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 .
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. Acknowledgment is made of applicants' claim for foreign priority to Chinese applications CN 202210536327.5 filed on 05/17/2022. Certified copies of the foreign priority document(s) are present in the application file.
Drawings
The application contains at least one color drawing or color photograph. Color photographs and color drawings are not accepted in utility applications unless a petition filed under 37 CFR 1.84(a)(2) is granted. Any such petition must be accompanied by the appropriate fee set forth in 37 CFR 1.17(h), one set of color drawings or color photographs, as appropriate, if submitted via the USPTO patent electronic filing system or three sets of color drawings or color photographs, as appropriate, if not submitted via the via USPTO patent electronic filing system, and, unless already present, an amendment to include the following language as the first paragraph of the brief description of the drawings section of the specification:
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Color photographs will be accepted if the conditions for accepting color drawings and black and white photographs have been satisfied. See 37 CFR 1.84(b)(2).
Information Disclosure Statement
The information disclosure statements (IDS) submitted on 12/17/2025 and 05/08/2024 are in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statements are being considered by the examiner.
Election/Restrictions
Applicant’s election without traverse of species B drawn to 930th amino acid cleavage site; NpuDnaE or RmaDnaB intein in the reply filed on 12/18/2025 is acknowledged. Election was made without traverse in the reply filed on 12/18/2025.
Status of Claims
Claims 1-20 are pending and under exam.
Claim Interpretation
Claims 14 and 16 are directed to a product that is obtained by a certain process. “[E]ven though product-by-process claims are limited by and defined by the process, determination of patentability is based on the product itself. The patentability of a product does not depend on its method of production. If the product in the product-by-process claim is the same as or obvious from a product of the prior art, the claim is unpatentable even though the prior product was made by a different process.” See MPEP 2113. As such the specification does not teach how a drug composition obtained from the dual vector system of claim 1 for treatment of hearing deficiencies is different from a drug obtained using any other methods. As such claim 16 is evaluated based on the resulting drug composition comprising the dual-vector OTOF system of claim 1, regardless of the steps to produce it. Similar analysis applies to the adenovirus of claim 14.
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1-8, and 10-20 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.
Regarding claim 1: The claim covers a dual vector system for expressing OTOF with:
a first nucleotide sequence comprises two first ITR sequences and an expression cassette inserted between the two first ITR sequences, a promoter, an N-terminal coding sequence of OTOF, an N-terminal coding sequence of intein, and a polyA and a
second nucleotide sequence comprises two second ITR sequences, an expression cassette inserted between the two second ITR sequences, a promoter, an N-terminal coding sequence of OTOF, an N-terminal coding sequence of intein, and a polyA; and wherein
the N-terminal coding sequence of the OTOF is a nucleotide coding sequence from an N-terminal of the OTOF amino acid sequence to the OTOF cleavage site, and the C-terminal coding sequence of OTOF is a nucleotide coding sequence from an amino acid next to the OTOF cleavage site to a C-terminal of the OTOF amino acid sequence.
The scope of the claim encompasses (a) OTOF cleavage site anywhere in the OTOF protein; (b) any N-term or C-term intein.
The specification summarizes number of possible constructs such as amino acids for N-term of OTOF and C-term part of OTOF (See specification Table 3). [0126] of the specification states that “A cleavage site was arranged on an OTOF amino acid sequence, an N-terminal coding sequence of OTOF was a nucleotide coding sequence from an N-terminal of the OTOF amino acid sequence to the cleavage site, and a C-terminal coding sequence of OTOF was a nucleotide coding sequence from an amino acid next to the cleavage site to a C-terminal of the OTOF amino acid sequence.” Further the same paragraph states that “[t]here were many choices for cleavage sites of the OTOF, some of which were listed in Table 3.” At [0127] the specification taught that “In an amino acid sequence of an OTOF protein shown in SEQ ID NO: 2, a 827th amino acid residue was used as a cleavage site (S1), a 930th amino acid residue was used as a cleavage site (S2), and a 1,130th amino acid residue was used as a cleavage site (S4) respectively; and NpuDnaE was used as intein and a pAAV-CMV plasmid (FIG. 1 and a sequence shown as SEQ ID NO: 9) as a vector to construct a first/second nucleotide sequence.”
It is submitted that while the specification described a general method of constructing a dual vector system expressing OTOF by splitting the protein at a cleavage site and fusing the N- and C-terms to inteins, only cleavage sites 827th, 930th and 1130th amino acid residues of SEQ ID NO: 2 using NpuDnaE intein and a pAAV-CMV vector were exemplified. While Table 3 lists numerous N-term and C-term fragments, the specification does not indicate that each of these sites were actually tested. It is submitted that the specification does not show that the inventors possessed all embodiments encompassed by the claim.
