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 and Election
Claims 1, 5-7, 10, 12-13, 18, 20, 24-28, 30-31, 36-37, 41-42, 45, 51-53, 55-57, 60-65, 68-69, 74-76, 85-87, 89, 91, 93-95, 97-98, 100-108 are pending.
Applicant’s election without traverse of Group I in the reply filed October 21, 2025 is acknowledged. It is noted that in the Restriction Requirement mailed August 21, 2025, claim 101 was mistakenly placed in Group I. However, claim 101 depends from claim 37, which is Group 2. Claims 37, 41-42, 45, 51-52, 57, 62, 64-65, 69, 74-76, 93, 95, 97, 101 and 105-108 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected group there being no allowable generic or linking claim.
Claims 1, 5-7, 10, 12-13, 18, 20, 24-28, 30-31, 36, 53, 55-56, 60-61, 63, 68, 85-87, 89, 91, 94, 98, 100 and 102-104 are under examination.
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 – Nucleotide and/or amino acid sequences appearing in the drawings, FIGs 1, 4, 13-19 and 23A, are not identified by sequence identifiers in accordance with 37 CFR 1.821(d). Sequence identifiers for nucleotide and/or amino acid sequences must appear either in the drawings or in the Brief Description of the Drawings. Due to the vast number of unidentified sequences in the Drawings, Examiner has not confirmed that all the sequences are present in the sequencing listing. If not, a new sequencing listing will need to be filed.
Required response #1 – Applicant must provide:
Replacement and annotated drawings in accordance with 37 CFR 1.121(d) inserting the required sequence identifiers;
AND/OR
A substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3) and 1.125 inserting the required sequence identifiers into the Brief Description of the Drawings, consisting of:
A copy of the previously-submitted specification, with deletions shown with strikethrough or brackets and insertions shown with underlining (marked-up version);
A copy of the amended specification without markings (clean version); and
A statement that the substitute specification contains no new matter.
Required response #2 – If the sequences in FIGs 1, 4, 13-19 are not in the sequence listing, Applicant must also provide:
A "Sequence Listing" part of the disclosure, as described above in item 1); as well as
An amendment specifically directing entry of the "Sequence Listing" part of the disclosure into the application in accordance with 1.825(b)(2);
A statement that the "Sequence Listing" includes no new matter in accordance with 1.825(b)(5); and
A statement that indicates support for the amendment in the application, as filed, as required by 37 CFR 1.825(b)(4).
If the "Sequence Listing" part of the disclosure is submitted according to item 1) a) or b) above, Applicant must also provide:
A substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3) and 1.125 inserting the required incorporation-by-reference paragraph, consisting of:
A copy of the previously-submitted specification, with deletions shown with strikethrough or brackets and insertions shown with underlining (marked-up version);
A copy of the amended specification without markings (clean version); and
A statement that the substitute specification contains no new matter;
If the "Sequence Listing" part of the disclosure is submitted according to item 1) b), c), or d) above, Applicant must also provide:
A replacement CRF in accordance with 1.825(b)(6); and
Statement according to item 2) a) or b) above.
Drawings
The drawings are objected to for two reasons.
First, in the partial views of FIGs 1, 4 and 12, which appears on several sheets, have not been identified by the same number followed by a capital letter. 37 C.F.R. 1.84(u)(1) requires that “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.” See MPEP 608.02. To overcome the objection, sheets 1-3 should be labeled FIG. 1A, FIG. 1B and FIG. 1C, respectively. Sheets 6 and 7 should be labeled FIG. 4A and FIG. 4B, respectively. Sheets 13 and 14 should be labeled FIG. 12A and FIG. 12B, respectively
Second, the lines, shadings, numbers and letters of FIGs 3 and 24A are not sufficient to provide satisfactory reproduction characteristics. 37 CFR 1.84(l) states that “all drawings must be made by a process which will give them satisfactory reproduction characteristics. Every line, number, and letter must be durable, clean, black (except for color drawings), sufficiently dense and dark, and uniformly thick and well-defined.” In the instant case, the text in the circles in FIG. 3 and the text below the vector map in FIG 24A is too small or of too low resolution to permit satisfactory reproduction characteristics; and the text over shading in the vector FIG 24A is not sufficiently dark for reproduction.
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.
Specification
The use of the terms NEB® Stable, Nucleobond®, NucleoSpin®, CaptoTM Core, Turboluc®, NanoLight®, and jetPRIME®, which are trade names or marks used in commerce, has been noted in this application. The term should be accompanied by the generic terminology; furthermore the term should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term.
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.
Claim Objections
Claims 24-25 are objected to because of the following informalities:
Claim 24 recites “wherein the plasmid further comprises a fifth and a sixth restriction site for nicking endonuclease…” There needs to be an article “a” or “the” before “nicking endonuclease”.
Claim 25 recites “wherein the fifth and sixth nick are 1, 2…” “Nick” should be pluralized.
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 1, 5-7, 10, 18, 20, 24-28, 30-31, 36, 53, 55-56, 60-61, 63, 68, 85-87, 89, 91, 94, 98, 100 and 102-104 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.
