged, including mutants and spectral variants that “retain the ability to fluoresce” (pg. 36, lines 28-29), e.g., mCherry, mOrange, tdTomato and many more (paragraph bridging pg. 36 and 37); (ii) a variant of supercharged eGFP (i.e., +scGFP of SEQ ID NO: 2 (pg. 27)) that contemplates surface charges of +11, +15, +20, +25, +27, +30, +32, +35, 30 +36, or +38 (pg. 25, lines 26-30), which covers variants of SEQ ID NO: 2 only. Dependent claim 6 is drawn to specific +scmCerulean3 proteins sharing at least 95% sequence identity to SEQ ID NO:4, one of ordinary skill in the art could reasonably appraise of the metes and bound of the claim. Therefore, claim 6 remedies the indefiniteness therein as opposed to claim 7.
Claim 7 recites “wherein the +scGFP is at least 95% identical to SEQ ID NO: 4”, (emphasis added) which renders the claim indefinite due to ambiguity between protein identity and amino acid sequence. The specification teaches a positively supercharged eGFP (i.e., +scGFP) consisting of the amino acid sequence set forth in SEQ ID NO: 2, while SEQ ID NO: 4 corresponds to +scmCerulean3 protein (pg. 27). It is unclear whether the claim requires +scGFP or +scmCerulean3 positively supercharged proteins. It is unclear whether the claim requires proteins that share at least 95% sequence identity to SEQ ID NO: 4 or SEQ ID NO: 2.
Claim 11 recites “wherein the protein is a genome editing or epigenome modulating protein” (emphasis added). Claim 11 depends on claim 10, which recited two proteins (i) wherein the cargo is a protein, and (ii) wherein the cargo is a nucleic acid encoding a protein. The recitation of “the protein” in subsequent claim is unclear as it is uncertain which of the two proteins was intended (see MPEP 2173.05(e)). It is unclear whether the protein of claim 11 is referring to the protein of (i) or the protein of (ii) recited in claim 10.
Claim 14 recites “wherein the protein is or comprises a CRISPR based nuclease” (emphasis added). Claim 14 depends from claim 10, similarly to claim 11. See discussion above as applied to claim 11. It is unclear whether the protein of claim 14 is referring to the protein of (i) or the protein of (ii) recited in claim 10.
Those claims included in the statement of rejection but not otherwise discussed are rejected for depending from a rejected claim but failing to remedy the indefiniteness therein.
Claim Interpretation
Claim 1 recites “a positively supercharged protein” which is interpreted to encompass proteins “which have, or have been engineered to have, a surface charge of at least +11” as disclosed in the specification (pg. 25, lines 27-29).
Claim 5 recites “wherein the positively supercharged protein comprises [a] supercharged eGFP or a variant thereof”, which renders the claim indefinite as discussed above. The recitation “a variant thereof” is interpreted as any variants of eGFP that retain the ability to fluoresce and are then supercharged in light of the specification (pg. 36, lines 27-28) teaching numerous fluorescent proteins comprising different chromophores (paragraph bridging pg. 36 and 37). This interpretation is also consistent with dependent claim 6 reciting +scmCerulean3 that are variants of eGFP and are supercharged rather than variants of supercharged eGFP.
Claim 7 recites “wherein the +scGFP is at least 95% identical to SEQ ID NO: 4”, which renders the claim indefinite as discussed above. In view of the specification teaching a positively supercharged eGFP (i.e., +scGFP) consisting of the amino acid sequence set forth in SEQ ID NO: 2 (pg. 27), and instant claim 6 reciting a positively supercharged mCerulean3 (i.e., +scmCerulean3) consisting of the amino acid sequence set forth in SEQ ID NO: 4 which is supported by the specification (pg. 27), instant claim 7 is interpreted as “wherein the +scGFP is at least 95% identical to SEQ ID NO: 2” for compact prosecution.
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.
Claim 14 is rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention. This is a NEW MATTER rejection.
MPEP 2163.II.A.3.(b) states, “when filing an amendment an applicant should show support in the original disclosure for new or amended claims” and “[i]f the originally filed disclosure does not provide support for each claim limitation, or if an element which applicant describes as essential or critical is not claimed, a new or amended claim must be rejected under 35 U.S.C. 112a, as lacking adequate written description". According to MPEP § 2163.I.B, "While there is no in haec verba requirement, newly added claim limitations must be supported in the specification through express, implicit, or inherent disclosure" and "The fundamental factual inquiry is whether the specification conveys with reasonable clarity to those skilled in the art that, as of the filing date sought, applicant was in possession of the invention as now claimed. See, e.g., Vas-Cath, Inc., 935 F.2d at 1563-64, 19 USPQ2d at 1117".