Applicant’s claims do not provide support for all the claimed embodiments of the claim. The specification as filed does not provide sufficient evidence that Applicants were in possession of the full scope of the claimed invention at the time of filing of the instant invention.
Claims 2-8 and 10-20 are rejected for their dependency.
Claim 1-8, and 10-20 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 enablement requirement.
The claim(s) contains subject matter which was not described in the specification in such a way as to enable one skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention.
Applicant's specification is found enabling for dual-vector OTOF constructs where the OTOF protein is split at 827, 930, 1130, using intein NpuDnaE and at positions 827, 930, 954, and 1130 using intein RmaDnaB.
Applicant's specification is not found to be enabling for all possible OTOF cleavage sites outside the ones actually tested in Examples 2 and 3.; and all possible N-term and C-term inteins. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to carry out the method of the invention commensurate in scope with the current claims.
Analysis of whether a particular claim is supported by the disclosure in an application requires a determination of whether that disclosure, when filed, contained sufficient information regarding the subject matter of the claims as to enable one skilled in the pertinent art to make and use the claimed invention without undue or unreasonable experimentation. See Mineral Separation v. Hyde, 242 U.S. 261, 270 (1916). The key word is 'undue,' not experimentation.' " (Wands, 8 USPQ2d 1404). The factors to be considered in determining whether undue experimentation is required are summarized In re Wands 858 F.2d 731, 8 USPQ2nd 1400 (Fed. Cir, 1988). The factors to be considered in determining whether undue experimentation is required include: (1) the quantity of experimentation necessary, (2) the amount or direction or guidance presented, (3) the presence or absence of working examples, (4) the nature of the invention, (5) the state of the prior art, (6) the relative skill of those in the art, (7) the predictability or unpredictability of the art, and (8) the breadth of the claims. While all these factors are considered, a sufficient number are discussed below so as to create a prima facie case.
Applicants' claims are directed to a dual vector system for expressing OTOF with:
a first nucleotide sequence comprises two first ITR sequences and an expression cassette inserted between the two first ITR sequences, a promoter, an N-terminal coding sequence of OTOF, an N-terminal coding sequence of intein, and a polyA and a
second nucleotide sequence comprises two second ITR sequences, an expression cassette inserted between the two second ITR sequences, a promoter, an N-terminal coding sequence of OTOF, an N-terminal coding sequence of intein, and a polyA; and wherein
the N-terminal coding sequence of the OTOF is a nucleotide coding sequence from an N-terminal of the OTOF amino acid sequence to the OTOF cleavage site, and the C-terminal coding sequence of OTOF is a nucleotide coding sequence from an amino acid next to the OTOF cleavage site to a C-terminal of the OTOF amino acid sequence.
The scope of the claim encompasses (a) OTOF cleavage site anywhere in the OTOF protein; (b) any intein site.
The specification provides support for a general concept of dual-vector OTOF split as disclosed in Example 1. Example 2 provides construction of first and second nucleotide sequences using intein NpuDnaE at cleavage sites 827, 930, 1130 as well as using intein RmaDnaB at cleavage sites 827, 930, 954 and 1130 as shown in Example 3. Additional examples teach construction of recombinant plasmids, preparation of AAVs, recombination of plasmids various ratios , transfection efficiencies comparison of OTOF intein recombination vs DNA recombination and in vivo testing.
At the time the invention was made it was known construct design of a dual vector system is critical and that splitting proteins outside structural domains, selecting appropriate inteins and balancing vector sizes were required for optimization. Due to variable splicing efficiency and protein stability, and co-infection efficiency meant that success cannot be assumed for arbitrary split sites. For example, Tornabene taught that “splitting points for each protein were selected taking into account both amino acid residue requirements at the junction points for efficient protein trans-splicing, as well as preservation of the integrity of critical protein domains, which should favor proper folding and stability of each independent polypeptide, and thus, of the final reconstituted protein. Additional split-inteins were also considered.” (See Tornabene p. 5, para 4). It was known from Al-Moyed that otoferlin coding sequence (CDS) can be split into two AAV by using the first half of the otoferlin coding sequence (CDS), and a splice donor sequence in the first AAV half vector and a splice acceptor sequence and the second half of the Otoferlin in the second half vector. However, Al-Moyed did not demonstrate use of any site in OTOF as the split site as currently claimed. It is submitted that there was a recognized level of unpredictability with regards to choice of cleavage sites and selection of intein. It is pointed out that Stevens et al (Proc. Natl. Acad. Sci. U.S.A; “Stevens;” See PTO-892) taught that “ currently used naturally split inteins suffer from an “extein dependence,” whereby residues surrounding the splice junction strongly affect splicing efficiency, limiting the general applicability of many PTS-based methods.” (See Stevens Abstract). Therefore, it was known that splicing depends on local sequence context and is not predictable.