Regarding claim 1, the term “in proximity” is a relative term which renders the claim indefinite. The term “proximity” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. The Specification does not define “in proximity” and does not give an indication of how far the nicking endonuclease site can be from the inverted repeats (IRs) for the site to be still considered “in proximity”.
Claims 5-7, 10, 18, 20, 24-28, 30-31, 53, 55-56, 60-61, 63, 68, 85-87, 98, 100 and 102-104 are rejected from depending from claim 1 and not remedying the indefiniteness.
Note that claims 12-13 are not included in this rejection because they recite specific distances between the nick site and the ITR closing base.
Claim 36 recites “The double-stranded DNA molecule of, further comprising…” Claim 36 is rejected as being incomplete because it starts with dependent claim language, but does not recite a claim upon which it is dependent. Claims 91 and 94 depend from claim 36 and are also rejected as it is not clear what additional limitations are in the claims. Claims 36, 91 and 94 have not been further treated because they are incomplete.
Claim 89 recites “The double-stranded DNA molecule of claim 88”. Claim 88 is a cancelled claim. Claim 89 is rejected as being incomplete because it depends directly from a canceled base claim. See MPEP 608.01(n)(V). Claim 89 has not been further treated because it is incomplete.
Claim Rejections - 35 USC § 103
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.
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, 10, 12-13, 18, 24-27, 30-31, 53, 55-56, 60-61, 63, 68, 85-87, 89, 98, 100 and 102-104 are rejected under 35 U.S.C. 103 as being unpatentable over Alkan (US 20210071197 A1, priority to at least January 18, 2019) in view of Wang (US 20090305272 A1, published December 10, 2009) and Jack (US 20030022317 A1, published January 30, 2003). Claim 27 is evidenced by NEB (Nt.BstNBI, neb.com/en-us/products/r0607-ntstnbi [retrieved January 22, 2026]).
Note on nicking enzyme nomenclature: It appears in the art that “N.BstNBI and Nt.BstNBI are used interchangeably in the art to refer to the same nicking enzyme that originates from Bacillus stereothermophilus. “N” refers to nicking and “Nt” refers to nicking the top strand. See NEB websites: https://www.neb.com/en-us/products/restriction-endonucleases/hf-nicking-master-mix-time-saver-other/nicking-endonucleases [retrieved January 23, 2026] and https://www.neb.com/en-us/tools-and-resources/feature-articles/everything-you-ever-wanted-to-know-about-type-ii-restriction-enzymes [retrieved January 23, 2026].
Regarding claim 1, Alkan teaches the synthesis of DNA vectors, particularly closed-ended DNA vectors for gene expression in vivo, ex vivo or in vivo (Abstract). Alkan teaches the structure of the ceDNA vectors comprising an expression cassette that is flanked by two AAV ITRs (Fig 1, [0061[[0067]]). Alkan teaches assembling the ceDNA vectors by creating a left ITR (i.e., a 5’ ITR) with a 5’ overhang that comprises an AvrII restriction endonuclease recognition sequences (FIG 6B; [0072]). Alkan teaches assembling the ceDNA vectors by creating a left ITR (i.e., a 3’ ITR) with a 3’ overhang that comprises an Sbf1 restriction endonuclease recognition sequences (FIG 6A; [0072]). Alkan teaches once assembled, the ITR’s with 5’ and 3’ overhangs can anneal with the double-stranded DNA expression cassette to create a double-stranded DNA molecule (FIG 13; [0531]). Alkan teaches assembling a ceDNA with the Left ITR comprising an overhang comprising the AvrII restriction site cleavage overhang (i.e., a 5’ overhang) and the Right ITR comprising an overhang with the Sbf1 restriction site cleavage overhang (i.e., a 3’ overhang) ([0532]). Thus, when assembled, the ceDNA would comprise the following structure (adapted from Alkan FIG. 13A):
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Thus, Alkan teaches a double-stranded DNA molecule comprising (a) a left ITR (i.e., a first ITR) with restriction sites in proximity with cleavage sites on opposite strands such that when cleavage occurs it would create an ITR-containing segment with a bottom strand with a 3’ single stranded DNA overhang, and (b) a right ITR (i.e., a second ITR) with restriction sites in proximity with cleavage sites on opposite strands such that when cleavage occurs it would create an ITR-containing segment with a bottom strand with a 5’ single stranded DNA overhang. Alkan teaches the purpose of choosing the AvrII and SfbI sites is so that the segments would not homodimerize ([0536]).
Alkan does not teach the restriction sites are nicking endonucleases.
Wang teaches methods for cloning genes of interest into AAV vectors comprising inverted terminal repeats (ITRs) (Fig 1A and 7). Wang teaches using the nicking endonuclease, Nt. BbvCI, and compatible single-stranded overhangs to facilitate cloning of ITRs and expression constructs (Fig 7A).
Jack teaches site-specific nucleic acid nicking enzymes to create single-stranded regions in duplex nucleic acids that can be useful in ordered, oriented assembly of DNA modules (Abstract). Jack teaches the recognition sequence and cleavage position of Nt. BstNBI is 5’- GATCNNNN↓N-3’ and 3’ – CTCAGNNNNN-5’ ([0024]). Jack teaches using two Nt. BstNBI sites on opposite strands in relation to each other to create double-stranded DNA molecules with single-stranded DNA overhangs (Fig 2, [0027]), which can be either 5’ overhangs or 3’ overhangs ([0028]). Jack teaches that the single-strand cohesive ends can be defined by the user ([0031]). Jack teaches that using nicking endonucleases instead of restriction enzymes that produce overhang allows for longer cohesive ends to produce stable base pairing during cloning reactions ([0031]).
