DETAILED ACTION
Applicant’s amendment and Arguments/Remarks received on 02 January 2026 have been entered. Claims 1-6, 8-10, 18, 20-22, and 36-45 were previously pending in the application. Claims 8 and 10 have been cancelled by Applicant. Claims 1-6, 9, 18, 20-22, and 36-45 are currently pending in the application. Claims 1, 2, 18, 36, 37, 38, 39, 40, 42, and 43 are independent claims.
The election of Group I, drawn a first dual-vector system, a second dual-vector system, a vector system for expressing a coding sequence of a STRC gene, a cell containing the vector system, a pharmaceutical composition the vector system, a first method for treating autosomal recessive hearing loss in a subject comprising administering the first dual-vector system, a second method for treating autosomal recessive hearing loss in a subject comprising administering the pharmaceutical composition, a method comprising contacting at least one cell of a subject with the pharmaceutical composition, and a method for treating and/or preventing a pathology or disease characterized by a hearing loss, remains in effect in the instant application.
The following election of species remains in effect in the instant application: STRC sequences: SEQ ID NO: 1.
Claims 39 remain withdrawn from consideration as being directed to a nonelected invention, there being no allowable generic or linking claim.
Claims 1-6, 9, 18, 20-22, 36-38, and 40-45 are currently pending and under examination in the instant application. An action on the merits follows.
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA . The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Priority
The present application is a 35 U.S.C. 371 national stage filing of International Application No. PCT/US2021/016720, filed 05 February 2021, which claims priority to U.S. provisional application 62/971,555, filed 07 February 2020.
Thus, the earliest possible priority for the instant application is 07 February 2020.
Information Disclosure Statement
The information disclosure statements filed 09 December 2025 and 02 January 2026 have been considered by the Examiner. Examiner notes the filing of IDS Size Fee assertions for the IDSs filed 09 December 2025 and 02 January 2026, as required under 37 CFR 1.98, indicating that no IDS size fee is required under 37 CFR 1.17(v) at this time.
Specification
The objection to the specification of the disclosure for reciting trade names and/or marks used in commerce and for misattribution of figures is withdrawn in view of the amendment to the specification.
37 CFR 1.121(c)
The claim amendments filed 02 January 2026 is objected to under 37 CFR 1.121(c) because Applicant’s claim listing is not in compliance with 37 CFR 1.121(c) which states that the claim listing must provide the status of all claims, that all claims being currently amended in an amendment paper shall be presented in the claim listing, indicate a status of "currently amended," and be submitted with markings to indicate the changes that have been made relative to the immediate prior version of the claims. The text of any added subject matter must be shown by underlining the added text. The text of any deleted matter must be shown by strike-through except that double brackets placed before and after the deleted characters may be used to show deletion of five or fewer consecutive characters. The text of any deleted subject matter must be shown by being placed within double brackets if strike-through cannot be easily perceived.
Specifically, claim 3 is marked “Currently amended” and comprises some markings indicating insertions and deletions from the previous version. However, amended claim 3 omits the deleted text “of interest” from lines 4-5 the prior version of the claim (i.e., between “protein” and “sequence” in line 5 of the current version of the claim filed 02 January 2026) which should be present and marked with a strikethrough.
In the interests of compact prosecution, the claim listing has been entered. However, future claim listings must include the correct status of all claims, along with the appropriate mark-ups for changes to the claims, in order for the claim listing to meet the requirements for entry under 37 CFR 1.121(c) or a Notice of Non-Compliant Amendment will be mailed to applicant.
Claim Objections
The objection to amended claims 18 and 20-21 for reciting “STRC” without first writing out the term for which is an abbreviation, is withdrawn in view of the amendments to the claims writing out “stereocilin”.
**The following new rejection is necessitated by Applicant’s amendments to the claims.
Amended claim 20 is newly objected to because of the following informalities: amended claim 20 newly recites, “coding C-intein”, which appears to be a typographical spelling error for “encoding C-intein”. Appropriate correction is required.
Claim Rejections - 35 USC § 112(b)
The rejection of amended, previously presented, and cancelled claims 1-6, 8-10, 18, 20-22, 36-38, and 40-45 under 35 U.S.C. 112(b) as failing to particularly point out and distinctly claim the subject matter which the inventor(s) regards as the invention for multiple issues of indefiniteness is withdrawn in view of Applicant’s amendments to the claims addressing the issues of indefiniteness.
**The following new rejection is necessitated by Applicant’s amendments to the claims.
Amended and previously presented claims 1-6, 9, 18, 20-22, 36-38, and 40-45 are newly 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. Claims 3-6, 9, 22, 36-38, and 40-45 are included in this rejection due to their dependence on and/or encompassing of claims 1, 2, 18, 20, and/or 21.
Amended claims 1, 2, and 20 newly recite, “encoded by SEQ ID NO: 13” in line 12, lines 15-16, and line 18, respectively, and “encoded by SEQ ID NO:21” in line 18, lines 28-29, and line 30, respectively, which are indefinite because an amino acid sequence may be encoded by nucleotide sequences but not my sequence identifiers themselves. The phrases are additionally indefinite because it is unclear whether the N-intein and the C-intein must comprise or consist of the full sequence encoded by the sequences of SEQ ID NOs: 13 and 21, respectively, or whether they merely need to comprise any portion of the full sequence encoded by the sequences of SEQ ID NOs: 13 and 21. As such, the metes and bounds of the claims cannot be determined.
Amended claims 1, 2, and 20 also newly recite, “wherein the first STRC signal sequence and the second STRC signal sequence are the same sequence selected from SEQ ID NO:9 or SEQ ID NO:11” in lines 30-31, lines 40-42, and lines 39-40, respectively, which is indefinite because it is unclear whether the first STRC signal sequence and the second STRC signal sequences are meant to comprise or consist of the sequence according to SEQ ID NOs: 9 or 11. Additionally, recitation of “selected from” is indefinite because the phrase does not indicate whether the group is an open or a closed group. As such, the metes and bounds of the claims cannot be determined.
Amended claim 2 further newly recites, “the first signal sequence” in line 6. There is insufficient antecedent basis for this limitation in the claim. Claim 2 has a prior recitation of “a first stereocilin (STRC) signal sequences” but does not have a prior recitation of “a first signal sequence”. As such, the metes and bounds of the claims cannot be determined.
Amended claim 18 newly recites, “wherein the vector system comprises at least one vector comprising the coding sequence of the STRC gene according to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:38, SEQ ID NO:30, or SEQ ID NO:32”, which is indefinite because it is unclear whether the group of coding sequences is an open or a closed group. As such, the metes and bounds of the claim cannot be determined.
Amended claim 21 recites, “a signal peptide sequence” in line 9, which is indefinite because it is unclear whether Applicant means to refer to the second STRC signal peptide sequence encoded by the second STRC signal sequence recite in claim 20, upon which claim 21 depends, or whether Applicant intends to claim any signal peptide sequence. As such, the metes and bounds of the claims cannot be determined.