Due to the lack of teachings in the art regarding all the split sites in OTOF coding sequence, and the recognized unpredictability in choosing inteins, a large amount of guidance and teachings would be necessary in order to be enabling for methods of such.
Guidance and teachings provided by Applicants in the instant specification is limited to use of intein NpuDnaE at cleavage sites 827, 930, 1130 as well as using intein RmaDnaB at cleavage sites 827, 930, 954 and 1130 as shown in Examples 2 and 3. The Examiner acknowledges that the Office does not require the presence of working examples to be present in the disclosure of the invention (see MPEP §2164.02). However, in light of the state of the art, discussed above, which recognizes a high level of unpredictability in the field of molecular engineering, and limited teachings with regards to OTOF protein expression, the Office would require appropriate disclosure to support the breadth of the currently claimed composition. The amount of guidance or direction needed to enable the invention is inversely related to the amount of knowledge in the state of the art as well as the predictability in the art. In re Fisher, 427 F.2d 833, 839, 166 USPQ 18, 24 (CCPA 1970). Thus, due to the high level of unpredictability in the art, the current specification would have to provide greater amounts of teachings and guidance directed to methods of carrying out the claimed invention.
Therefore, due to the sum of all the aforementioned factors, one of ordinary skill in the art, at the time the invention was made, would not expect to arrive at all the encompassed claimed composition. It is submitted that the skilled artisan would be faced with the impermissible burden of undue experimentation in order to practice the claimed invention using any species of stem cell. Accordingly, claims 1-8, 10-20 are deemed properly rejected.
Claims 2-8 and 10-20 are rejected for their dependency.
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 10, 15, and 19-20 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), s-second paragraph, as failing to set forth the subject matter which the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the applicant regards as the invention.
Claim 10 recites “a vector carrying AAV rep and cap genes, and a helper virus vector, and the vector carrying the AAV rep and cap genes and the helper virus vector are packaged into AAV vectors.” It is unclear if the vector carrying AAV rep and cap genes and the helper virus vector are themselves intended to be packaged into AAV particles or whether they are merely provided during vector production. Claims 19-20 are rejected for their dependency.
Claim 15 recites a method of use, however fails to indicate any active steps constituting the method of use. As such the scope of the claim is unclear.
Claims 19-20 recite a packaging vector, but claims an OTOF sequence. It is known that packaging vectors have the rep/cap genes and helper genes, but not the gene of interest. As such the metes and bounds of this claim is unclear.
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claim 14 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
The claims are directed to an AAV obtained by a packaging method. As indicated above, products-process claims are evaluated based on their composition. As such the AAV does not further limit the packaging method of claim 12. Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claim 15 is rejected under 35 U.S.C. 101 because the claim does not recite a statutory subject matter.
The claim(s) does/do not fall within at least one of the four categories of patent eligible subject matter because it is directed to a use of a dual vector system or an adeno-associated virus. The claimed recitation of a use, without setting forth any steps involved in the process, results in an improper definition of a process, i.e. results in a claim which is not a proper process under 35 U.S.C. 101. See for example Ex parte Dunki, 153 USPQ678 (Bd.App.1967) and Clinical Products, Ltd. v. Brenner, 255 F. Supp.131, 149 USPQ 475 (D.D.C.1966).
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.
This rejection under 35 U.S.C. 103 might be overcome by: (1) a showing under 37 CFR 1.130(a) that the subject matter disclosed in the reference was obtained directly or indirectly from the inventor or a joint inventor of this application and is thus not prior art in accordance with 35 U.S.C.102(b)(2)(A); (2) a showing under 37 CFR 1.130(b) of a prior public disclosure under 35 U.S.C. 102(b)(2)(B); or (3) a statement pursuant to 35 U.S.C. 102(b)(2)(C) establishing that, not later than the effective filing date of the claimed invention, the subject matter disclosed and the claimed invention were either owned by the same person or subject to an obligation of assignment to the same person or subject to a joint research agreement. See generally MPEP § 717.02.
