DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Priority
Applicant’s claim to priority from International Application PCT/US2021/033491 filed 05/20/2021 and from Provisional Applications Nos. 63/027,868 and 63/027,866 both filed 05/20/2020, is hereby acknowledged.
Application Status
This Application is a National entry application of PCT/US2021/033491 filed 05/20/2021 under 35 U.S.C. § 371.
Amendments to claims filed 03/25/2026 are hereby acknowledged.
Claims 1, 4, 8, 15, 18, 28, 33 and 50 are currently amended. Claims 2 and 7 are cancelled.
Claims 1, 3-6, and 8-140 are currently pending.
Claims 6,10-11,14,16-17,19, 21-27, 29, 34-49 and 54-140 are withdrawn from consideration.
Therefore, claims 1, 3-5, 8-9, 12-13, 15, 18, 20, 28, 30-33, and 50-53 are under consideration in this office action.
Any objection or rejection not reiterated herein has been overcome by amendments and is therefore withdrawn.
Applicant’s amendments and arguments have been thoroughly reviewed, but are not persuasive to place the claims in condition for allowance for the reasons that follows.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 03/25/2026 was filed after the mailing date of the Office Action on 12/29/2025. The submission is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Specification
Replacement marked-up and clean copies of the Specification are hereby acknowledged and are acceptable.
Claim Objections
Claim 5 is objected to because of the following informalities: A colon “:” is missing between “PINK1” and the start of the sequence “1 MAVR…”.
Claim 30 is objected to, as it does not include the amendments dated 04/10/2023: “A vector, comprising the polynucleotide of claim1”. Claim 30 now recites, as in the ordinal presentation dated 11/17/2022, “A vector, comprising the polynucleotide of any one of claims 1-29”.
Appropriate correction is required.
NOTE: For the purpose of examination, the claim is interpreted as being dependent solely upon claim 1.
The following rejections are new or modified as necessitated by Applicant’s amendments:
Claim Rejections - 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 30-33, 50 and 52-53 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Regarding claim 30, it depends from any one claim from claim 1 to claim 29. However, claims 2 and 7 are cancelled. The metes and bounds of the claim are unclear, therefore, the scope of the claim is indefinite.
Claims 31-33 and 52-53 are dependent on claim 30. Therefore, they are rejected as well.
Regarding claim 50, the amended claim recites “A mammalian cell comprising the polynucleotide of claim 1.” The metes and bounds of the claim are unclear. The claim encompasses any mammalian cell, e.g., a human cell. Since the construct comprising the polynucleotide can be used for gene therapy (see § [0330]), the claim encompasses any cell in the brain and/or body of a subject after gene therapy. Therefore, as written, Applicant is claiming possession of any cell in a human body, of any subject after receiving the composition as a gene therapy drug.
A potential issue/rejection under 35 U.S.C. §101 section 33(a) of the America Invents Act as being directed to or encompassing a human organism can be remediated by reciting instead: “ An isolated mammalian cell comprising the polynucleotide of claim 1.”
The following rejection is maintained from Office Action dated 12/29/2025:
Claim Rejections - 35 USC § 112(d)
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 53 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.
Regarding claim 53, it recites “A kit comprising the vector of claim 30 and instructions for use”. A kit can be a collection of empty tubes and is merely the sum of parts comprising said kit. Therefore, claim 53 is drawn to a vector already claimed in claim 30 and does not further limits the claim it depends on. The instructions do not add patentable value to the product. See MPEP 2112.01, section III.
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.
Response to Arguments
Applicant's arguments filed 03/25/2026 have been fully considered but they are not persuasive.
In response to Applicant’s argument on pages 34 and 35 regarding Claim 53’s rejection under 35 U.S.C. §112 (d), it is clear that claim 53 does not add any structural limitation to the composition from claim 30 itself. Therefore, there is no additional element further limiting the claim. A written document explaining the use of the product does not have patentable weight. The rejection is maintained.