Claim Rejections - 35 USC § 112(a)
The rejection of amended, previously presented, and cancelled claims 1-6, 8-10, 18, 20-22, 36-38, and 40-45 under 35 U.S.C. 112(a) for:
while being enabling for:
a recombinant adeno-associated virus (rAAV) comprising a dual-vector system for expressing a protein of interest in a cell, the dual-vector system comprising both a first vector and a second vector according to claim 2;
wherein the protein of interest is a human STRC protein, wherein the sequence encoding the N-terminal portion of the protein of interest is nucleotides 1-2175 or 1-2843 of the murine STRC coding sequence according to SEQ ID NO: 31, or the corresponding position in a human STRC gene according to SEQ ID NO: 1, wherein the sequence encoding the C-terminal portion of the protein of interest is nucleotides 2173-5436 or 2842-5361 of the murine STRC coding sequence according to SEQ ID NO: 31, or the corresponding position in a human STRC gene according to SEQ ID NO: 1;
wherein the N-intein and C-intein are components of the same split intein pair, such that the N-intein and C-intein are capable of protein trans-splicing with each other when contacted with each other, and wherein sequence encoding the N-intein is according to SEQ ID NO: 13 and the sequence encoding the C-intein is according to SEQ ID NO: 21;
wherein the signal sequence is according to the sequence of SEQ ID NO: 9 or SEQ ID NO: 11;
wherein the promoter is capable of driving expression in cochlear inner hair cells and/or cochlear outer hair cells;
wherein the serotype(s) of the rAAV are AAV2, (AAV9)-Php.B, and/or Anc80;
and
a method for treating autosomal recessive hearing loss associated with a loss-of-function mutation in STRC, the method comprising:
administering the recombinant adeno-associated virus (rAAV) comprising a dual vector system, or a pharmaceutical composition comprising the rAAV comprising a dual vector system;
wherein the administering is by injecting the rAAV virus comprising the dual vector system into the cochlea of the subject, thereby contacting at least one cell of the subject, wherein the contacting delivers the vector system comprising the first nucleotide sequence and the second nucleotide sequence into the at least one cell of the subject, wherein the at least one cell of the subject is a cochlear inner hair cell or a cochlear outer hair cell, wherein the contacted at least one cell of the subject expresses the N-terminal portion of the human STRC protein and the C-terminal portion of the STRC protein joined by a peptide bond to form a full-length protein;
does not reasonably provide enablement for:
the dual-vector system according to claims 1, 2, or 18, wherein the dual-vector system according to claim 1, 2, or 18 expresses any protein of interest; wherein the first vector comprises a sequence encoding any N-terminal portion of the protein of interest and the second vector comprises a sequence encoding any C-terminal portion of the protein of interest; wherein the first vector comprises a sequence encoding any N-intein and the second vector comprises a sequence encoding any C-intein; wherein first vector and second vector each comprise any signal sequence; wherein the N-terminal portion and the C-terminal portion are operably linked and under control of any promoter;
nor
a method for treating any autosomal recessive hearing loss or for treating and/or preventing any pathology or disease characterized by a hearing loss, the method comprising administering to a subject in need thereof an effective amount of the dual-vector system according to claim 1 or the pharmaceutical composition according to claim 37 (which comprises the vector system according to claim 1); and wherein the administering is to any cell/tissue/organ of the subject via any delivery mechanism,
is withdrawn over cancelled claims 8 and 10 and maintained over amended and previously presented claims 1-6, 9, 18, 20-22, 36-38, and 40-45. Applicant's amendments to the claims and arguments have been fully considered but have not been found persuasive in overcoming the rejection for reasons of record as discussed in detail below.
Applicant has amended independent claim 1 to incorporate that the protein of interest is specifically STRC, that the signal sequences are specifically STRC signal sequences are the same and selected from the sequences according to SEQ ID NOs: 9 or 11, that the N-intein is specifically the sequence of SEQ ID NO: 13, and that the C-intein is specifically the sequence of SEQ ID NO: 21.
However, independent claim 1 does not recite that the viral vectors are AAV vectors capable of transducing cochlear hair cells, wherein the vectors comprise 5’ and 3’ ITRs, promoter sequences capable of driving expression in expression in cochlear inner hair cells and/or cochlear outer hair cells, polyadenylation sequences, and specific STRC sequences. Independent claim 1 has not been amended to recite that the STRC protein is split into nucleotides 1-2175 and 2173-5436 or 1-2843 and 2842-5361 of the murine STRC coding sequence according to SEQ ID NO: 31 or into the corresponding fragments in a human STRC gene according to SEQ ID NO: 1.
Similarly, independent claim 2 does not recite that the vectors are AAV vectors capable of transducing cochlear hair cells, that the vector comprises any specific STRC sequences, nor that the STRC protein is split into nucleotides 1-2175 and 2173-5436 or 1-2843 and 2842-5361 of the murine STRC coding sequence according to SEQ ID NO: 31 or the corresponding positions in a human STRC gene according to SEQ ID NO: 1.
Independent claim 18 merely recites a vector system for expressing a coding sequence of a STRC gene in a host cell, wherein the vector system comprises one or more vectors comprising the coding sequence of the STRC gene according to one of the recites sequences. As such, independent claim 18 does not require a dual vector system nor any of the components of the dual vector system described in and enabled by the instant disclosure for expressing the STRC gene in a host cell.
Further, the method claims 38, 40, 42, and 43 have been amended to require the subject to have a mutated STRC gene, but have not been amended to recite the specific administration routes nor specific target cells for delivery and expression of the STRC sequence within the subject.
Therefore, Applicant’s amendments do not overcome the scope of enablement rejection under 35 U.S.C. 112(a).
Applicant argues that the claims have been amended to the STRC protein as the protein of interest, specific sequences of N-intein and C-intein, and STRC signal sequences to address the rejection. However, while the amendments address most of the issues addressed by the scope of enablement rejection, it has not addressed all of the issues. For example, as discussed above, independent claims 1 and 2 do not recite specific STRC sequence nor the specific break points in the STRC sequence which Applicant has demonstrated are functional for reconstituting a full-length STRC from a split STRC via protein trans-splicing. Additionally, independent claim 18 has been amended to address issues of indefiniteness but has not incorporated any of the limitations lacking prior to the current amendment.
Therefore, Applicant’s arguments do not overcome the scope of enablement rejection under 35 U.S.C. 112(a), and the rejection is maintained.
Claim Rejections - 35 USC § 102
The rejection of amended claims 1, 3-6 and 36 under 35 U.S.C. 102(a)(1) as being anticipated by Moll et al. [2017, ACS Synthetic Biology¸6, 2260-2272], is withdrawn in view of Applicant’s claims which now recite expressing a stereocilin protein, such that the first viral vector encodes an amino terminal portion of the STRC protein and the second viral vector encodes a C-terminal portion of the STRC protein.