Claims 1, 3, 5-8 and 15-18 are rejected under 35 U.S.C. 103 as being obvious over Al-Moyed et al (EMBO Mol Med. 2019 Jan; "Al-Moyed;" See PTO-892) in view of Tornabene et al (Sci Transl Med. 2019 May 15; " Tornabene;" See IDS filed on 12/17/2025).
Regarding claims 1, 5, 7 and 15-18: Al-Moyed “generated two adeno-associated viruses (AAVs), each containing half of the otoferlin (OTOF) cDNA.” (See Al-Moyed Abstract). Al-Moyed indicated that “the limited AAV cargo capacity of approximately 4.7–5 kb presents an obstacle for the transfer of large coding sequences (CDS) such as the 6 kb-long otoferlin cDNA. Split-AAV vectors, each containing a fragment of the large transgene expression cassette, have been developed to circumvent this problem. This technique takes advantage of the intrinsic ability of the AAV genome to form tail-to-head concatemers by end-joining of its inverted terminal repeats (ITRs)” (See Al Moyed p. 2, col. 1, para 1). As such it was previously known that the 6 kb long cDNA of Otoferlin presented significant obstacle for the transfer into AAV vector. Further Al-Moyed demonstrated use of the first half of the otoferlin coding sequence (CDS), and a splice donor sequence in the first AAV half vector and the second half of the Otoferlin in the second half vector. It is noted that Al Moyed did not teach the claimed AAV construct particularly involving inteins.
However, Tornabene taught that “delivery of multiple AAV vectors each encoding one of the fragments of target proteins flanked by short split-inteins results in protein trans-splicing and full-length protein reconstitution in the retina of mice, pigs and in human retinal organoids.“ Tornabene “took advantage of the intrinsic ability of split-inteins to mediate protein trans-splicing to reconstitute large full-length proteins following their fragmentation into either two or three split-intein-flanked polypeptides whose sequences fit into single AAV vectors” and “tested the efficiency of AAV intein in the retina by delivering the enhanced green fluorescent protein (EGFP) gene and the large ATP binding cassette subfamily A member 4 (ABCA4) and centrosomal protein 290 (CEP290) genes, which are defective in two common forms of severe inherited retinal diseases, Stargardt disease (STGD1) and Leber congenital amaurosis type 10 (LCA10), respectively.” (See Tornabene p. 2 last para to p. 3 1st para). Tornabene used dual AAV vectors (or plasmids, as required by claim 7) each with its own ITRs, promoters, flag tag (as required by claim 5) and poly A and either a N-term or a C-term coding sequence. (See Tornabene Figure 1). With their design Tornabene demonstrated AAV intein-mediated protein trans-splicing reconstitutes large proteins both in vitro and in the retina of mouse, pig and in human retinal organoids. (See Tornabene p. 10, 5th para).
A person of ordinary skill in the art would have been motivated to combine the teachings of Al Moyed’s dual-AAV OTOF split concept with the split intein AAV approach taught by Tornabene, resulting in a system where both halves of OTOF are independently expressed with promoters and poly A signals and reconstitute via split intein trans-splicing. The person would have been motivated to combine and would have had a reasonable expectation of success as Tornabene shows successful intein-mediated reconstitution of large proteins in AAV systems.
It is noted that claim 15 does not recite any further patentably distinct technical limitations. As such the same rejection as those for claim 1 apply to claim 15.
It is also noted that Al-Moyed taught that “[a]uditory function in profoundly deaf otoferlin knock-out mice was partially rescued after split-AAV otoferlin treatment” as generated in their invention (See Al-Moyed p. 10, blue box, 2nd para) as required by claim 16. It is noted that their “[f]inal virus preparations were dialyzed against PBS, and their purity was confirmed by SDS–gel electrophoresis.” (See Al Moyed p. 9, col. 1, para 2). PBS reads on a neutral salt buffer as required by claim 17. They injected “The virus solution was injected through the auditory bulla covering the round window membrane (RWM) into the scala tympani of the left cochlea at postnatal day” (See Al-Moyed p. 9, col. 1, para 2), as required by claim 18. Al Moyed also used AAV2/6.eGFP (1.44 × 1010 vg/µl), otoferlin dual-AAV2/6-TS half-vectors (1:1) (1.2 × 1010 vg/µl), and otoferlin dual-AAV2/6-Hyb half-vectors (1:1) (1.38 × 1010 vg/µl).