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have replaced the AvrII and SfbI restriction sites in Alkan’s AAV vectors with ITRs with Nt. BstNBI nicking sites such that the orientation of the overhangs is maintained. The skilled artisan would have predicted that nicking endonucleases could be used in place of the AvrII and SfbI sites because Wang teachings cloning AAV vectors (i.e., vectors comprising ITRs) with the use of nicking endonucleases. The skilled artisan would have been motivated to use nicking endonuclease sites so that the overhang can be user-defined to engineer sequences into the AAV vectors and promote stable-base pairing during cloning.
Regarding claim 5, Alkan teaches assembling the ceDNA in vitro (i.e., an isolated DNA molecule) (Abstract).
Regarding claim 6, the obviousness of including a single nicking endonuclease like Jack’s Nt. BstNBI is recited above as for claim 1.
Regarding claims 7 and 10, Alkan teaches the ITRs are wild type ITRs from AAV2 (i.e., the ITRs are from a parvovirus that is a Dependoparvovirus) (Figs 2 and 6; [0068], [0073]).
Regarding claims 12 and 13, Alkan teaches the AvrII enzyme cleaves the within 6 nucleotides from the 5’ end of the closing base pair of the left ITR and within 1 nucleotide from the 3’ end of the closing base pair of the left ITR (Fig 6B). Alkan teaches the Sbf1 enzyme cleaves within 2 nucleotides from the 5’ end of the closing base pair of the right ITR and 6 nucleotides from the 3’ end of the closing base pair of the right ITR (Fig 6C). The obviousness of including restriction sites from Jack’s nicking endonuclease Nt. BstNBI in place of the AvrII and SbfI sites is recited above as for claim 1.
Regarding claim 18, Alkan teaches the components of the ceDNA construct can be cloned into a circular plasmid (Fig 17; [0545]). Wang teaches ITRs flanked with nicking endonuclease sites within the context of a plasmid (Fig 7).
Regarding claims 24-26 and 31, Jack also teaches the nicking endonucleases can be used for plasmid assembly (FIGs 3B and 6). Jack teaches incorporation of additional Nt. BstNBI sites in the plasmid backbone (FIG. 6). Jack teaches generating Nt. BstNBI 18-nucleotide overhangs that are not compatible with other Nt. BstNBI overhangs ([0069]).
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have additionally included an 3rd set of Nt.BstNBI sites (i.e., a 5th and 6th site on opposite strands) to create a double strand break with 1-20 base overhangs that are not compatible to the cohesive ends of the pairs of the 1st and 2nd or 3rd and 4th endonuclease sites in the plasmid backbone. It would have amounted to the simple combination of known elements by known means to yield predictable results. The skilled artisan would have been motivated to include an additional pair of nicking sites in the backbone for the purpose of assembling the entire ITR-containing plasmid by the method of Jack. It would have been entirely predictable to have done so because Jack teaches using different nicking endonuclease cohesive ends as a means for orderly plasmid assembly in vitro.
Regarding claim 27, the specification dose not define or describe “an endogenous nicking endonuclease”. Because the claims are directed to DNA molecules and not compositions or cells, the term is interpreted to mean that the nicking endonuclease must originate from some organism so that it is endogenous to some cell. NEB teaches that Nt. BstNBI originates from Bacillus stereothermophilus. Thus, the nicking endonuclease site rendered obvious for claim 1 is the target sequence for a nicking endonuclease endogenous to Bacillus stereothermophilus.
Regarding claim 30, the obviousness of including a single nicking endonuclease like Jack’s Nt. BstNBI is recited above as for claim 1.
Regarding claims 53, 56, 60, 61, and 63 Alkan teaches the sequences of the right and left ITRs (FIG 6B and 6C), and their relation to the WT AAV ITR (FIG 6A and 6D). Alkan teaches the sequences of the Rep-binding sequence (i.e., a RABS) and the TRS ([0222]). It is evident from the “oligo synthesis” diagram of FIGs 6A and 6D, and the disclosed sequences of the ITRs terminating in the 5’ and 3’ overhangs in FIG 6B and 6C, that both the right and left ITRs lack an RAB sequence and a TRS.
Regarding claim 55, Alkan teaches the vectors for carrying expression cassettes including Luciferase and a transgene (Fig 1), indicating the double stranded DNA molecule does not encoding a viral capsid protein.
Regarding claim 68, Alkan teaches the DNA purification scheme, which involves removal of unligated oligos and parental plasmid digestion products (i.e., the isolated double strand DNA molecules are free from fragments of the double stranded DNA molecule) (FIG. 12).