Claim Rejections - 35 USC § 103
The rejection of amended, previously presented, and cancelled claims 1-6, 8-10, 18, 20-22, 36-38 and 40-45 under 35 U.S.C. 103 as being unpatentable over Moll et al. [2017, ACS Synthetic Biology¸6, 2260-2272]; in view of Addgene [2013, Plasmid: pcDNA3.1(+), retrieved on 30 August 2025 from the Internet: <https://web.archive.org/web/20131125013217/https://www.addgene.org/vector-database/2093/>, archived 25 November 2013]; Truong et al. [2015, Nucleic Acids Research, 43(13), 6450-6458]; Simons [US20210330814A1, published 28 October 2021, filed 12 January 2021, with priority to U.S. Provisional application No. 62/697,652, filed 13 July 2018]; and GenBank [2008, Homo sapiens stereocilin (STRC), mRNA, NCBI Reference Sequence: NM_153700.2, retrieved on 30 August 2025 from the Internet: <https://www.ncbi.nlm.nih.gov/nucleotide/NM_153700.2?report=genbank&log$=nuclalign&blast_rank=1&RID=B7NJDF82014&from=142&to=5403>, sequence version last updated on or before 29 March 2008, IDS], is withdrawn in view of Applicant’s claims which now recite nucleotide sequences according to SEQ ID NOs: 13 and 21 for encoding the N-intein and C-intein, respectively.
**The following new rejection is necessitated by Applicant’s amendments to the claims.
Amended and previously presented claims 1-6, 9, 18, 20-22, 36-38 and 40-45 are newly rejected under 35 U.S.C. 103 as being unpatentable over Moll et al. [2017, ACS Synthetic Biology¸6, 2260-2272, cited in a prior action]; in view of Truong et al. [2015, Nucleic Acids Research, 43(13), 6450-6458, cited in a prior action, including Supplemental Data]; Simons [US20210330814A1, published 28 October 2021, filed 12 January 2021, with priority to U.S. Provisional application No. 62/697,652, filed 13 July 2018, cited in a prior action]; Liu [US20180127780A1, published 10 May 2018]; Addgene [2013, Plasmid: pcDNA3.1(+), retrieved on 30 August 2025 from the Internet: <https://web.archive.org/web/20131125013217/https://www.addgene.org/vector-database/2093/>, archived 25 November 2013, cited in a prior action]; and GenBank [2008, Homo sapiens stereocilin (STRC), mRNA, NCBI Reference Sequence: NM_153700.2, retrieved on 30 August 2025 from the Internet: <https://www.ncbi.nlm.nih.gov/nucleotide/NM_153700.2?report=genbank&log$=nuclalign&blast_rank=1&RID=B7NJDF82014&from=142&to=5403>, sequence version last updated on or before 29 March 2008, IDS, cited in a prior action].
Regarding independent claim 1, Moll teaches a dual-vector system for expressing a protein of interest (e.g., an IL-6 Hyper-cytokine) in a cell, the dual vector system comprising:
a first vector comprising a first nucleotide sequence comprising, in a 5’ to 3’ direction:
a first signal sequence at the 5’ end of a partial coding sequence encoding an amino terminal (N-terminal) portion of the protein of interest;
the partial coding sequence encoding the N-terminal portion of the protein of interest;
a sequence encoding an amino terminal fragment of intein (N-intein) adjacent to and downstream of the partial coding sequence; and
a second vector comprising a second nucleotide sequence comprising, in a 5’ to 3’ direction:
a second signal sequence;
a sequence encoding a carboxy terminal fragment of intein (C-intein), wherein the C-intein is flanked by the second signal sequence and a partial coding sequence encoding a C-terminal portion of the protein of interest;
wherein the sequence encoding the C-intein is downstream of and adjacent to the second signal sequence; and
the partial coding sequence encoding the C-terminal portion of the protein of interest; wherein the sequence encoding the C-intein is immediately adjacent to the partial coding sequence encoding the C-terminal portion of the protein of interest; and wherein the partial coding sequence encoding the C-terminal portion of the STRC protein is downstream of the sequence encoding the C-intein [column 7 ¶ 3, Figure 1, 4, S1].
Moll does not teach 1) wherein the first vector and the second vector are viral vectors; 2) wherein the protein of interest is an STRC protein; 3) wherein the first signal sequence and the second signal sequence are an STRC signal sequence according to SEQ ID NOs: 9 or 11; nor 4) wherein the N-intein is encoded by a nucleotide sequence according to SEQ ID NO: 13 and the C-intein is encoded by a nucleotide sequence according to SEQ ID NO: 21.
Regarding 1), Truong teaches that the current standard delivery system for gene therapy used in humans is the recombinant adeno-associated virus (rAAV) due to their high infection efficiency and very low immune response [column 2 ¶ 2- column 3 ¶ 1]. Truong also teaches that the split intein system, with its intein-mediated trans-splicing, allows for bypassing the packaging limit of rAAV to express large proteins for gene therapy, wherein the packaging capacity of AAV is confined to a maximum of ~4.7-5 kb [abstract, column 3 ¶ 1]. Truong also teaches that the split intein system permits the precise spaciotemporal control of the split-protein activity [column 3 ¶ 3]. Therefore, given the teachings of Truong that rAAV is a preferred vector for gene therapy due to high infection efficiency and very low immune response, along with the teachings of Truong that the split intein system allows for overcoming the rAAV packaging limit and for allowing precise spaciotemporal control of a split-protein activity, an ordinarily skilled artisan at the time of filing the instant application would have been motivated to utilize an rAAV viral vector for the delivery of split-intein gene therapy products.
Regarding 2), Simons teaches that a long-felt need remains for agents and methods for preventing or reversing non-syndromic deafness [0003]. Simons also teaches that the STRC gene encodes stereocilin, a protein that is normally expressed in the inner ear and is associated with stereocilia of specialized hair cells in the inner ear [0069]. Simons further teaches that the full-length human wildtype STRC protein is 1775 residues in length, including the signal peptide [0070], which corresponds to 5,325 nucleotides of coding nucleic acid sequence. As such, the coding sequence for stereocilin itself exceeds the packaging capacity of rAAV vectors of 4.7-5kb, as taught by Truong and discussed above, before inclusion of additional sequences needed for expression. Simons also teaches administering a nucleic acid encoding stereocilin to treat non-syndromic sensorineural hearing loss in a subject in need thereof [0005].
To facilitate the administration of the STRC encoding sequence, Simons teaches a dual vector system, wherein each of the two vectors comprise a portion of a stereocilin protein coding sequence which can be used to generate a sequence encoding an active stereocilin protein (e.g., a full-length stereocilin protein) in a mammalian cell, and thereby treat autosomal recessive non-syndromic sensorineural hearing loss (e.g., DFNB16) in a subject in need thereof [0005, 0071]. Simons also teaches that each of the two vectors include a different segment of an intron, and wherein the two different segments overlap in sequence, thereby facilitating homologous recombination with each other, and thereby forming a recombined nucleic acid that encodes a full-length stereocilin protein [0006, 0008].