Regarding claim 3: Tornabene taught that “[i]ntein activity is context-dependent, with certain peptide sequences surrounding their ligation junction (called N- and C-exteins) that are required for efficient trans-splicing to occur, of which the most important is an amino acid containing a thiol or hydroxyl group (Cys, Ser or Thr) as first residue in the C-extein” (See Tornabene p. 2, 3rd para). As such one of ordinary skill would have been motivated to choose a cleavage site which is immediately before S, T or C as required by the claim.
Regarding claim 6: Tornabene used “DnaE split-intein from Nostoc punctiforme [Npu Fig. 1A]” (See Tornabene p. 3, para 2).
Regarding claim 8: Tornabene used a pAAV plasmid for expression of split GFP (See Tornabene Fig. 1)
Claim 2 is rejected under 35 U.S.C. 103 as being obvious over Al-Moyed et al (EMBO Mol Med. 2019 Jan; "Al-Moyed;" See PTO-892) in view of Tornabene et al (Sci Transl Med. 2019 May 15; "Tornabene;" See IDS filed on 12/17/2025) and Yasunaga et al (Nat Genet. 1999 Apr; hereinafter "Yasunaga;" See PTO-892).
Regarding claim 2: The teachings of Al Moyed in view of Tornabene are set forth above. However, none of the references explicitly taught Otoferlin of SEQ ID NO: 1 or SEQ ID NO: 2. However, it is submitted that the sequence of SEQ ID NO: 1 was well known before the filing date of instant application, for example as shown by the alignment below by Yasunaga. It would have been obvious for a person of ordinary skill in the art to arrive at the claimed AAC dual vector system with the OTOF sequence taught by Yasunaga. The person would have had reasonable expectation of success would have had a reasonable expectation of success as the prior art showed successful intein-mediated reconstitution of large proteins in AAV systems and the sequence of the claimed protein of interest was known.
Query Match 100.0%; Score 10533; Length 1997;
Best Local Similarity 100.0%;
Matches 1997; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MALLIHLKTVSELRGRGDRIAKVTFRGQSFYSRVLENCEDVADFDETFRWPVASSIDRNE 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MALLIHLKTVSELRGRGDRIAKVTFRGQSFYSRVLENCEDVADFDETFRWPVASSIDRNE 60
Qy 61 MLEIQVFNYSKVFSNKLIGTFRMVLQKVVEESHVEVTDTLIDDNNAIIKTSLCVEVRYQA 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 MLEIQVFNYSKVFSNKLIGTFRMVLQKVVEESHVEVTDTLIDDNNAIIKTSLCVEVRYQA 120
Qy 121 TDGTVGSWDDGDFLGDESLQEEEKDSQETDGLLPGSRPSSRPPGEKSFRRAGRSVFSAMK 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 TDGTVGSWDDGDFLGDESLQEEEKDSQETDGLLPGSRPSSRPPGEKSFRRAGRSVFSAMK 180
Qy 181 LGKNRSHKEEPQRPDEPAVLEMEDLDHLAIRLGDGLDPDSVSLASVTALTTNVSNKRSKP 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 LGKNRSHKEEPQRPDEPAVLEMEDLDHLAIRLGDGLDPDSVSLASVTALTTNVSNKRSKP 240
Qy 241 DIKMEPSAGRPMDYQVSITVIEARQLVGLNMDPVVCVEVGDDKKYTSMKESTNCPYYNEY 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 DIKMEPSAGRPMDYQVSITVIEARQLVGLNMDPVVCVEVGDDKKYTSMKESTNCPYYNEY 300