Regarding claims 85-87, Alkan teaches the sequences of the right and left ITRs (FIG 6B and 6C), and their relation to the WT AAV ITR (FIG 6A and 6D). Alkan teaches the sequences of the Rep-binding sequence (i.e., a RABS) and the TRS ([0222]). Alkan teaches the overall structure of the double-stranded DNA molecule with the ITRs and transgenes (Fig 1). It is evident from the “oligo synthesis” diagram of FIGs 6A and 6D, the disclosed sequences of the ITRs terminating in the 5’ and 3’ overhangs in FIG 6B and 6C, and the overall vector diagram that Alkan’s ceDNA lacks an RBS and TRS because there were not included in the oligo synthesis (i.e., they were deleted), but includes the ITRs (i.e., does not delete an entire stem/loop structure).
Regarding claim 98, based on FIGs 1, 3, 3 and 6, concerning the cloning and components of the ITRs, there is no evidence in Alkan that the ITRs have any transcriptional activity.
Regarding claim 100, Alkan teaches the advantages of using ceDNA over plasmid-based expression is that ceDNA vectors do not have over-representation of CpG dinucleotides often found in plasmids ([0213]). Alkan teaches removal of part of the ITR structure including several CpG dinucleotides (compare FIG. 6C and 7B). Therefore, Alkan teaches engineering the ceDNA with ITRs such that they have low or reduced CpG motifs.
Regarding claim 102, Alkan teaches the left ITR and the right ITR form hairpins indicated that each comprise a palindromic sequence that is 100% identical to a sequence a complementary strand when read backwards (FIG 6B and 6C). Jack also teaches that the recognition sequences for the nicking endonuclease sites can be as far apart as needed to create cohesive ends as desired by the user ([0031]). Jack teaches a working example in which the cohesive ends are 18 nucleotides long ([0059]-[0070]). Jack teaches the nick site is 4 nucleotides downstream of the recognition site ([0069]). Thus, in Jack’s working example, the recognition sequences are 22 nucleotides apart to create the cohesive ends.
It would have been obvious to one skilled in the art to place the first and second Nt. BstNBI recognition sequences 22 nucleotides apart (i.e., at least 19, at least 20 nucleotides apart) in the DNA molecule rendered obvious above for claim 1. It would have amounted to designing the Nt. BstNBI-mediated cloning reaction by known means to yield predictable results. The skilled artisan would have been motivated to place the Nt. BstNBI recognition sequences at least 22 nucleotides apart, with a reasonable expectation of success because 1) Jack demonstrates that 18 base cohesive ends are sufficient for cloning and 2) Jack teaches that longer cohesive strands can promote cohesive-end hybridization stability.
Regarding claim 103, Alkan teaches the Gibb’s free energy in modified ITRs of Fig 10 ([0077]) and Fig 3 ([0069]). Using the online tool at https://en.vectorbuilder.com/tool/dna-secondary-structure.html, it is evident that the Gibbs free energies of Alkan’s left ITR and right ITR from the two terminal bases of the stem are -50.7 kcal/mol and -46.9 kcal/mol (i.e., no more than -10 kcal/mol) (See OA Appendix pages 1-2).
Regarding claim 104, Jack teaches that the recognition sequences for the nicking endonuclease sites can be as far apart as needed to create cohesive ends as desired by the user ([0031]). Jack teaches in a working example cohesive ends that are 18 nucleotides in length ([0059]-[0070]). Jack teaches the cohesive ends can be longer than 18 nucleotides ([0031]). Jack teaches lengths of the region should be long enough to allow unique stable pairing under the reaction conditions used in the joining reaction ([0031]).
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have placed the first and second Nt. BstNBI recognition sequences such that the single stranded region is at least 20 nucleotides in the DNA molecule rendered obvious above for claim 1. It would have amounted to optimizing the Nt. BstNBI-mediated cloning reaction by known means to yield predictable results. The skilled artisan would have been motivated to optimize the length of the single-stranded region to arrive at 20 nucleotides or more because Jack teaches optimization is necessary. A 20-nucleotide overhang would have been completely predictable because Jack demonstrates that 18 base cohesive ends are sufficient for cloning and teaches that longer overhangs are within the scope of the method.
Claim 20 is rejected under 35 U.S.C. 103 as being unpatentable over Alkan (US 20210071197 A1, priority to at least January 18, 2019), Wang (US 20090305272 A1, published December 10, 2009) and Jack (US 20030022317 A1, published January 30, 2003), as applied to claims 1, 5-7, 10, 12-13, 18, 24-27, 30-31, 53, 55-56, 60-61, 63, 68, 85-87, 89, 98, 100 and 102-104 above, and further in view of D’Costa (D’Costa et al., Molecular Therapy — Methods & Clinical Development (2016), 5: 16019, pages 1-9).
The teachings of Alkan, Wang and Jack are recited above and applied as for claims 1, 5-7, 10, 12-13, 18, 24-27, 30-31, 53, 55-56, 60-61, 63, 68, 85-87, 89, 98, 100 and 102-104. Briefly Alkan teaches ceDNA molecules with ITRs cloned using restriction enzymes leaving 5’ and 3’ overhangs for cloning with transgene expression cassettes. Alkan also teaches the components of the ceDNA can be in a plasmid. Wang teaches cloning ITRs and transgenes into an AAV plasmid using nicking endonucleases and Jack teaches the utility of nicking endonucleases. Wang also teaches transfecting the AAV plasmid into HEK 293 T virus-producing cells ([0092]). Wang teaches collecting the viral supernatant and determining the viral titer ([0092]).