The following rejections are new as necessitated by Applicant’s amendments:
Claim Rejections - 35 USC § 102
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 the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claims 1, 4-5, 8-9, 12-13, 15, 30 and 50-51 are rejected under 35 U.S.C. §102(a)(1) as being anticipated by Zheng (Zheng, X. et al. "Parkin mitochondrial translocation is achieved through a novel catalytic activity coupled mechanism." Cell Research, Vol. 23 (2013), pp: 886-897), as evidenced by Hauser (Hauser, D.N. et al. "The effects of variants in the PARK2 (parkin), PINK1, and PARK7 (DJ-1) genes along with evidence for their pathogenicity". Curr. Protein. Pept. Sci. Vol. 18, No. 7 (2017), pp: 702-714.), NM_032409.3 (Homo sapiens PTEN induced kinase 1 (PINK1), mRNA pdf, NCBI reference sequence: NM_032409.3 version updated and published November 22, 2018; cited previously) and Yamano (Yamano, K. et al. “PINK1 is degraded through the N-end rule pathway”. Autophagy, Vol. 9, No. 11 (2013), pp: 1758-1769).
Regarding claim 1, it recites “A polynucleotide, comprising a polynucleotide sequence encoding a fusion protein comprising a mitochondrial targeting sequence (MTS) of PINK1 or a functional variant thereof; a transmembrane domain (TMD) of PINK1 or a functional variant thereof; and a Parkin protein Parkin protein or functional variant or fragment thereof.”
Zheng teaches that cDNAs for human Parkin and PINK1 were cloned from the MGC human cDNA library, mutations were generated using QuickChange® site-directed mutagenesis kit from STRATAGENE and expressed in pEGFP CLONTECH vectors and Flag-tagged versions were expressed in pCMV-3tag from AGILENT vectors (see page 895, left column, “Materials and Methods” section). Zheng teaches that recombinant fusion proteins comprising a fragment of Parkin and a fragment of PINK1 were constructed and expressed. Zheng teaches a new chimeric protein constructed by replacing the PINK1 kinase domain (PINK1, 157-581 aa) with the Parkin RBR domain (216-465 aa), and adding a flag tag sequence between them, i.e., PINK1Δkinase-Flag-RBR protein (see page 890, left column, second paragraph; and see Figure 2).
According to Hauser, in human PINK1, the MTS corresponds to amino acids 1 to 77 and the transmembrane domain corresponds to amino acids 94 to 110 (see Figure 2, and see below).
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Therefore, the remainder of human PINK1 within the chimeric protein is aa 1-156, since only the kinase domain (157-581 aa) is removed. Parkin RBR domain corresponds to amino acids 216 to 465. Therefore, inherently, to express this chimeric protein, a polynucleotide comprising the sequence encoding a fusion protein comprising a MTS of PINK1 and a TMD of PINK1, fused to a fragment of Parkin protein, had to be cloned by Zheng. Zheng teaches transfecting HeLa cells with constructs comprising the recombinant chimeric protein (see page 891, Figure 2), therefore, Zheng teaches the polynucleotide as claimed.
Regarding claims 4 and 5, Zheng teaches a human PINK1 cDNA, therefore the residues 1-34 of the human PINK1 is included within the new chimeric protein, PINK1Δkinase-Flag-RBR protein (see page 890, left column, second paragraph; and see Figure 2), as evidenced by Figure 2B of Hauser. Also, this chimeric protein as taught by Zheng comprises 100% of the MTS of the human PINK1, and thus, should be at least 95% identical to SEQ ID NO: 66.