The new limitation of “the cargo further comprises a guide RNA” in claim 14 appears to represent new matter. The claim previously recites the cargo further comprises a guide RNA “complexed with the CRISPR based nuclease” (quoted limitation is presently deleted). Accordingly, the claim requires a cargo comprising a guide RNA wherein the guide RNA is complexed with the CRISPR based nuclease, but the amended claim merely requires the cargo to further comprise a guide RNA.
Claim 1 recites “one or more cargo molecules complexed with the positively supercharged protein”, dependent claim 10 further limits the cargo to be (i) a protein or (ii) a nucleic acid encoding a protein. Accordingly, claim 14 that depends on claim 10 encompasses embodiment (i) wherein the cargo is or comprises a CRISPR based nuclease, and the cargo further comprises a guide RNA. The specification teaches actual reduction to practice of extracellular vesicles (EV) complexed with Cas9 recombinant protein and synthetic sgRNA (brief description of FIG. 18A). Claim 14 additionally encompass embodiment (ii) wherein the cargo is a nucleic acid encoding a CRISPR based nuclease (e.g., DNA or mRNA), and the cargo further comprises a guide RNA. The specification discloses EV complexed with synthetic sgRNA alone (brief description of FIG. 18B). The specification also discloses contacting cells with a vector or vectors comprising sequences encoding the nuclease and gRNA, or nuclease/gRNA ribonucleoprotein (pg. 37, lines 14-17). These teachings are not insufficient to demonstrate possession of the new limitation, cargo(s) comprising a nucleic acid encoding a CRISPR based nuclease, and a guide RNA is complexed with a positively supercharged protein. There is no express, implicit, or inherent teaching from the current specification including any reduction to practice of claim 14’s embodiment (ii) that would support the new limitation.
No specific basis for this embodiment was identified in the specification, nor did a review of the specification by the examiner find any basis for the limitation. Since no basis has been identified, the claim is rejected as incorporating new matter.
Claims 1-4, 8-12, and 14 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.
MPEP 2163.II.A.3.(a).i) states, “Whether the specification shows that applicant was in possession of the claimed invention is not a single, simple determination, but rather is a factual determination reached by considering a number of factors. Factors to be considered in determining whether there is sufficient evidence of possession include the level of skill and knowledge in the art, partial structure, physical and/or chemical properties, functional characteristics alone or coupled with a known or disclosed correlation between structure and function, and the method of making the claimed invention”. MPEP § 2163 further states the written description requirement for a claimed genus may be satisfied through sufficient description of a representative number of species.
Claim 1 is directed to a genus of "positively supercharged proteins" complexed with one or more cargo molecules. The specification discloses that "positively supercharged proteins" have or have been engineered to have a surface charge of at least =11, optionally =15 to +38, with surface charges predicted in silico using bioinformatics (pg. 25, lines 27-30). The specification discloses "in some embodiments, the cargo comprises a nucleic acid, a protein, a ribonucleoprotein, a combination of DNA and protein, or a small molecule" (pg. 2, lines 10-13). Thus, the breadth of claim 1 is drawn to possession of a genus of any “positively supercharged proteins” defined by a biophysical property and claimed function of complexing with one or more cargo molecules identified above.
The specification teaches one generic rule of designing these proteins, which is adding lysine, arginine or histidine at positions exposed on the surface of the protein (pg. 26, lines 4-5). The specification further lists limited number of beta-barrel proteins that can be engineered to contain increased levels of positively charged amino acids, including human retinol-binding protein, porins, lipocalins, and translocases (pg. 26, lines 6-10). In addition, Cas9, Cre, Talen and zinc finger nucleases can be positively supercharged if distal sites to the catalytic cleft on the solvent exposed protein surface are modified to positively charged amino acids (pg. 26, lines 10-13). However, the specification is limited to two species, green and mCerulean3 fluorescent proteins of SEQ ID NOs: 2 and 4, respectively (pg. 27). While MPEP 2163 acknowledges that in certain situations “one species adequately supports a genus”, it also acknowledges that “[f]or inventions in an unpredictable art, adequate written description of a genus which embraces widely variant species cannot be achieved by disclosing only one species within the genus.” In view of the wide variant species encompassed by the genus, the one example described above is not enough and does not constitute a representative number of species to describe the whole genus.