Therefore, given the teachings of Simons to deliver a nucleic acid sequence encoding stereocilin to treat non-syndromic sensorineural hearing loss in a subject in need thereof, the teaching of Simons that stereocilin comprises 1775 amino acids (corresponding to 5,325 nucleotides of coding nucleic acid sequence), the teaching of Truong that the packaging capacity for rAAV gene therapy vectors is 4.7-5 kb, and the teachings of Simons to deliver a dual vector system encoding as split stereocilin in a mammalian cell, wherein the full-length stereocilin is reconstituted from the split coding sequence within the cell, to treat the hearing loss, an ordinarily skilled artisan at the time of filing the instant application would have been motivated to deliver a split stereocilin to a subject having non-syndromic sensorineural hearing loss as a treatment for the hearing loss within that subject.
Regarding 3), Moll teaches to use the endogenous IL-6 signal peptide for the C-terminal construct expressing the IL-6 component of the fusion/trans-spliced protein and the endogenous IL-6R signal peptide for the N-terminal construct expression the sIL-6R component of the fusion/trans-spliced protein [Figure S1]. Moll also teaches wherein the protein ligation occurs within the secretory compartment (i.e., in the EF-Golgi compartment), which necessarily requires that both the N-terminal portion and the C-terminal portion are present in the compartment together [column 7 ¶ 1-3, column 10 ¶ 2].
Further, Simons teaches that stereocilin is associated with the stereocilia of specialized hair cells in the inner ear, which are important for hearing and balance [0069]. Simon teaches that mutations in the STRC gene cause non-syndromic sensorineural hearing loss and that delivery of vectors to generate a sequence encoding an active, full-length stereocilin protein is used to treat the non-syndromic sensorineural hearing loss in a subject [0005, abstract].
Simons also teaches that the human STRC signal sequence is MALSLWPLLLLLLLLLLLSFAV (Simons SEQ ID NO: 10), which is encoded by the first 66 nucleotides of a human wildtype stereocilin cDNA sequence according to Simons SEQ ID NO: 2 [page 17 lines 6-7 and 33-34], wherein nucleotides 4-63 have 100% sequence identity to instant SEQ ID NO: 9, as seen in the alignment below.
PNG
media_image1.png
188
640
media_image1.png
Greyscale
Therefore, given the teachings of Simon that the sequence of instant SEQ ID NO: 9 is the wildtype STRC gene sequence encoding the wildtype STRC signal peptide, to include the signal peptide sequence in a vector encoding STRC protein for gene therapy, that stereocilin localizes to stereocilia, and to express the wild-type stereocilin to replace defective stereocilin to treat hearing loss associated with STRC mutations; and the teachings of Moll to use the endogenous signal peptide for the protein to be expressed in each of the N-terminal and C-terminal intein vectors and that protein splicing occurs within a secretory system compartment within a cell, an ordinarily skilled artisan would have been motivated to include the endogenous wildtype STRC signal sequence with the STRC gene when substituting the STRC gene for the sIL-6R/IL-6 protein in the split intein vectors of Moll to allow both the co-localization of the two STRC fragments to facilitate protein splicing as well as the proper localization of the stereocilin protein within the stereocilia.
Regarding 4), as discussed above, Truong teaches that the split intein system, with its intein-mediated trans-splicing, allows for bypassing the packaging limit of rAAV to express large proteins for gene therapy, and that the split intein system permits the precise spaciotemporal control of the split-protein activity [abstract, column 3 ¶ 1, 3]. Truong teaches the use of naturally occurring DnaE-n and DnaE-c split inteins, wherein the reconstituted spliced Cas9 exhibits nuclease activity comparable to wild-type Cas9 [abstract, column 5 ¶ 4-5].
Liu also teaches a split Cas9 system using DnaE-n intein-N and DnaE-c intein-C for AAV-mediated delivery of Cas9 split into two vectors, wherein the intein-N and intein-C allow reconstitution of a functional Cas9 nuclease via protein splicing in a cell [abstract, 0006, 0019, 0035, 0047, 0049-0050, 0105, Table 1]. Liu further teaches sequences encoding the DnaE-n intein-N (SEQ ID NO: 350) and the DnaE-c intein-C (SEQ ID NO: 352), such that SEQ ID NO: 350 of Liu is the same length as and 100% identical to instant SEQ ID NO: 13, as seen in the following alignment [00049, Table 1]:
PNG
media_image2.png
315
626
media_image2.png
Greyscale
; and such that SEQ ID NO: 352 of Liu comprises a sequence which is 100% identical to instant SEQ ID NO: 21, differing only by the presence of a start codon at the 5’ end, as seen in the following alignment:
PNG
media_image3.png
238
622
media_image3.png
Greyscale
.
Liu also teaches that the DnaE-n intein-N and DnaE-c intein-C pair encoded by the sequences according to SEQ ID NOs: 350 and 352 mediate protein splicing to join two fragments of a protein together, thereby reconstructing the full-length functional protein which has comparable activity to a nucleobase editor expressed as a whole [abstract, 0004, 0006, 0019, 0033, 0035, 0047, 0049-0050, 0105, 0232, Table 1, Figure 1C, 3].
Therefore, given the teachings of Truong and Liu to use the naturally occurring DnaE-n intein-N and DnaE-c intein-C split inteins for AAV-mediated delivery of large proteins to cells, wherein the intein-N and intein-C facilitate protein trans-splicing to produce a full-length protein with activity comparable to delivery of the protein expressed as a whole, and the teachings of Liu that the DnaE-n intein-N is encoded by a sequence (SEQ ID NO: 350) which is 100% identical to instant SEQ ID NO: 13 and that the DnaE-c intein-C is encoded by a sequence (SEQ ID NO: 352) which is 100% identical to instant SEQ ID NO: 21 other than the addition of a 5’ ATG encoding a start codon, an ordinarily skilled artisan at the time of filing the instant application would have been motivated to use the sequences of instant SEQ ID NOs: 13 and 21 as an N-intein and C-intein, respectively, to facilitate the trans-splicing of two fragments of a large protein, thereby allowing the AAV-mediated delivery of proteins with coding sequences too long to fit within the packaging limit of the AAV genome.
Regarding independent claim 2, Moll, Truong, Simons, and Lui teach all of the limitations of claim 1. Additionally, Moll teaches that the first and second vectors were produced from pcDNA3.1 expression plasmids and that the proteins expressed from the vectors [column 7 ¶ 3, column 21 ¶ 4]. Moll also teaches detailed plasmid construction information indicating restriction sites used for insertion of the signal peptide, intein, and partial POI coding sequences (e.g., HindIII and BamHI) [column 21 ¶ 4 – column 22 ¶ 1], but does not explicitly teach that the vectors comprises promoters operably linked to the signal peptide, intein, and partial POI coding sequence nor that the vectors comprise polyadenylation signal sequences 3’ of the signal peptide, intein, and partial POI coding sequence. However, Addgene teaches that the pcDNA3.1 vector comprises a CMV promoter immediately upstream of a multiple coding site (MCS), which comprises HindIII and BamHI sites, and which is operably linked to control sequences inserted into the MCS [page 2]. Addgene also teaches that the vector comprises a polyadenylation signal sequence (e.g., bGH polyA) 3’ of the MCS [page 2]. As such, Moll teaches a first and second vector which comprise promoters operably linked to the recited coding sequences and which comprise polyadenylation sequences 3’ of the recited coding sequence.