As evidenced by NM_032409.3 pdf, the sequence SEQ ID NO: 66 claimed in claim 4 is 100% identical to residues 1-34 of amino acids sequence described in NM_032409.3, as shown in the alignment below (Query , Qy = SEQ ID NO: 66; Database, Db = NM_032409.3):
Query Match 100.0%; Score 172; DB 1; Length 581;
Best Local Similarity 100.0%;
Matches 34; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MAVRQALGRGLQLGRALLLRFTGKPGRAYGLGRP 34
||||||||||||||||||||||||||||||||||
Db 1 MAVRQALGRGLQLGRALLLRFTGKPGRAYGLGRP 34
As evidenced by the alignment below, SEQ ID NO: 65 claimed in claim 5 is 100% identical to NM_032409.3 (Query, Qy = SEQ ID NO: 65; Database, Db = NM_032409.3):
Query Match 100.0%; Score 496; DB 1; Length 581;
Best Local Similarity 100.0%;
Matches 94; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MAVRQALGRGLQLGRALLLRFTGKPGRAYGLGRPGPAAGCVRGERPGWAAGPGAEPRRVG 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MAVRQALGRGLQLGRALLLRFTGKPGRAYGLGRPGPAAGCVRGERPGWAAGPGAEPRRVG 60
Qy 61 LGLPNRLRFFRQSVAGLAARLQRQFVVRAWGCAG 94
||||||||||||||||||||||||||||||||||
Db 61 LGLPNRLRFFRQSVAGLAARLQRQFVVRAWGCAG 94
Therefore, Zheng teaches a chimeric protein with a MTS sequence from PINK1 comprising a sequence 100% identical to SEQ ID NOs: 65 and 66.
Regarding claim 8, Zheng teaches a polynucleotide encoding for a chimeric protein PINK1Δkinase-Flag-RBR protein comprising the TMD (94-110 aa) from PINK1 (see page 890, left column, second paragraph; and see Figure 2). As evidenced by Yamano, Presenilin-associated rhomboid-like (PARL) cleaves PINK1 protein between Alanine 103 and Phenylalanine 104 , which corresponds to a cleavage site located within the TMD (see page 1759, right column, first paragraph).
Regarding claim 9, as evidenced by NM_032409.3 pdf, and the alignment below, the TMD sequence, i.e. SEQ ID NO: 67 (Qy) claimed in claim 9 is 100% identical to NM_032409.3 (Db):
Query Match 100.0%; Score 85; DB 1; Length 581;
Best Local Similarity 100.0%;
Matches 16; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 PCGRAVFLAFGLGLGL 16
||||||||||||||||
Db 95 PCGRAVFLAFGLGLGL 110
Therefore, Zheng teaches a chimeric protein comprising a sequence 100% identical to the sequence of SEQ ID NO: 67.
Regarding claim 12, Zheng teaches a polynucleotide constructed for transfecting HeLa cells expressing the chimeric protein PINK1Δkinase-Flag-RBR protein, which comprises a MTS-TMD fragment of PINK1 protein (see page 890, left column, second paragraph; and Figure 2).
Regarding claim 13, Zheng teaches a chimeric protein comprising a MTS-TMD fragment from PINK1, that is 100% identical to SEQ ID NO: 70. As evidenced by NM_032409.3 pdf, and the alignment below, the claimed sequence in SEQ ID NO:70 (Qy) is 100% identical to the residues 1-110 of human PINK1 protein of NM_032409.3 (Db):
Query Match 100.0%; Score 581; DB 1; Length 581;
Best Local Similarity 100.0%;
Matches 110; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MAVRQALGRGLQLGRALLLRFTGKPGRAYGLGRPGPAAGCVRGERPGWAAGPGAEPRRVG 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MAVRQALGRGLQLGRALLLRFTGKPGRAYGLGRPGPAAGCVRGERPGWAAGPGAEPRRVG 60
Qy 61 LGLPNRLRFFRQSVAGLAARLQRQFVVRAWGCAGPCGRAVFLAFGLGLGL 110
||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 LGLPNRLRFFRQSVAGLAARLQRQFVVRAWGCAGPCGRAVFLAFGLGLGL 110
Regarding claim 15, Zheng teaches a chimeric protein PINK1Δkinase-Flag-RBR protein comprising the Parkin RBR domain, corresponding to amino acids 216 to 465. As evidenced by Hauser, the chimeric protein has a fragment of Parkin comprising only the RBR domain, and lacking the Ubl domain (1-76 aa) and the RING0 domain (145-215 aa). Therefore, Zheng teaches a polynucleotide encoding for a ΔParkin protein comprising (i) a deletion of the N-terminal ubiquitin-like (Ubl) domain, and (ii) a deletion of the Ubl-RING0 interdomain linker sequence (76-145 aa), and (iii) a deletion of the N- terminal Ubl domain and a deletion of the Ubl-ING0 interdomain (figure 2A, and as shown above).