The specification fails to disclose (i) any common structural features shared among the members of the genus, particularly, sequence, positive charge, and/or mutation map for any other species within the claimed genus besides instant +scGFP and +scmCerulean3. The breadth of the claim encompasses any protein engineered to have any positive net charge equal to or above 11, with not upper limit. It also fails to disclose (ii) sufficient relevant identifying characteristics including but not limited to complete or partial structure of representative species. It also fails to disclose (iii) functional characteristics coupled with a known correlation between function and structure, for instance, (a) surface-exposed amino acids may be mutated in any of the listed candidates without disrupting folding, stability, and/or function; and (b) whether proteins exhibiting a surface charge of +11 is indeed sufficient to associate with one or more claimed cargo molecules as claimed.
The state of the art recognizes the claimed genus of proteins covers structurally unrelated proteins, rather than a defined pool of species. Ma (Adv. Mat.; 2020, 32(20):e1905309, 21 pages; IDS received on 04/15/2024; Cite No. 60) teaches supercharged proteins could categorized as (i) folded supercharged proteins in nature collectively perform diverse functions, suggesting that these proteins are structurally different (pg. 3, left-column, para. 2; Table 1); (ii) unfolded supercharged proteins in nature, which are largely disordered (pg. 3, left-column, para. 3; Table 2); (iii) engineered supercharged folded proteins by mutagenesis or post-translational chemical modifications to replace solvent-exposed amino acids with arginine or lysine (pg. 5, left-column, para 1), but its applicability needs to be evaluated for individual proteins (pg. 9, right-column, para. 2). Ma discusses one engineered supercharged protein, a positively supercharged GFP variant comprising 29 mutations, yielding a theoretical net charge of +36, which is +15 from the minimum claimed threshold (pg. 5, left-column, para. 1). Further, Ma discloses “supercharged natural proteins are challenging to use for the design of advanced functional materials due to their rather complex primary structure” (pg. 9, right-column, para. 3).
The claimed genus reads on every natural occurring protein, engineered protein of any fold, disordered proteins, and every engineered variant with the mere requirement that they meet the claimed threshold of at least +11 surface charge. Members of the claimed genus share no common motif, fold, structural features. The only thing they allegedly share is a biophysical property. The state of the art also establishes designing members of the claimed genus for the claimed function is challenging and unpredictable.
In contrast, dependent claim 5 is drawn to possession of a subgenus of eGFP variants that are supercharged (see claim interpretation above). The specification numerous fluorescent proteins which structure and surface-exposed residues in comparison to eGFP are known in the art (paragraph bridging pg. 36 and 37). Further, dependent claims 6 and 7 are drawn to possession of subgenus +scmCerulean3 proteins sharing at least 95% sequence identity to SEQ ID NO: 4 and +scGFP proteins sharing at least 95% sequence identity to SEQ ID NO: 2 (see claim interpretation section below). The 95% sequence identity threshold allows 12 amino acid modifications including mutations, insertions, and deletions. Simon (Nat. Chem.; 2019, 11:204-212) teaches an engineered mCerulean variant carrying over 12 amino acid substitutions on surface residues, resulting in a net charge of +32 (FIG. 1b). Liu (US 9,221,886; Pub Date: Dec 29, 2015; IDS received on 04/17/2024; Cite No. 1) teaches engineered GFP variants comprising over 12 amino acid substitutions on surface residues, resulting in net charges of +15, +25, and +36 (col. 145). Thus, the state of the art establishes over 12 surface-exposed residues that can be substituted to positively charged amino acids to increase positive net charge of eGFP variants. Dependent claims 5-7 remedy the lack of written description therein.
Accordingly, claim 1’s dependent claims 2-4, 8-12, and 14 are rejected for depending from a rejected claim and failing to remedy the lack of written description therein.
Based on the preponderance of the evidence, including the relevant teachings of the specification, the absence of working examples, and the state of prior art including the knowledge of positively supercharged proteins, one skilled in the art would conclude that Applicant was not in possession of the claimed genus of positively supercharge proteins required in claim 1.
Claim 11 recites a genus of epigenome modulating proteins, which encompass any proteins not defined by structure rather by a claimed function. Dependent claim 12 further recites a genus of “epigenome modulators”, which encompass any modulators not defined by structure rather by a claimed function that (i) directly modulate epigenome and (ii) has upstream and/or downstream effects capable of indirectly modulate epigenome.