Moll also does not teach that the vectors comprise a 5’-ITR nor a 3’-ITR sequence at the 5’ and 3’ ends flanking the recited promoters, coding sequences, and polyadenylation sequences.
However, Truong teaches a dual vector system comprising:
a first vector comprising a first nucleotide sequence comprising, in a 5’ to 3’ direction:
a 5’ inverted terminal repeat (5’-ITR) sequence;
a first promoter sequence (e.g., CBh);
a first signal sequence (e.g., nuclear localization signal sequence (NLS)), wherein the NLS is operably linked to and under control of the CBh promoter;
a partial coding sequence encoding an N-terminal portion of the protein of interest (e.g., N-Cas9 (2-573)), wherein the partial coding sequence is operably linked to and under control of the CBh promoter; and
a sequence encoding a split intein-N, wherein the sequence encoding the split intein-N is operably linked to and under control of the promoter;
a poly-adenylation (polyA) signal sequence (e.g., bGH polyadenylation site); and
a 3’-inverted terminal repeat (3’-ITR) sequence; and
a second vector comprising a second nucleotide sequence comprising, in a 5’ to 3’ direction:
a 5’-inverted terminal repeat (5’-ITR) sequence;
a second promoter sequence (e.g., a CBh promoter);
a sequence encoding a split intein-C, wherein the sequence encoding the split intein-C is operably linked to and under control of the CBh promoter;
a partial coding sequence encoding a C-terminal portion of the protein of interest (e.g., C-Cas9 (574-1368)), wherein the partial coding sequence encoding a C-terminal portion of the protein is operably linked to and under control of the promoter;
a second signal sequence (e.g., NLS), wherein the NLS is operably linked to and under control of the promoter;
a poly-adenylation (polyA) signal sequence (e.g., bGH polyadenylation site); and
a 3’-inverted terminal repeat (3’-ITR) sequence [Figure 2, 3].
Truong teaches that the current standard delivery system for gene therapy used in humans is the recombinant adeno-associated virus (rAAV) due to their high infection efficiency and very low immune response [column 2 ¶ 2- column 3 ¶ 1]. Truong also teaches that the split intein system, with its intein-mediated trans-splicing, allows for bypassing the packaging limit of rAAV to express large proteins for gene therapy, wherein the packaging capacity of AAV is confined to a maximum of ~4.7-5 kb [abstract, column 3 ¶ 1]. Truong also teaches that that split intein system permits the precise spaciotemporal control of the split-protein activity [column 3 ¶ 3]. Therefore, given the teachings of Truong that rAAV is a preferred vector for gene therapy due to high infection efficiency and very low immune response, along with the teachings of Truong that the split intein system allows for overcoming the rAAV packaging limit and for allowing precise spaciotemporal control of a split-protein activity, an ordinarily skilled artisan at the time of filing the instant application would have been motivated to utilize an rAAV viral vector for the delivery of split-intein gene therapy products, and in so doing would include the viral 5’-ITR and 3’-ITRs in the viral vector.
Moll additionally does not teach wherein the first promoter sequence and the second promoter sequence direct expression of the first nucleotide sequence and the second nucleotide sequence in an inner hair cell or an outer hair cell.
However, Simons teaches that the vector comprises a promoter to express the RNA encoding the stereocilin protein, wherein the promoter is a cochlear hair cell-specific promoter, or wherein the promoter is an outer hair cell-specific promoter [0011, 0131-0132]. Simons also teaches to administer a composition comprising the dual vector system for expressing STRC to a subject having hearing loss, including administration into the cochlea of a subject to introduce a therapeutically effective amount of the composition to a mammalian (e.g., human) cell present in a mammal (e.g., human), including a cochlear inner hair cell or a cochlear outer hair cell, to express a full-length stereocilin protein and thereby to treat non-syndromic sensorineural hearing loss in the subject identified as having a defective stereocilin gene [0118, 0167-0169, 0179, 0181]. Therefore, given the teachings of Simons to include a cochlear hair cell-specific promoter, including a cochlear outer hair cell-specific promoter, and to deliver a split STRC gene to the cochlear inner hair cells and/or outer hair cells in a subject to express the full-length STRC protein and thereby treat hearing loss, an ordinarily skilled artisan at the time of filing the instant application would have been motivated to administer a pharmaceutical composition comprising the dual vector split-intein system for expressing STRC to an inner ear cell, including an inner hair cell or an outer hair cell, wherein the dual vector system comprises promoters capable of directing expression in an inner hair cell or an outer hair cell, such as a cochlear hair cell-specific promoter.
Regarding claim 3, Moll, Truong, Simons, and Lui teach all of the limitations of claim 1. Moll further discloses wherein the first vector in a cell expresses a first protein sequence comprising in an N-terminal to C-terminal direction: a first signal peptide sequence linked to an N-terminal portion of the protein of interest sequence fused at its C-terminal end to an N-intein protein sequence; and wherein the second vector in a cell expresses a second protein sequence comprising in an N-terminal to C-terminal direction: a second signal peptide sequence linked to a C-intein protein sequence fused to the N-terminal end of a C-terminal portion of the protein of interest sequence [column 7 ¶ 3, Figure 1, 4, S1]. Therefore, in combining the teachings of Moll with the teachings of Truong, Simons, and Liu as discussed above for claim 1, the ordinarily skilled artisan would arrive at a dual vector system wherein the first vector in a cell expresses a first protein sequence comprising in an N-terminal to C-terminal direction: a first STRC signal peptide sequence linked to a sequence of the N-terminal portion of the STRC protein sequence fused at its C-terminal end to an N-intein protein sequence; and wherein the second vector in a cell expresses a second protein sequence comprising in an N-terminal to C-terminal direction: a second STRC signal peptide sequence linked to a C-intein protein sequence fused to the N-terminal end of a sequence of the C-terminal portion of the STRC protein sequence.
Regarding claim 4, Moll, Truong, Simons, and Lui teach all of the limitations of claim 1. Moll discloses wherein the N-terminal portion of the protein of interest (POI) and the C-terminal portion of the POI are configured to form a full-length protein of interest [column 10 ¶ 3, Figure 1, 4, S1].
Regarding claims 5-6, Moll, Truong, Simons, and Lui teach all of the limitations of claim 1. Moll teaches wherein the signal peptide sequence of the first protein sequence and the signal peptide sequence of the second protein sequence are configured to transport the first protein sequence and the second protein sequence to the same cellular compartment of the cell (e.g., the ER-Golgi secretory system) [column 7 ¶ 3, Figure S1].