Regarding claim 30, Zheng teaches vectors comprising the polynucleotide as claimed in claim 1 (see page 895, left column, “Materials and Methods” section).
Regarding claim 50, Zheng teaches expressing/transfecting the constructs into HeLa cells, which is a human cell line (see Figure 2).
Regarding claim 51, Zheng teaches recombinant fusion protein PINK1Δkinase-Flag-RBR expressed in HeLa cells (see figure 2).
Claim 3 is rejected under 35 U.S.C. §102(a)(1) as being anticipated by Zheng (Zheng, X. et al. "Parkin mitochondrial translocation is achieved through a novel catalytic activity coupled mechanism." Cell Research, Vol. 23 (2013), pp: 886-897), as evidenced by Hauser (Hauser, D.N. et al. "The effects of variants in the PARK2 (parkin), PINK1, and PARK7 (DJ-1) genes along with evidence for their pathogenicity". Curr. Protein. Pept. Sci. Vol. 18, No. 7 (2017), pp: 702-714.) and Bayne (Bayne, A.N. et al. “Substrate recognition by the human mitochondrial processing peptidase and its processing of PINK1”. Journal of Biological Chemistry, Vol. 301, No. 12 (2025), p: 110860).
It is noted that claim 3 depends upon claim 1. The rejection of claim 1 is described above, as anticipated by Zheng as evidenced by Hauser.
Regarding claim 3, Zheng teaches that cDNAs for human Parkin and PINK1 were cloned from the MGC human cDNA library, mutations were generated using QuickChange® site-directed mutagenesis kit from STRATAGENE and expressed in pEGFP CLONTECH vectors and Flag-tagged versions were expressed in pCMV-3tag from AGILENT vectors (see page 895, left column, “Materials and Methods” section). Zheng teaches that recombinant fusion proteins comprising a fragment of Parkin and a fragment of PINK1 were constructed and expressed. Zheng teaches a new chimeric protein constructed by replacing the PINK1 kinase domain (PINK1, 157-581 aa) with the Parkin RBR domain (216-465 aa), and adding a flag tag sequence between them, i.e., PINK1Δkinase-Flag-RBR protein (see page 890, left column, second paragraph; and see Figure 2).
According to Hauser, in human PINK1, the MTS corresponds to amino acids 1 to 77 and the transmembrane domain corresponds to amino acids 94 to 110 (see Figure 2, and see below).
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232
768
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Therefore, the remainder of human PINK1 within the chimeric protein is aa 1-156, since only the kinase domain (157-581 aa) is removed. Parkin RBR domain corresponds to amino acids 216 to 465. Therefore, inherently, to express this chimeric protein, a polynucleotide comprising the sequence encoding a fusion protein comprising a MTS of PINK1 and a TMD of PINK1, fused to a fragment of Parkin protein, had to be cloned by Zheng. Zheng teaches transfecting HeLa cells with constructs comprising the recombinant chimeric protein (see page 891, Figure 2), therefore, Zheng teaches the polynucleotide as claimed.
In sum, Zheng teaches a polynucleotide constructed to encode for a chimeric PINK1Δkinase-Flag-RBR protein comprising the MTS (1-77 aa) from PINK1 (see page 890, left column, second paragraph; and see Figure 2). As evidenced by Bayne, the MTS of PINK1 comprises a site for substrate recognition by the human mitochondrial processing peptidase (MPP) between Alanine 28 and Tyrosine 29 (see title and abstract).
NOTE: According to MPEP §2124, “Exception to the rule that the reference must be Prior Art”, in some circumstances a factual reference need not antedate the filing date.