The specification mentions “epigenome modulating protein” twice (pg. 2 lines, 13-16): “in some embodiments the genome editing or epigenome modulating protein is or comprises a CRISPR based nuclease, zinc finger or a TALE, optionally fused to an epigenome modulator”. Regarding the genus of “epigenome modulator”, the specification also discloses cargos designed for purposed of epigenome modulation “can include CRISPR base nuclease, zinc fingers and TALEs fused to an epigenome modulator or combination of epigenome modulators or a functional derivative thereof connected together by” linkers (pg. 30, lines 27-32). This teaching identifies gene editing proteins or DNA-binding domains and does not teach what an epigenome modulating protein or an epigenome modulator is. The specification fails to disclose (i) any common structural features shared among the members of the genus, (ii) sufficient relevant identifying characteristics, (iii) representative species, and/or (iv) functional characteristics coupled with a known correlation between function and structure.
Regarding the state of the art, Madvedeva (Database; 2015, pg: 1-10; doi: 10.1093/database/bav067) teaches epigenetics cover (i) DNA and histone modifications, (ii) chromatin remodeling, and (iii) DNA methylation. Further, epigenetic factors (i.e., epigenome modulating proteins or epigenome modulator) are defined to perform diverse functions including but not limited to proteins (i) acting as histones, (ii) performing post-translational modifications of histones or recognizing such modifications, (iii) changing general structure of chromatin, (iv) assisting histone folding and assembly, and (v) acting upon modifications of DNA or RNA such that it affects gene expression (pg. 4, left-column). Exemplary proteins falling within these definitions including (i) histone modifiers such as writers, erasers, and readers; (ii) ATP-dependent complexes that remodel chromatin; (iii) and histone chaperones in nucleosome assembly (pg. 2, paragraph bridging left and right column). Madvedeva further teaches epigenetic proteins expands to transcription factors that regulate genes coding for epigenetic factors, TF-like proteins that recruit epigenetic proteins to genomic sites, and microRNAs that act upon mRNAs for epigenetic proteins (pg. 2, right-column, para. 1). Thus, the claimed genu covers proteins on hundreds of structurally distinct proteins, plus their variants, fusions, and functional derivatives. Further, the claimed genus is not confined to proteins that add or remove modifications but extend to upstream and downstream proteins and factors. Accordingly, one of ordinary skill in the art cannot reasonably determine whether a protein falls within the claimed genus.
Based on the preponderance of the evidence, including the relevant teachings of the specification, the absence of working examples, and the state of prior art including the knowledge of epigenome modulating proteins and epigenome modulators, one skilled in the art would conclude that Applicant was not in possession of the claimed genus in claims 11 and 12.
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.
Claims 1-5, 7-12, and 14 are rejected under 35 U.S.C. 103 as being unpatentable over Evox Therapeutics Ltd (US 2019/0388347 A1; Pub Date: Dec 26, 2019; hereinafter as Evox), in view of Liu (US 9,221,886; Pub Date: Dec 29, 2015; IDS received on 04/17/2024; Cite No. 1).
Regarding claim 1, Evox teaches a composition comprising extracellular vesicles (EV) (i.e., biovesicles) comprising at least one pharmaceutical agents (i.e., cargo molecules) complexed with a cell-penetrating peptide (CPP) via covalent or non-covalent interactions ([0006], [0021]). Evox further teaches EVs encompass microvesicles, exosomes, cell membrane-based vesicles, and any type of lipid-based structure acting as a delivery vehicle for pharmacological agents of interest ([0024]). Accordingly, Evox teaches wherein the EV comprise a lipid bilayer surrounding an aqueous lumen.
However, Evos does not teach a positively supercharged protein and wherein the cargo is complexed with the positively supercharged protein.
Liu teaches positively supercharged proteins engineered by replacing solvent-exposed charged or polar residues with lysine or arginine amino acids that are positively charged at physiological pH (col. 4, lines 56-64; col. 5, lines 8-10) to increase the proteins’ overall net charge to at least +5 and up to +50 (col. 6, lines 20-23). Further, the positively supercharged proteins are associated with therapeutic agents (i.e., cargo) via electrostatic, covalent, or non-covalent interactions to form complexes to deliver the therapeutic agents into a cell (col. 3, lines 35-40; col. 6, lines 45-67). Liu teaches these complexes “may be contained within or associated with a micelle, liposome, or other drug delivery system” (col. 4, lines 10-14), and the cell-penetration potency of positively supercharged proteins, particularly GFP with an overall net charge of +36, “exceeds that of known cell-penetrating peptides and proteins, especially at low concentration” (FIG. 20, 33, 38, 44; col. 30: brief description of FIG. 44).