Also, as discussed above, Simons teaches the motivation to include the endogenous wildtype STRC signal sequence with the STRC gene when substituting the STRC gene for the sIL-6R/IL-6 protein in the split intein vectors of Moll to allow both the co-localization of the two STRC fragments to facilitate protein splicing as well as the proper localization of the stereocilin protein within the stereocilia. As such, signal sequences with identical nucleic acid sequences will encode signal peptide sequences with identical amino acid sequences.
Regarding claim 9, Moll, Truong, Simons, and Lui teach all of the limitations of claim 1. Additionally, as discussed above, Truong teaches the motivation to utilize an rAAV viral vector for the delivery of split-intein gene therapy products.
Regarding claim 18, as discussed above Moll, Truong, Simons, and Lui teach a vector system for expressing coding sequences in a host cell. Additionally, Simons teaches that the stereocilin protein produced by the joining of the split-STRC has a sequence according to SEQ ID NO: 11 [0010], which is the same length as and 100% identical to the stereocilin protein recited in instant SEQ ID NO: 25, and which comprises the 1755 amnio acid sequence encoded by instant SEQ ID NO: 1 (corresponding to amino acids 22-1775 of Simons SEQ ID NO: 11) along with a 21-amino acid signal sequence (as taught in Simons SEQ ID NO: 10 and corresponding to amino acids 1-21 of Simons SEQ ID NO: 11). Simons also teaches a nucleic acid sequence encoding STRC (Simons SEQ ID NO: 2), which is 5433 nucleotides long and has 97.4% identity with SEQ ID NO: 1 of the instant application. Simons further teaches to express wildtype human stereocilin protein [0040, 0070].
However, Simons does not explicitly recite an STRC-encoding nucleotide sequence according to SEQ ID NO: 1 of the instant application.
GenBank teaches a human wildtype STRC gene coding sequence which is the same length as and which is 100% identical to the sequence of instant SEQ ID NO: 1 [pages 5-6].
Therefore, given the teachings of Simons of a vector system comprising a coding sequence of a STRC gene for expression of a full-length, wildtype human STRC protein to treat a subject having hearing loss, and the teachings of GenBank of a coding sequence encoding a full-length, wildtype human STRC protein which is 100% identical to the sequence of instant SEQ ID NO: 1, an ordinarily skilled artisan would have been motivated to utilize the nucleic acid sequence of GenBank to encode an STRC protein for gene therapy applications.
Regarding claim 20-21, as discussed above, Moll, Truong, Simons, Lui, Addgene, and GenBank teach all of the limitations of claims 1, 2, 3, and 18, including:
a first vector comprising a first nucleotide sequence comprising, in a 5’ to 3’ direction:
a 5’ inverted terminal repeat (5’-ITR) sequence;
a first promoter sequence;
a first STRC signal sequence, wherein the first STRC signal sequence is operably linked to and under control of the first promoter sequence;
a partial coding sequence encoding an N-terminal portion of the STRC protein (i.e., a 5’ end fragment of the coding sequence of the STRC gene), wherein the ., a 5’ end fragment is operably linked to and under control of the first promoter sequence;
a sequence encoding an amino terminal fragment of intein (N-intein), wherein the sequence encoding the N-intein is operably linked to and under control of the first promoter sequence and is adjacent to and downstream of the 5’ end fragment of the coding sequence of the STRC gene, wherein the N-intein is encoded by the nucleotide sequence of SEQ ID NO: 13;
a poly-adenylation (polyA) signal sequence; and
a 3’-inverted terminal repeat (3’-ITR) sequence; and
a second vector comprising a second nucleotide sequence comprising, in a 5’ to 3’ direction:
a 5’-inverted terminal repeat (5’-ITR) sequence;
a second promoter sequence;
a second STRC signal sequence, wherein the second STRC signal sequence is operably linked to and under control of the second promoter sequence;
a sequence encoding a carboxy terminal fragment of intein (C-intein), wherein the sequence encoding the C-intein is operably linked to and under control of the second promoter sequence and is adjacent to and downstream of the second STRC signal sequence, wherein the C-intein is encoded by SEQ ID NO: 21;
a partial coding sequence encoding a C-terminal portion of the STRC protein sequence (i.e., a 3’ end fragment of the coding sequence of the STRC gene), wherein the 3’ end fragment is operably linked to and under control of the second promoter sequence, wherein the sequence encoding C-intein is immediately adjacent to the 3’ end fragment of the coding sequence of the STRC gene, wherein the 3’ end fragment of the coding sequence of the STRC gene is downstream of the sequence encoding C-intein;
a poly-adenylation (polyA) signal sequence; and
a 3’-inverted terminal repeat (3’-ITR) sequence
wherein the first STRC signal sequence and the second STRC signal sequence are the same sequence selected from the nucleotide sequences of SEQ ID NO: 9 or SEQ ID NO: 11.
As such, as discussed above, Moll, Truong, Simons, Lui, Addgene, and GenBank teach all of the limitations of dependent claim 20. Additionally, the teachings to express the vectors taught by Moll, Truong, Simons, Lui, Addgene, and GenBank, as discussed above, are teachings to express in the cell a) a first protein sequence comprising in an N-terminal to C-terminal direction: a first STRC signal peptide sequence linked to an N-terminal portion of the STRC protein sequence fused at its C-terminal end to an N-intein protein sequence; and b) a second protein sequence comprising in an N-terminal to C-terminal direction: a second STRC signal peptide sequence linked to a C-intein protein sequence fused to the N-terminal end of a C-terminal portion of the STRC protein sequence.
Regarding claim 22, as discussed above, Moll, Truong, and Lui teach to configure split inteins with N-terminal portions and C-terminal portions of proteins of interest to form a full-length, functional protein. Additionally, Simons teaches to configure the vectors expressing split STRC such that a full-length STRC protein is expressed.
Regarding claim 36, Moll, Truong, Simons, and Lui teach all of the limitations of claim 1. Moll also teaches a cell containing the vector system [abstract, column 7 ¶ 3, Figure 1-4].
Regarding claim 37, Moll, Truong, Simons, and Lui teach all of the limitations of claim 1. However, Moll does not teach to formulate the vector system in a pharmaceutical composition.
Truong teaches the use of the rAAV dual-vector system comprising the split inteins for efficient delivery of a POI for gene therapy [abstract], but does not explicitly recite that the vector system is formulated as a pharmaceutical composition. Simons teaches to formulate the split STRC vector system into a pharmaceutical composition to administer to a subject having hearing loss [0012, 0183-0192]. Therefore, given the teachings of Simons to administer pharmaceutical composition comprising a dual-vector system comprising split-STRC coding sequences to reconstitute full-length STRC within the cells of a patient in need, an ordinarily skilled artisan at the time of filing the instant application would have been motivated to formulate the dual vector system in a pharmaceutical composition for gene therapy.