Qy 301 FVFDFHVSPDVMFDKIIKISVIHSKNLLRSGTLVGSFKMDVGTVYSQPEHQFHHKWAILS 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 FVFDFHVSPDVMFDKIIKISVIHSKNLLRSGTLVGSFKMDVGTVYSQPEHQFHHKWAILS 360
Qy 361 DPDDISSGLKGYVKCDVAVVGKGDNIKTPHKANETDEDDIEGNLLLPEGVPPERQWARFY 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 DPDDISSGLKGYVKCDVAVVGKGDNIKTPHKANETDEDDIEGNLLLPEGVPPERQWARFY 420
Qy 421 VKIYRAEGLPRMNTSLMANVKKAFIGENKDLVDPYVQVFFAGQKGKTSVQKSSYEPLWNE 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 VKIYRAEGLPRMNTSLMANVKKAFIGENKDLVDPYVQVFFAGQKGKTSVQKSSYEPLWNE 480
Qy 481 QVVFTDLFPPLCKRMKVQIRDSDKVNDVAIGTHFIDLRKISNDGDKGFLPTLGPAWVNMY 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 481 QVVFTDLFPPLCKRMKVQIRDSDKVNDVAIGTHFIDLRKISNDGDKGFLPTLGPAWVNMY 540
Qy 541 GSTRNYTLLDEHQDLNEGLGEGVSFRARLLLGLAVEIVDTSNPELTSSTEVQVEQATPIS 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 541 GSTRNYTLLDEHQDLNEGLGEGVSFRARLLLGLAVEIVDTSNPELTSSTEVQVEQATPIS 600
Qy 601 ESCAGKMEEFFLFGAFLEASMIDRRNGDKPITFEVTIGNYGNEVDGLSRPQRPRPRKEPG 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 601 ESCAGKMEEFFLFGAFLEASMIDRRNGDKPITFEVTIGNYGNEVDGLSRPQRPRPRKEPG 660
Qy 661 DEEEVDLIQNASDDEAGDAGDLASVSSTPPMRPQVTDRNYFHLPYLERKPCIYIKSWWPD 720
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 661 DEEEVDLIQNASDDEAGDAGDLASVSSTPPMRPQVTDRNYFHLPYLERKPCIYIKSWWPD 720
Qy 721 QRRRLYNANIMDHIADKLEEGLNDIQEMIKTEKSYPERRLRGVLEELSCGCCRFLSLADK 780
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 721 QRRRLYNANIMDHIADKLEEGLNDIQEMIKTEKSYPERRLRGVLEELSCGCCRFLSLADK 780
Qy 781 DQGHSSRTRLDRERLKSCMRELENMGQQARMLRAQVKRHTVRDKLRLCQNFLQKLRFLAD 840
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 781 DQGHSSRTRLDRERLKSCMRELENMGQQARMLRAQVKRHTVRDKLRLCQNFLQKLRFLAD 840
Qy 841 EPQHSIPDIFIWMMSNNKRVAYARVPSKDLLFSIVEEETGKDCAKVKTLFLKLPGKRGFG 900
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 841 EPQHSIPDIFIWMMSNNKRVAYARVPSKDLLFSIVEEETGKDCAKVKTLFLKLPGKRGFG 900
Qy 901 SAGWTVQAKVELYLWLGLSKQRKEFLCGLPCGFQEVKAAQGLGLHAFPPVSLVYTKKQAF 960
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 901 SAGWTVQAKVELYLWLGLSKQRKEFLCGLPCGFQEVKAAQGLGLHAFPPVSLVYTKKQAF 960
Qy 961 QLRAHMYQARSLFAADSSGLSDPFARVFFINQSQCTEVLNETLCPTWDQMLVFDNLELYG 1020
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 961 QLRAHMYQARSLFAADSSGLSDPFARVFFINQSQCTEVLNETLCPTWDQMLVFDNLELYG 1020
Qy 1021 EAHELRDDPPIIVIEIYDQDSMGKADFMGRTFAKPLVKMADEAYCPPRFPPQLEYYQIYR 1080
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1021 EAHELRDDPPIIVIEIYDQDSMGKADFMGRTFAKPLVKMADEAYCPPRFPPQLEYYQIYR 1080
Qy 1081 GNATAGDLLAAFELLQIGPAGKADLPPINGPVDVDRGPIMPVPMGIRPVLSKYRVEVLFW 1140
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1081 GNATAGDLLAAFELLQIGPAGKADLPPINGPVDVDRGPIMPVPMGIRPVLSKYRVEVLFW 1140
Qy 1141 GLRDLKRVNLAQVDRPRVDIECAGKGVQSSLIHNYKKNPNFNTLVKWFEVDLPENELLHP 1200
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1141 GLRDLKRVNLAQVDRPRVDIECAGKGVQSSLIHNYKKNPNFNTLVKWFEVDLPENELLHP 1200