Thus, it would have been obvious to one of ordinary skill in the art before the effective filling date of the invention to have modified Evox’s cell-penetrating peptide complexed with cargos to positively supercharged proteins as taught by Liu because it would have merely amounted to a simple substitution of prior art elements according to known methods to yield predictable results. The modification further would have been obvious because it would have merely amounted to applying a known technique to a known device ready for improvement to yield predictable results. It would have been obvious to substitute one positively charged molecule that forms complexes with cargos via electrostatic interactions to another positively charged molecule that serves the same purpose via a similar mechanism. One would have been motivated to have done so for the advantage of delivering therapeutic cargos with significantly greater cell-penetration potency as demonstrated by Liu (col. 38, lines 56-59). One would have had a reasonable expectation of success in doing so because both Evox and Liu both teach lipid-based structure vesicles comprising a positively charged molecule complexed with cargos, and Liu further demonstrates this design works with positively supercharged proteins.
Regarding claim 2, Evox teaches EVs are obtained from bone marrow, adipose tissue, or perinatal tissue ([0028])
Regarding claim 3, Evox teaches wherein the EVs are obtained from media in a cell culture ([0054]).
Regarding claim 5, Liu teaches wherein positively supercharged protein comprises a supercharged eGFP or variants of the supercharged eGFP, particularly variants with net charges of +15, +36, +42, +48, and +49 (col. 39 and 40; FIG. 1).
Regarding claim 7, it is interpreted as wherein the +scGFP is at least 95% identical to SEQ ID NO: 2 (see claim interpretation above). Liu further teaches wherein the positively supercharged GFP with net charge +36 referred to as GFP-POS36 comprises the amino acid sequence of SEQ ID NO: 7 that is 99.6% identical to instant SEQ ID NO: 2. It is noted SEQ ID NO: 7 contains a GHHHHHHG tag inserted on the N-terminal between the first and second residue of the positively supercharged GFP protein, resulting in 0.4% sequence mismatch. This polyhistidine tag is added by Liu for ease in purification of the protein (col. 13, lines 41-41).
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Regarding claims 8-10, Evox teaches wherein the cargo comprises “nucleic acid-based compounds” including but not limited to mRNA ([0025]).
Claims 11-12, and 14 is rejected under 35 U.S.C. 103 as being unpatentable over Evox and Liu (cited supra) as applied to claim 10 above, and further in view of Zhang (WO 2014/204729 A1; Pub Date: Dec 24, 2014).
The teachings of Evox and Liu are discussed above.
Regarding claims 11-12, and 14, Liu teaches wherein the cargo comprises a nucleic acid, including but not limited to DNA, RNA, plasmid, and "nucleotide sequence that encode proteins" (col.5, lines 56-64; col.16, line 57). Liu further demonstrates delivering plasmid DNA encoding β-galactosidase complexed with the same positively supercharged +36GFP protein into mammalian cells (col. 26, lines 10-17; FIG. 21).
However, Liu does not explicitly teach wherein the cargo is a nucleic acid encoding a genome editing protein, specifically, a CRISPR based nuclease, and that the cargo further comprises a guide RNA.
Zhang teaches “the methods of the supercharged proteins may be used for delivery of the CRISPR Cas system” by complexing plasmid DNA encoding the CRISPR Cas system with positively supercharged proteins ([00520]). Positively supercharged +36GFP protein is an “effective plasmid delivery reagent in a range of cells” ([00512]) and that it is predictable to associate the +36 GFP protein with plasmids encoding CRISPR Cas system for delivery into cells by following the disclosed optimized protocol ([00513]-[00519]). The sole parameter to optimize is molar ratios of plasmid DNA and positively supercharged protein given that plasmid DNA is larger than siRNA, proportionately more +36 GFP protein would be required for effective complexation ([0512]). Zhang further teaches the CRISPR Cas system is a two-component system consisting of a Cas9 nuclease and a guide RNA that targets the Cas9 nuclease to different loci for gene editing ([001133]).