Although Alkan and Wang teach ITRs and transgenes cloned into a plasmid, Alkan, Wang and Jack do not teach the plasmids have additional recognition sequences for cleaving or nicking restriction endonucleases in the plasmid backbone, i.e., outside of the ITR-transgene-ITR cassette. Wang does not teach by what means the rAAV was titered.
D’Costa teaches rAAV stocks need to be tittered before they can be administered in the clinic (Abstract). D’Costa teaches qPCR using a linearized version of the AAV plasmid (i.e., comprising the ITRs and transgene DNA) as a standard is routine in the art (Abstract). D’Costa teaches incorporating two restriction enzyme recognition sequences in the AAV plasmid just outside the ITRs so that the ITR-transgene-ITR cassette can be used as a standard, which gives results with less error (Fig 1).
Regarding claim 20, It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have incorporated one or more additional restriction endonuclease sites in the backbone of the AAV plasmid rendered obvious above for claim 18. It would have been amounted to the simple combination of elements by known means to yield predictable results. The skilled artisan would have been motivated to incorporate a different restriction enzyme site different from the nicking sites for the purpose of being able to utilize the plasmid as a standard to titer the rAAV stock after the plasmid has been used to generate the rAAV in helper cells as taught in D’Costa and Wang. By using a different restriction enzyme site, the ITR-transgene-ITR cassette would be intact and could be used as the standard. It would have been entirely predictable to have done so because engineering restriction enzyme sites into plasmids is well known in the art, as evidenced by each of the Alkan, Wang, Jack and D’Costa.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
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Claims 1, 5-7, 10, 12-13, 18, 20, 24-27, 30-31, 53, 55-56, 60-61, 63, 68, 85-87, 98, 100 and 102-104 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-24 of U.S. Patent No. 11634742. Claims 24-26 and 30-31 are rejected in view of Jack (US 20030022317 A1, published January 30, 2003). Claims 55 and 98 are rejected in view of Alkan (US 20210071197 A1, priority to at least January 18, 2019).
Patented claim 1 recites A method for preparing a hairpin-ended DNA molecule in vitro, wherein the method comprises: a. providing a double-stranded DNA molecule, wherein the double-stranded DNA molecule comprises in 5′ to 3′ direction of the top strand: i. a first inverted repeat, wherein a first and a second restriction site for nicking endonuclease are arranged on opposite strands wherein nicking results in: (1) a top strand 5′ single strand DNA overhang comprising the first inverted repeat or a fragment thereof upon separation of the top from the bottom strand of the first inverted repeat; or (2) a bottom strand 3′ single strand DNA overhang comprising the first inverted repeat upon separation of the top from the bottom strand of the first inverted repeat; and… iii. a second inverted repeat, wherein a third and a fourth restriction site for nicking endonuclease are arranged on opposite strands wherein nicking results in: (1) a top strand 3′ single strand DNA overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand of the second inverted repeat; or (2) a bottom strand 5′ single strand DNA overhang comprising the second inverted repeat upon separation of the top from the bottom strand of the second inverted repeat… wherein the length of each single strand DNA overhang is independently at least 20 nucleotides in length. Patented claim 5 recites The double-stranded DNA molecule of claim 1, wherein the first, second, third, and fourth restriction sites for nicking endonuclease are all restriction sites for the same nicking endonuclease. Patented claims 6 and 7, wherein the double-stranded DNA molecule of step a is characterized in that: a. the first and/or the second inverted repeat has a nucleotide sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or at least 99% identical to the ITR of a parvovirus; and/or b. the first and/or the second inverted repeat is a modified ITR of a parvovirus, wherein the parvovirus is a Dependoparvovirus, a Bocaparvovirus, an Erythroparvovirus, a Protoparvovirus, or a Tetraparvovirus. Patented claim 8 recites wherein the double-stranded DNA molecule of step (a) is characterized in that the first nick is within 1-50 nucleotides from the 5′ nucleotide of the ITR closing base pair of the first inverted repeat; and/or the second nick is within 1-50 nucleotides from the 3′ nucleotide of the ITR closing base pair of the first inverted repeat; and/or the third nick is within 1-50 nucleotides from the 5′ nucleotide of the ITR closing base pair of the second inverted repeat and the fourth nick is within 1-50 nucleotides from the e′ nucleotide of the ITR closing base pair of the second inverted repeat. Patented claim 9 recites wherein the double-stranded DNA molecule of step (a) is characterized in that the first nick is within 1-50 nucleotides from the 3′ nucleotide of the ITR closing base pair of the first inverted repeat; and/or the second nick is within 1-50 nucleotides from the 5′ nucleotide of the