Qy 1201 PLNIRVVDCRAFGRYTLVGSHAVSSLRRFIYRPPDRSAPSWNTTVRLLRRCRVLCNGGSS 1260
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1201 PLNIRVVDCRAFGRYTLVGSHAVSSLRRFIYRPPDRSAPSWNTTVRLLRRCRVLCNGGSS 1260
Qy 1261 SHSTGEVVVTMEPEVPIKKLETMVKLDATSEAVVKVDVAEEEKEKKKKKKGTAEEPEEEE 1320
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1261 SHSTGEVVVTMEPEVPIKKLETMVKLDATSEAVVKVDVAEEEKEKKKKKKGTAEEPEEEE 1320
Qy 1321 PDESMLDWWSKYFASIDTMKEQLRQQEPSGIDLEEKEEVDNTEGLKGSMKGKEKARAAKE 1380
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1321 PDESMLDWWSKYFASIDTMKEQLRQQEPSGIDLEEKEEVDNTEGLKGSMKGKEKARAAKE 1380
Qy 1381 EKKKKTQSSGSGQGSEAPEKKKPKIDELKVYPKELESEFDNFEDWLHTFNLLRGKTGDDE 1440
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1381 EKKKKTQSSGSGQGSEAPEKKKPKIDELKVYPKELESEFDNFEDWLHTFNLLRGKTGDDE 1440
Qy 1441 DGSTEEERIVGRFKGSLCVYKVPLPEDVSREAGYDSTYGMFQGIPSNDPINVLVRVYVVR 1500
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1441 DGSTEEERIVGRFKGSLCVYKVPLPEDVSREAGYDSTYGMFQGIPSNDPINVLVRVYVVR 1500
Qy 1501 ATDLHPADINGKADPYIAIRLGKTDIRDKENYISKQLNPVFGKSFDIEASFPMESMLTVA 1560
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1501 ATDLHPADINGKADPYIAIRLGKTDIRDKENYISKQLNPVFGKSFDIEASFPMESMLTVA 1560
Qy 1561 VYDWDLVGTDDLIGETKIDLENRFYSKHRATCGIAQTYSTHGYNIWRDPMKPSQILTRLC 1620
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1561 VYDWDLVGTDDLIGETKIDLENRFYSKHRATCGIAQTYSTHGYNIWRDPMKPSQILTRLC 1620
Qy 1621 KDGKVDGPHFGPPGRVKVANRVFTGPSEIEDENGQRKPTDEHVALLALRHWEDIPRAGCR 1680
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1621 KDGKVDGPHFGPPGRVKVANRVFTGPSEIEDENGQRKPTDEHVALLALRHWEDIPRAGCR 1680
Qy 1681 LVPEHVETRPLLNPDKPGIEQGRLELWVDMFPMDMPAPGTPLDISPRKPKKYELRVIIWN 1740
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1681 LVPEHVETRPLLNPDKPGIEQGRLELWVDMFPMDMPAPGTPLDISPRKPKKYELRVIIWN 1740
Qy 1741 TDEVVLEDDDFFTGEKSSDIFVRGWLKGQQEDKQDTDVHYHSLTGEGNFNWRYLFPFDYL 1800
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1741 TDEVVLEDDDFFTGEKSSDIFVRGWLKGQQEDKQDTDVHYHSLTGEGNFNWRYLFPFDYL 1800
Qy 1801 AAEEKIVISKKESMFSWDETEYKIPARLTLQIWDADHFSADDFLGAIELDLNRFPRGAKT 1860
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1801 AAEEKIVISKKESMFSWDETEYKIPARLTLQIWDADHFSADDFLGAIELDLNRFPRGAKT 1860
Qy 1861 AKQCTMEMATGEVDVPLVSIFKQKRVKGWWPLLARNENDEFELTGKVEAELHLLTAEEAE 1920
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1861 AKQCTMEMATGEVDVPLVSIFKQKRVKGWWPLLARNENDEFELTGKVEAELHLLTAEEAE 1920
Qy 1921 KNPVGLARNEPDPLEKPNRPDTSFIWFLNPLKSARYFLWHTYRWLLLKLLLLLLLLLLLA 1980
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1921 KNPVGLARNEPDPLEKPNRPDTSFIWFLNPLKSARYFLWHTYRWLLLKLLLLLLLLLLLA 1980
Qy 1981 LFLYSVPGYLVKKILGA 1997
|||||||||||||||||
Db 1981 LFLYSVPGYLVKKILGA 1997
Claim 4 is rejected under 35 U.S.C. 103 as being obvious over Al-Moyed et al (EMBO Mol Med. 2019 Jan; "Al-Moyed;" See PTO-892) in view of Tornabene et al (Sci Transl Med. 2019 May 15; " Tornabene;" See IDS filed on 12/17/2025) and Proudfoot (Genes Dev. 2011 Sep 1; See PTO-892).