Regarding claim 38, 40-41, and 43 Moll, Truong, Simons, and Lui teach all of the limitations of claim 1. Simons teaches to deliver an effective amount of the split STRC vector system to a subject in need for the treatment of autosomal recessive non-syndromic sensorineural deafness-16 (DFNB16), which is associated with mutations which cause a defective STRC gene [0070-0071, 0170]. Therefore, an ordinarily skilled artisan would have been motivated to administer a therapeutically effective amount of the dual-vector system expressing split STRC and/or a pharmaceutical composition comprising the dual vector system expressing split STRC for the treatment of autosomal recessive hearing loss, which is a pathology or disease characterized by a hearing loss, and wherein the autosomal recessive hearing loss is DFNB16.
Regarding claim 42, as discussed above, Moll, Truong, Simons, and Lui teach all of the limitations of claim 1. Additionally, Moll teaches a method comprising contacting at least one cell with the dual vector system, wherein the contacting delivers the vector system comprising the first nucleotide sequence and the second nucleotide sequence into the at least one cell, wherein the contacted at least one cell expresses an N-terminal portion of the protein and a C-terminal portion of the to form a full-length protein [column 7 ¶ 3, column 10 ¶ 2-3, column ¶ , Figure 1, 4, S1].
Moll does not explicitly teach contacting a subject with a pharmaceutical composition comprising the dual vector system nor that the protein trans-splicing results in a protein joined by a peptide bond.
However, as described above, Simons teaches to formulate the split STRC vector system into a pharmaceutical composition to administer to a subject having hearing loss [0012, 0183-0192], and Truong teaches the use of the rAAV dual-vector system comprising the split inteins for efficient delivery of a POI for gene therapy [abstract]. Therefore, Simons and Truong provide the motivation to formulate the dual vector system in a pharmaceutical composition for gene therapy and to deliver the system to at least one cell of a subject to treat hearing loss in the subject.
Truong also teaches that the protein trans-splicing results in the joining of the N-terminal portion and the C-terminal portion of the POI via a peptide bond [column 5 ¶ 4, Figure 1].
Regarding claims 44-45, Moll, Truong, Simons, and Lui teach all of the limitations of claim 1. Simons additionally teaches to administer a composition comprising the dual vector system for expressing STRC to a subject having hearing loss, including administration into the cochlea of a subject to introduce a therapeutically effective amount of the composition to a mammalian (e.g., human) cell present in a mammal (e.g., human), including a cochlear inner hair cell or a cochlear outer hair cell, to treat non-syndromic sensorineural hearing loss in the subject identified as having a defective stereocilin gene [00167-0169]. Therefore, given the teachings of Simons to deliver a split STRC gene to the cochlear inner hair cells and/or outer hair cells in a subject to treat hearing loss, an ordinarily skilled artisan at the time of filing the instant application would have been motivated to administer a pharmaceutical composition comprising the dual vector split-intein system for expressing STRC to an inner ear cell, including an inner hair cell or an outer hair cell.
Given the motivation taught by Truong to utilize an rAAV viral vector for the delivery of split-intein gene therapy products, and in so doing to include the viral 5’-ITR and 3’-ITRs in the viral vector; the motivation taught by Simons to deliver a split stereocilin to a subject having non-syndromic sensorineural hearing loss as a treatment for the hearing loss within that subject; the motivation taught by Simons to include the endogenous wildtype STRC signal sequence with the STRC gene when substituting the STRC gene for the sIL-6R/IL-6 protein in the split intein vectors of Moll to allow both the co-localization of the two STRC fragments to facilitate protein splicing as well as the proper localization of the stereocilin protein within the stereocilia; the motivation taught by Truong and Liu motivated to use the sequences of instant SEQ ID NOs: 13 and 21 as an N-intein and C-intein, respectively, to facilitate the trans-splicing of two fragments of a large protein, thereby allowing the AAV-mediated delivery of proteins with coding sequences too long to fit within the packaging limit of the AAV genome; the motivation taught by Simons to administer a pharmaceutical composition comprising the dual vector split-intein system for expressing STRC to an inner ear cell, including an inner hair cell or an outer hair cell, wherein the dual vector system comprises promoters capable of directing expression in an inner hair cell or an outer hair cell, such as a cochlear outer hair cell-specific promoter; the teachings and motivation taught by Simons and GenBank to utilize the nucleic acid sequence of GenBank to encode a full-length wildtype human STRC protein for gene therapy applications; the motivation taught by Simons to formulate the dual vector system in a pharmaceutical composition for gene therapy; the further motivation taught by Simons to administer a therapeutically effective amount of the dual-vector system expressing split STRC and/or a pharmaceutical composition comprising the dual vector system expressing split STRC for the treatment of autosomal recessive hearing loss, which is a pathology or disease characterized by hearing loss, and wherein the autosomal recessive hearing loss is DFNB16; and the additional motivation taught by Simons to administer a pharmaceutical composition comprising the dual vector split-intein system for expressing STRC to an inner ear cell, including an inner hair cell or an outer hair cell; it would have been prima facie obvious to an ordinarily skilled artisan at the time of filing the instant application to modify the dual-vector system and methods of Moll to express an N-terminal portion of a human STRC gene and a C-terminal portion of a human STRC gene, wherein the N-terminal portion and C-terminal portion together comprise an STRC gene according to instant SEQ ID NO: 1, to include STRC signal sequences according to SEQ ID NO: 9, to include an N-intein coding sequence according to SEQ ID NO: 13, to include a C-intein coding sequence according to SEQ ID NO: 21, to formulate the dual-vector system into a pharmaceutical composition, and to administer the pharmaceutical composition to at least one cell of a subject having autosomal recessive sensorineural hearing loss (e.g., DFNB16), wherein the at least one cell comprises inner ear cells, including cochlear inner hair cells and/or cochlear outer hair cells, with a reasonable expectation of success.
Insofar as Applicant’s arguments apply to this new grounds of rejection, Applicant argues that:
the proposed combination of Moll in view of Addgene, Truong, Simons, and GenBank fails to teach or suggest replacing Simons’ DNA strategy with protein trans-splicing for STRC;
Examiner relies on information gleaned solely from Applicant’s specification, and so the rejection is improper;
the cited references do not teach or suggest (i) the STRC signal peptide on both construct fragments, (ii) the STRC signal peptide is the same, and (iii) the first STRC signal sequence and the second STRC signal sequence are selected from SEQ ID NO: 9 or SEQ ID NO: 11; and
the split intein pair and configuration are not taught by the cited references as claimed, in that neither Truong nor Moll teaches applying the specific intein pair to STRC.
However, this is not agreed.