ITR closing base pair of the first inverted repeat; and/or the third nick is within 1-50 nucleotides from the 5′ nucleotide of the ITR closing base pair of the second inverted repeat and the fourth nick is within 1-50 nucleotides from the e′ nucleotide of the ITR closing base pair of the second inverted repeat. Patented claim 10 recites The method of claim 1, wherein the double-stranded DNA molecule of step a is a plasmid, wherein the plasmid is circular; or is linear. Patented claim 11 recites The method of claim 10, wherein the plasmid further comprises a restriction enzyme site in the region 5′ to the first inverted repeat and 3′ to the second inverted repeat wherein the restriction enzyme site is not present in any of the first inverted repeat, second inverted repeat, and/or the region between the first and second inverted repeats; and a. wherein cleavage with said restriction enzyme results in single strand DNA overhangs that do not anneal under annealing conditions of the first and/or second inverted repeat. Patented claim 14 recites, wherein the first and second inverted repeat of the double-stranded DNA molecule of step a each lack a functional RABS and a functional TRS. Patented claim 19 recites wherein the double-stranded DNA molecule of step a is characterized in that the first inverted repeat and the second inverted repeat each comprise a palindromic sequence that is 78%-99% identical when read forwards as when read backwards on the complementary strand, and wherein the first and the second restriction site for nicking endonuclease are at least 19 to at least 200 nucleotides apart; and/or the third and the fourth restriction sites for nicking endonuclease at least 19 to at least 200 nucleotides apart. Patented claim 22 recites wherein the first and second inverted repeat of the double-stranded DNA molecule of step a each further comprise a modification (e.g., deletion, substitution or addition) of at least one nucleotide in order to replace or deplete the occurrence of CpG motifs. Patented claim 23 recites wherein the first and second inverted repeat of the double-stranded DNA molecule of step d comprises the following: The sequence from one nucleotide of the ITR closing base pair to the other nucleotide of the ITR closing base pair has a Gibbs free energy of unfolding under physiological conditions of no more than −10 kcal/mol. Patented claim 24 recites wherein the length of each single strand DNA overhang is independently at least 21 to at least 100 nucleotides in length. Therefore, the patented claims anticipate examined claims 1, 5-7, 10, 12-13, 18, 20, 27, 53, 56, 60-61, 63, 68, 85-87, 100 and 102-104.
The patented claims do not recite the restriction site in outside of the ITR-expression cassette-ITR is a nicking site (claims 24-26) or the species of nicking endonucleases (claims 30-31). The patented claims do not recite the DNA molecule lacks a viral capsid protein encoding sequence (claim 55) or that the ITRs lack transcriptional activity (claim 98).
Regarding claims 24-26 and 30-31, the teachings of Jack are recited above in paragraphs 29, 36, and 49 and are incorporated here.
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have 1) specifically chosen the N.BstNBI enzyme and 2) to have included an 3rd set of N.BstNBI sites (i.e., a 5th and 6th site on opposite strands) to create a double strand break with 1-20 base overhangs that are not compatible to the cohesive ends of the pairs of the 1st and 2nd or 3rd and 4th endonuclease sites in the plasmid backbone of the patented plasmid. It would have amounted to 1) choosing a known nicking endonuclease for a generic disclosure, and 2) the simple combination of known elements by known means to yield predictable results. The skilled artisan would have been motivated to have chosen N. BstNBI because Jack teaches it is sufficient and exemplary for plasmid cloning purposes. Additionally, Jack teaches including an additional pair of nicking sites in the backbone for the purpose of assembling the entire ITR-containing plasmid. It would have been entirely predictable to have done so because Jack teaches using different nicking endonuclease cohesive ends as a means for orderly plasmid assembly in vitro.
Regarding claims 55 and 98, the teachings of Alkan are recited above in paragraphs 26, 31, 33-35, 40-46 and 49 and incorporated here.
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have modified the patented plasmid by ensuring the ITRs did not have transcriptional activity and the plasmid did not comprise the coding sequence for a viral capsid. It would have amounted to the simple combination of elements by known means to yield predictable results. The skilled artisan would have been motivated to make the changes with a reasonable expectation of success because Alkan teaches such features of double stranded DNA molecules with ITRs are sufficient for gene delivery in vivo.
Claims 1, 5-7, 10, 12-13, 18, 20, 24-28, 30-31, 53, 55-56, 60-61, 63, 68, 85-87, 98, 100 and 102-104 provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 5-13, 18-31, 36, 68, 71 of copending Application No. 18056931. Claims 53, 55-56, 60-81, 85-87 98, 100 and 102-103 are rejected in view of Alkan (US 20210071197 A1, priority to at least January 18, 2019).