Regarding claim 4: The teachings of Al Moyed in view of Tornabene are set forth above. It is noted that Tornabene used a CMV promoter for expression of EGFP and confirmed expression by infecting HEK293 cells with either AAV2/2-EGFP intein. (See Tornabene p. 3, last para; Figure 1). While none of the references taught a AATAA poly A, the sequence is well known as taught by Proudfoot “The central sequence motif AAUAAA was identified in the mid-1970s and subsequently shown to require flanking, auxiliary elements for both 3′-end cleavage and polyadenylation of premessenger RNA (pre-mRNA) as well as to promote downstream transcriptional termination.” (See Proudfoot Abstract). It would have been obvious for a person of ordinary skill in the art to have used a poly A comprising AATAAA motif as taught by Proudfoot as it was well known at the time of filing of the instant application.
Claims 10-14 and 20 are rejected under 35 U.S.C. 103 as being obvious over Al-Moyed et al (EMBO Mol Med. 2019 Jan; "Al-Moyed;" See PTO-892) in view of Tornabene et al (Sci Transl Med. 2019 May 15; "Tornabene;" See IDS filed on 12/17/2025) and Dieter et al (Nat Commun 10, 1962 (2019); See PTO-892); further evidenced by US7115391B1 (published Oct 03, 2006; see PTO-892).
Regarding claims 10-14 and 20: The teachings of Al-Moyed in view of Tornabene are set forth above. It is noted that Al-Moyed used otoferlin dual-AAV constructs and an eGFP-expressing control construct, containing an hbA promoter, a CMVe enhancer, a WPRE element, and a pA sequence packaged into an AAV2/6 serotype. (See Al-Moyed, p.9, col. 1, 2nd para) It is noted that Al-Moyed used transfection HEK293 cells in the presence of the helper plasmid pDP. (See Al Moyed, p.9, col. 1, 2nd para). Al-Moyed did not teach or suggest pHelper plasmid. However, use of pHelper plasmid was routinely used in AAV mediated delivery of genetic material to cells of the ear. For example, Dieter taught the use of HEK-293T cells to generate Adeno-associated viruses (AAVs). Dieter taught, triple transfection of HEK-293T cells using pHelper plasmid, trans-plasmid providing viral capsid PHP.B and cis-plasmid containing gene of interest flanked by two ITRs in the ends. (As required by claims 10-13). Dieter taught that “trans-plasmid provided the viral capsid PHP.B.” It is known and further evidenced by US7115391B1 that the trans plasmid comprises re/cap genes. For example US7115391B1 taught that “[o]ne method that has been used to produce recombinant AAV (rAAV) vectors comprises co-transfecting eukaryotic cells with a plasmid containing rAAV sequences (the cis plasmid) and a plasmid containing rep and cap (the trans plasmid).“ Using this method, Dieter manufactured an rAAV carrying a gene of interest. For example, Dieter mentioned that harvested viral particles 72 h after transfection from the medium and 120 h after transfection from cells and the medium. It would have been obvious for a person of ordinary skill in the art to have used the packaging vector as taught by Dieter as it was well known at the time of filing of the instant application and was known to produce functional adeno associated viruses.
Claim 19 is rejected under 35 U.S.C. 103 as being obvious over Al-Moyed et al (EMBO Mol Med. 2019 Jan; "Al-Moyed;" See PTO-892) in view of Tornabene et al (Sci Transl Med. 2019 May 15; "Tornabene;" See IDS filed on 12/17/2025) and Dieter et al (Nat Commun 10, 1962 (2019); See PTO-892); further evidenced by US7115391B1 (published Oct 03, 2006; see PTO-892) and further in view of Yasunaga et al (Nat Genet. 1999 Apr; hereinafter "Yasunaga;" See PTO-892).
Regarding claim 19: The teachings of each of the cited prior art are set forth above. It is submitted that it would have been obvious for a person of ordinary skill in the art to arrive at the claimed packaging vector as the prior art showed successful intein-mediated reconstitution of large proteins in AAV systems and the sequence of the claimed protein of interest was known. The person would have also had a reasonable expectation of success in view of teaching of Dieter that Adeno-associated viruses (AAVs can be generated by triple transfection of HEK-293T cells using pHelper plasmid, trans-plasmid providing viral capsid PHP.B and cis-plasmid containing gene of interest flanked by two ITRs in the ends. As such a person will have a reasonable expectation of success in arriving at the claimed packaging vector system.
Claim Objections
Claim 9 is objected to for being dependent on a rejected base claim but will be allowable if written in an independent form.
Conclusion
Claim 9 is found to be free of art. No claim is allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to JAGAMYA VIJAYARAGHAVAN whose telephone number is (703)756-5934. The examiner can normally be reached 9:00a-5:00p.
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/JAGAMYA NMN VIJAYARAGHAVAN/Examiner, Art Unit 1633
/EVELYN Y PYLA/Primary Examiner, Art Unit 1633