It would have been obvious to one of ordinary skill in the art before the effective filling date of the invention to have modified Liu’s cargo comprising a nucleic acid encoding β-galactosidase to a nucleic acid encoding CRISPR Cas system as taught by Zhang because it would have merely amounted to a simple combination of prior art elements according to known methods to yield predictable results. It would further be obvious to modify the cargo not only to encode the CRISPR based nuclease, but also to further comprise a guide RNA as these two components are required to function as a CRISPR Cas system. This substitution is merely swapping one nucleic acid cargo, carrying a net negative charge, to another that serves the same purpose: associate with the positively supercharged +36GFP protein via electrostatic interactions to form a complex. This modification does not change the principle of operation of the complex or render the complex unsatisfactory for tis intended purpose. One would have had a reasonable expectation of success in doing so because Liu demonstrates nucleic acid encoding a protein complexed with positively supercharged protein and delivery into a cell. Liu also teaches nucleic acid cargos of various sizes, including viral genome and artificial chromosome, positively indicating that cargo size is not a constrain (col. 5, lines, 62). Further, Zhang teaches an established and optimized protocol complexing plasmid DNA encoding CRISPR Cas system with positively supercharged protein.
Claim 6 is rejected under 35 U.S.C. 103 as being unpatentable over Evox and Liu (cited supra) as applied to claim 5 above, and further in view of Rizzo (WO 2011/043814 A2; Pub Date: Apr 14, 2011), Simon (Nat. Chem.; 2019, 11:204-212 and supplementary materials; IDS received on 04/15/2024; Cite No. 101).
The teachings of Evox and Liu are discussed above.
Regarding claim 6, Liu teaches exemplary positively supercharged GFP proteins origin from Aequorea Victoria (col. 38, lines 10-13) and that “particularly useful supercharged proteins are proteins sharing a structural feature with a supercharged protein herein” ([col. 39, lines 4-6).
However, Liu does not teach a positively supercharged mCerulean3 protein that is at least 95% identical to instant SEQ ID NO: 4, and has a predicted surface charge of at least +11.
Rizzo teaches a cyan fluorescent protein, mCerulean2, of SEQ ID NO: 6 that shares 86.1% amino acid sequence identity to instant +scmCerulean3 of SEQ ID NO: 4 (see sequence alignment below). Rizzo further teaches mCerulean2 is a mutant of GFP origin from Aequorea Victoria (pg. 10, lines 26-29).
In addition, Simon teaches a positively supercharged mCerulean protein with a net charge of +32 (Fig. 1b). Simon also discloses the supercharged mCerulean protein contains more than twenty substitutions at solvent-exposed residues (Suppl. Fig. 1). These mutations correspond to the surface mutations of instant SEQ ID NO: 4 as annotated with * in the attached sequence alignment, resulting in a predicted surface charge of at least +11.
To the extent that Simon teaches nearly all mutations in instant SEQ ID NO: 4, except at position 40 and 105, Liu discloses these two surface-exposed residues in supercharged GFP variants are substituted to arginine and lysine, respectively, to increase net surface charges (see Liu’s SEQ ID NO: 7 above).
Thus, it would have been obvious to one of ordinary skill in the art before the effective filling date of the invention to have applied Simon’s teachings to generate an additional positively supercharged GFP mutant protein in view of Rizzo and Liu’s teachings because it would have merely amounted to a simple combination of prior art elements according to known methods to yield predictable results. One would have been motivated to do so because Liu discloses supercharged proteins sharing a structural feature with supercharged GFP proteins herein are particularly useful for delivering therapeutic cargos into cells, and charge distribution plays a role in enhanced cell-penetration potency of positively supercharged GFP proteins (col. 38, lines 52-59). Further, Rizzo supplies the scaffold of a GFP mutant from the same origin, mCerulean2. In view of Simon’s teachings, one would have modified mCerulean2’s surface-exposed residues accordingly to increase net positive charge to associate with cargos via electrostatic interactions. One further would have modified residues at positions 40 and 105 to arginine and lysine in view of substitutions on Liu’s supercharged GFP mutant proteins. One would have had a reasonable expectation of success in doing so because Simon teaches cerulean3 protein tolerates more than twenty substitutions on surface-exposed residues, specifically to increase the net charge with folding correctly. Further, Liu’s supercharged GFP and Rizzo’s mCerulean2 origin from A. Victoria, sharing beta-barrel structures that differ principally at the chromophore, which makes transferring substitutions at the same positions routine and predictable.
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Conclusion
No claims are allowable.
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/QIWEN SU-TOBON/
Examiner
Art Unit 1636
/NEIL P HAMMELL/Supervisory Patent Examiner, Art Unit 1636
Prosecution Insights