Copending claim 1 recites A double-stranded DNA molecule comprising in 5' to 3' direction of the top strand: a. a first viral inverted terminal repeat (ITR), wherein a first and a second restriction site for nicking endonuclease are arranged on opposite strands within 20 nucleotides from ITR closing base pairs of the first viral ITR such that nicking results in: i. a top strand 5' single strand DNA overhang comprising the first viral ITR or a functional fragment thereof upon separation of the top from the bottom strand of the first viral ITR; or ii. a bottom strand 3' single strand DNA overhang comprising the first viral ITR or a functional fragment thereof upon separation of the top from the bottom strand of the first viral ITR; and b. an expression cassette; and c. a second viral ITR, wherein a third and a fourth restriction site for nicking endonuclease are arranged on opposite strands within 20 nucleotides from ITR closing base pairs of the second viral ITR such that nicking results in:i. a top strand 3' single strand DNA overhang comprising the second viral ITR or a functional fragment thereof upon separation of the top from the bottom strand of the second viral ITR; or ii. a bottom strand 5' single strand DNA overhang comprising the second viral ITR or a functional fragment thereof upon separation of the top from the bottom strand of the second viral ITR. Copending claim 5 recites wherein the double-stranded DNA molecule is an isolated DNA molecule. Copending claims 6 recites wherein the first, second, third, and fourth restriction sites for nicking endonuclease are all restriction sites for the same nicking endonuclease. Copending claims 8-11 recite wherein the first and/or the second viral ITR is an ITR of a parvovirus, including a dependoparvovirus. Copending claims 12 and 13 recite the limitations of examined claims 12-13. Copending claim 18 recites wherein the double-stranded DNA molecule is a plasmid. Copending claims 20-21 recite wherein the plasmid further comprises a restriction enzyme site in the region 5' to the first inverted repeat and 3' to the second inverted repeat wherein the restriction enzyme site is not present in any of the first inverted repeat, second inverted repeat, and the region between the first and second inverted repeats, wherein the cleavage with the restriction enzyme results in single strand overhangs that do not anneal at detectable levels under conditions that favor annealing of the first and/or second inverted repeat. Copending claim 22 recites wherein the plasmid further comprises an open reading frame encoding the restriction enzyme. Copending claim 24 recites wherein the plasmid further comprises a fifth and a sixth restriction site for nicking endonuclease in the region 5' to the first inverted repeat and 3' to the second inverted repeat, wherein the fifth and sixth restriction sites for nicking endonuclease are: a. on opposite strands; and b. create a break in the double stranded DNA molecule such that the single strand overhangs of the break do not anneal at detectable levels inter- or intramolecularly under conditions that favor annealing of the first and/or second inverted repeat. Copending claim 25 recites wherein the fifth and the sixth nick are 1,2,3,4,5,6,7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or20 nucleotides apart. Copending claim 26 recites wherein the first, second, third, fourth, fifth, and sixth restriction sites for nicking endonuclease are all target sequences for the same nicking endonuclease. Copending claim 27 recites wherein one or more of the nicking endonuclease site(s) is a target sequence of an endogenous nicking endonuclease. Copending claim 28 recites wherein the plasmid further comprises an open reading frame encoding a nicking endonuclease that recognizes the first, second, third, fourth, fifth, and/or sixth restriction site for nicking endonuclease. Copending claims 30-31 recite wherein the nicking endonuclease that recognizes the first, second, third, fourth, fifth and/or sixth restriction site for nicking endonuclease is Nt. BsmAI; Nt. BtsCI; N. ALwl; N. BstNBI; N. BspD6I; Nb. BsrDI; Nt. BtsI; Nt. BsaI; Nt. BpulOI; Nt. BsmBI; Nb. BbvCI; Nt. BbvCI; or Nt. BspQI. Copending claims 68 and 71 recite wherein the isolated double-stranded DNA molecules are: a. free of fragments of the double-stranded DNA molecule; and/or b. no more than 1- 10% of the isolated double-stranded DNA molecules; and/or c. free of nucleic acid contaminants that are not fragments of the double- stranded DNA molecules; and/or d. free of baculoviral DNA. Therefore, the copending claims anticipate examined claims 1, 5-7, 10, 12-13, 18, 20, 24-28, 30-31, 68, and 104.
The copending claims do not recite features of the ITR such as lack of RABS, TRS, CpG motifs or promoters (claims 53, 56, 60-61, 63, 85-87, 100). The patented claims do not recite the DNA molecule lacks a viral capsid protein encoding sequence (claim 55) or that the ITRs lack transcriptional activity (claim 98). The copending claims do not recite the ITRs are palindromic or have a specific Gibbs free energy (claims 102-103).
The teachings of Alkan are recited above in paragraphs 26, 31, 33-35, 40-46 and 49 and incorporated here.
Regarding claims 53, 55-56, 60-81, 85-87 and 98, it would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have modified the copending plasmid by ensuring the ITRs did not have RABS, TRS, promoters or otherwise transcriptional activity and that the plasmid did not comprise the coding sequence for a viral capsid. It would have amounted to the simple combination of elements by known means to yield predictable results. The skilled artisan would have been motivated to make the changes with a reasonable expectation of success because Alkan teaches such features of double stranded DNA molecules with ITRs are sufficient for gene delivery in vivo.
Regarding claim 100, it would have been obvious to have modified the copending DNA molecule by shortening one of the ITRs to removed CpGs because 1) Alkan demonstrates removal of a part of a hairpin region still promotes gene delivery and 2) teaches that CpG motifs can bind Toll-like receptors to initiate immune responses in vivo. Thus, the skilled artisan would have been motivated to modify or remove any CpG motifs to limit the immune reaction in vivo, which would render gene delivery less effective.
Regarding claims 102-103, it would have been obvious to one skilled in the art to ensure that the ITRs are palindromic or nearly palindromic with a Gibbs free energy of less than -10 kcal/mol since that are the key features of inverted repeats as illustrated in Alkan.
This is a provisional nonstatutory double patenting rejection.
Claims 1, 5-7, 10, 12-13, 18, 20, 24-27, 30-31, 53, 55-56, 60-61, 63, 68, 85-87, 98, 100 and 102-104 provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 27, 33-34, 41, 43, 50, 54, 57, 69 and 73 of copending Application No. 18728735. Claims 12-13, 25, 100-104 are rejected in view of Alkan (US 20210071197 A1, priority to at least January 18, 2019). Claim 12-13, 25 and 104 are rejected further in view of Jack (US 20030022317 A1, published January 30, 2003).