In response to Applicant’s arguments against the references, it is noted that the test for obviousness is not whether the features of a secondary reference may be bodily incorporated into the structure of the primary reference; nor is it that the claimed invention must be expressly suggested in any one or all of the references. Rather, the test is what the combined teachings of the references would have suggested to those of ordinary skill in the art. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981). Further, the Examiner recognizes that obviousness may be established by combining or modifying the teachings of the prior art to produce the claimed invention where there is some teaching, suggestion, or motivation to do so found either in the references themselves or in the knowledge generally available to one of ordinary skill in the art. See In re Fine, 837 F.2d 1071, 5 USPQ2d 1596 (Fed. Cir. 1988), In re Jones, 958 F.2d 347, 21 USPQ2d 1941 (Fed. Cir. 1992), and KSR International Co. v. Teleflex, Inc., 550 U.S. 398, 82 USPQ2d 1385 (2007). In addition, it must be recognized that any judgment on obviousness is in a sense necessarily a reconstruction based upon hindsight reasoning. But so long as it takes into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made, and does not include knowledge gleaned only from the applicant's disclosure, such a reconstruction is proper. See In re McLaughlin, 443 F.2d 1392, 170 USPQ 209 (CCPA 1971).
Specifically, regarding Applicant’s argument 1), note that Moll, Addgene, Truong, and GenBank are not relied on to teach motivations to modify the dual vector system of Simons; but that Truong, Simons, Liu, Addgene, and GenBank are relied on to provide the teachings and motivations to modify the dual vector system of Moll to express an STRC protein as claimed. As discussed above, Simons teaches the motivation to deliver and express an STRC gene for the treatment of nonsyndromic sensorineural deafness associated with mutations in the STRC gene, and to deliver a split construct for expressing the STRC gene due to the large size of the coding sequence and the packaging limits of appropriate viral vectors. The combination of references further teaches the motivation to use the STRC signal sequences both for the appropriate localization of the STRC protein to the stereocilia as well as the co-localization of the dual fragments into the same compartment of the cell for the protein trans-splicing to occur.
Additionally, Truong teaches to use natural DnaE-n intein-N and DnaE-c intein-C sequences, wherein the intein-N sequence taught by Truong in the Supplemental Data is 100% identical to the N-intein amino acid sequence encoded by instant SEQ ID NO: 13 [page 1-2], and wherein the intein-C sequence taught by Truong is 96.7% identical to the C-intein amino acid sequence encoded by instant SEQ ID NO: 21 [Supplemental Data page 2]. Additionally, Liu teaches sequences encoding the DnaE-n intein-N (SEQ ID NO: 350) and the DnaE-c intein-C (SEQ ID NO: 352), such that SEQ ID NO: 350 of Liu is the same length as and 100% identical to instant SEQ ID NO: 13 and SEQ ID NO: 352 of Liu comprises a sequence which is 100% identical to instant SEQ ID NO: 21, differing only by the presence of a start codon at the 5’ end. Both Liu and Truong teach that the DnaE-n intein-N and the DnaE-c intein-C are effective for promoting protein trans-splicing to reconstitute full-length proteins with activities comparable to their wholly-expressed counterparts, thereby allowing the expression of long coding sequences which would otherwise exceed the packaging capacity of desirable viral vectors, such as AAV.
As such, the cited references provide the teachings and motivations for modifying the dual vector system of Moll to arrive at the instantly claimed invention.
Regarding Applicant’s argument 2), as discussed above, it must be recognized that any judgment on obviousness is in a sense necessarily a reconstruction based upon hindsight reasoning. But so long as it takes into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made, and does not include knowledge gleaned only from the applicant's disclosure, such a reconstruction is proper. See In re McLaughlin, 443 F.2d 1392, 170 USPQ 209 (CCPA 1971). As discussed above, the cited references provide the teachings and motivation to modify the dual vector system of Moll to arrive at the instantly claimed invention and takes into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made. Therefore, the combination of references was not an impermissible reconstruction based upon hindsight reasoning.
Regarding Applicant’s argument 3), as discussed above, the cited references provide the teachings and motivation to arrive at a dual vector system wherein (i) the STRC signal peptide is on both construct fragments, (ii) the STRC signal peptide is the same, and (iii) the first STRC signal sequence and the second STRC signal sequence are selected from SEQ ID NO: 9 or SEQ ID NO: 11. Moll teaches matching the signal peptide to the protein being expressed (i.e., an IL-6R signal peptide for IL-6R and an IL-6 signal peptide for IL-6).
Additionally, Simons teaches that stereocilin is localized to the stereocilia of cochlear hair cells. Simons also teaches the human STRC signal sequence (SEQ ID NO: 10) comprising the sequence of instant SEQ ID NO: 9 with 100% identity as well as the mouse STRC signal peptide sequence (amino acids 2-21 of SEQ ID NO: 8: ALSLQPQLLLLLSLLPQEVTS) which is 100% identical to the peptide encoded by instant SEQ ID NO: 11 [specification page 113].
Further, Moll teaches that protein trans-splicing occurs within cellular compartments (i.e., ER-Golgi) such that both protein fragments localize to the same cellular compartments. Truong and Lui additionally teach signal sequences (i.e., nuclear localization sequences) in each vector to target each fragment to the target cellular compartment to facilitate both trans-splicing and the desired protein function. Therefore, the cited references provide the teachings and motivations to use the native STRC signal sequence for expressing STRC to replace a defective STRC gene such that the STRC protein expressed must be transported through the cell to replicate the native localization of the STRC protein for the appropriate stereocilia-associated functions of STRC, and also so that the two fragments of the STRC protein will be present within the same cellular locality to undergo the trans-splicing process.
Regarding Applicant’s argument 4), as discussed above, the cited references provide the teachings and motivation to arrive at a dual vector system wherein the N-intein is a DnaE-n intein-N according to SEQ ID NO: 13 and the C-intein is a DnaE-c intein-C according to SEQ ID NO: 21. Specifically, Liu was cited for teaching sequences encoding the DnaE-n intein-N (SEQ ID NO: 350) and the DnaE-c intein-C (SEQ ID NO: 352), such that SEQ ID NO: 350 of Liu is the same length as and 100% identical to instant SEQ ID NO: 13 and SEQ ID NO: 352 of Liu comprises a sequence which is 100% identical to instant SEQ ID NO: 21, differing only by the presence of a start codon at the 5’ end. Both Liu and Truong teach that the DnaE-n intein-N and the DnaE-c intein-C are effective for promoting protein trans-splicing to reconstitute full-length proteins with activities comparable to their wholly-expressed counterparts, thereby allowing the expression of long coding sequences which would otherwise exceed the packaging capacity of desirable viral vectors, such as AAV.
Therefore, Applicant’s arguments do not overcome a finding of obviousness over Moll, Truong, Simons, Liu, Addgene, and GenBank under 35 U.S.C. 103.
Conclusion
No claim is allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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DR. KATIE L. PENNINGTON
Examiner
Art Unit 1634
/KATIE L PENNINGTON/Examiner, Art Unit 1634
Dr. A.M.S. Wehbé
/ANNE MARIE S WEHBE/Primary Examiner, Art Unit 1634
Prosecution Insights