Copending claim 27 recites A double-stranded DNA molecule comprising in 5' to 3' direction of the top strand: a. a first inverted repeat, wherein a first and a second restriction site for nicking endonuclease are arranged on opposite strands in proximity of the first inverted repeat such that nicking results in a top strand 5' overhang or a bottom strand 3' overhang comprising the first inverted repeat upon separation of the top from the bottom strand of the first inverted repeat; b. an expression cassette comprising a transgene encoding human FVIII or a catalytically active fragment thereof; and c. a second inverted repeat, wherein a third and a fourth restriction site for nicking endonuclease are arranged on opposite strands in proximity of the second inverted repeat such that nicking results in a top strand 3' overhang or a bottom strand 5' overhang comprising the second inverted repeat upon separation of the top from the bottom strand of the second inverted repeat. Copending claim 34 recites wherein the first and/or the second inverted repeat is an inverted terminal repeat (ITR) of a parvovirus2 wherein the parvovirus is a Dependoparvovirus, a Bocaparvovirus, an Erythroparvovirus, a Protoparvovirus, or a Tetraparvovirus. Copedning claim 41 recites wherein the ITR does not comprise a replication-associated protein binding sequence (RABS). Coepnding claim 50 recites wherein the DNA molecule is a plasmid. Copending claim 54 recites wherein the plasmid further comprises a fifth and a sixth restriction site for nicking endonuclease in the region 5' to the first inverted repeat and 3' to the second inverted repeat, wherein the fifth and sixth restriction sites for nicking endonuclease are: a. on opposite strands; and b. create a break in the double stranded DNA molecule such that the single strand overhangs of the break do not anneal at detectable levels inter- or intramolecularly under conditions that favor annealing of the first and/or second inverted repeat. Copending claims 57 recites wherein the nicking endonuclease that recognizes the first, second, third, fourth, fifth, and/or sixth restriction site for nicking endonuclease is Nt. BsmAI; Nt. BtsCI; N. ALwl; N. BstNBI; N. BspD6I; Nb. Mval269I; Nb. BsrDI; Nt. BtsI; Nt. BsaI; Nt. Bpul0I; Nt. BsmBI; Nb. BbvCI; Nt. BbvCI; or Nt. BspQI. Copending claim 73 recites wherein the DNA molecule: a. lacks any functional replication-associated protein binding sequence (RABS); and/or b. lacks any functional terminal resolution site (TRS}; and/or c. lacks or has reduced (by at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or at least 99.9%) PS AAV promoter activity; and/or d. lacks any other elements required for viral replication. Therefore, the copending claims anticipate examined claims 1, 5-7, 10, 18, 20, 24, 26-27, 30-31, 53, 56, 60-61, 63, 68, 85-87, 89 and 98.
The copending claims do not recite distances for the nicking sites (claims 12-13, 25 and 104). The patented claims do not recite the DNA molecule lacks a viral capsid protein encoding sequence (claim 55) or modified to remove CpG motifs (claim 100). The copending claims do not recite the ITRs are palindromic or have a specific Gibbs free energy (claims 102-103).
The teachings of Alkan are recited above in paragraphs 26, 31, 33-35, 40-46 and 49 and incorporated here. The teachings of Jack are recited above in paragraphs 29, 36, and 49 and are incorporated here.
Regarding claims 12-13, 25 and 104, it would have been obvious to one skilled in the art to have situated the nicking sites in the copending DNA molecule within 1-50 nucleotides of the ITR and to generate overhangs that are at least 20 nucleotides. It would have amounted to arranging known elements according to known cloning design parameters to yield expected results. The skilled artisan would have been motivated to include the nicking sites within 1-50 nucleotides of the ITR with a reasonable expectation of success because Alkan teaches other restriction endonuclease sites within that region promotes cloning and results in vectors that are efficient for gene delivery in vivo. The skilled artisan would have also been motivated to optimize the length of the single-stranded region to arrive at least 20 nucleotides because Jack teaches optimization is necessary. A 20-nucleotide overhang would have been completely predictable because Jack demonstrates that 18 base cohesive ends are sufficient for cloning and teaches that longer overhangs are within the scope of the method.
Regarding claim 100, it would have been obvious to have modified the copending DNA molecule by shortening one of the ITRs to remove CpGs because 1) Alkan demonstrates partial removal of a stem region still promotes gene delivery and 2) teaches that CpG motifs can bind Toll-like receptors to initiate immune responses in vivo. Thus, the skilled artisan would have been motivated to modify or remove any CpG motifs to limit the immune reaction in vivo, which would render gene delivery less effective.
Regarding claims 102-103, it would have been obvious to one skilled in the art to ensure that the ITRs are palindromic or nearly palindromic with a Gibbs free energy of less than -10 kcal/mol since that are the key features of inverted repeats as illustrated in Alkan.
This is a provisional nonstatutory double patenting rejection.
Conclusion
No claims are allowable.
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/CATHERINE KONOPKA/Examiner, Art Unit 1635