The following rejections are new as necessitated by Applicant’s amendments:
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 non-obviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claim 18 is rejected under 35 U.S.C. §103 as being unpatentable over Zheng (Zheng, X. et al. "Parkin mitochondrial translocation is achieved through a novel catalytic activity coupled mechanism." Cell Research, Vol. 23 (2013), pp: 886-897), in view of Hauser (Hauser, D.N. et al. "The effects of variants in the PARK2 (parkin), PINK1, and PARK7 (DJ-1) genes along with evidence for their pathogenicity". Curr. Protein. Pept. Sci. Vol. 18, No. 7 (2017), pp: 702-714.), NM_032409.3 (Homo sapiens PTEN induced kinase 1 (PINK1), mRNA pdf, NCBI reference sequence: NM_032409.3 version updated and published November 22, 2018; cited previously) and Yamano (Yamano, K. et al. “PINK1 is degraded through the N-end rule pathway”. Autophagy, Vol. 9, No. 11 (2013), pp: 1758-1769), as applied to claims 1 and 15 above, and in further view of NM_004562.3 (Homo sapiens parkin RBR E3 ubiquitin protein ligase (PRKN), transcript variant 1, mRNA pdf, NCBI reference sequence NM_004562.3 version updated and published November 23, 2018; cited previously) and Yi ( Yi, W. et al. “The landscape of Parkin variants reveals pathogenic mechanisms and therapeutic targets in Parkinson’s disease”. Human Molecular Genetics, Vol. 28, No. 17 (2019), pp: 2811-2825; cited previously).
The rejections of claims 1 and 15 are described above. It is noted that the claims are anticipated by Zheng as evidenced by Hauser.
Therefore, combination of Zheng, Hauser, NM_032409.3 and Yamano renders elements of claims 1 and 15 obvious. However, the combination of references does not render elements of claim 18 obvious, i.e., “The polynucleotide of claim 15, wherein the ΔParkin protein comprises a polypeptide sequence at least 95% identical to residues 76-465 of human Parkin further comprising F146A and W403 amino acid substitutions” as set forth in SEQ ID NO: 74.
Regarding claim 18, Zheng teaches that cDNAs for human Parkin and PINK1 were cloned from the MGC human cDNA library, mutations were generated using QuickChange® site-directed mutagenesis kit from STRATAGENE and expressed in pEGFP CLONTECH vectors and Flag-tagged versions were expressed in pCMV-3tag from AGILENT vectors (see page 895, left column, “Materials and Methods” section). Zheng teaches that recombinant fusion proteins comprising a fragment of Parkin and a fragment of PINK1 were constructed and expressed. Zheng teaches a new chimeric protein constructed by replacing the PINK1 kinase domain (PINK1, 157-581 aa) with the Parkin RBR domain (216-465 aa), and adding a flag tag sequence between them, i.e., PINK1Δkinase-Flag-RBR protein (see page 890, left column, second paragraph; and see Figure 2).
According to Hauser, in human PINK1, the MTS corresponds to amino acids 1 to 77 and the transmembrane domain corresponds to amino acids 94 to 110 (see Figure 2, and see below).
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232
768
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Therefore, the remainder of human PINK1 within the chimeric protein is aa 1-156, since only the kinase domain (157-581 aa) is removed. Parkin RBR domain corresponds to amino acids 216 to 465. Therefore, inherently, to express this chimeric protein, a polynucleotide comprising the sequence encoding a fusion protein comprising a MTS of PINK1 and a TMD of PINK1, fused to a fragment of Parkin protein, had to be cloned by Zheng. Zheng teaches transfecting HeLa cells with constructs comprising the recombinant chimeric protein (see page 891, Figure 2), therefore, Zheng teaches the polynucleotide as claimed.
Zheng also teaches a chimeric protein PINK1Δkinase-Flag-RBR protein comprising the Parkin RBR domain, corresponding to amino acids 216 to 465. As evidenced by Hauser, the chimeric protein has a fragment of Parkin comprising only the RBR domain, and lacking the Ubl domain (1-76 aa) and the RING0 domain (145-215 aa). Therefore, Zheng teaches a polynucleotide encoding for a ΔParkin protein comprising (i) a deletion of the N-terminal ubiquitin-like (Ubl) domain, and (ii) a deletion of the Ubl-RING0 interdomain linker sequence (76-145 aa), and (iii) a deletion of the N- terminal Ubl domain and a deletion of the Ubl-ING0 interdomain (figure 2A, and as shown above).
Zheng does not teach SEQ ID NO: 74.
However, NM_004562.3 (Db) teaches the sequence of human Parkin is 99.4% identical to the sequence claimed in claim 18, i.e. SEQ ID NO: 74 (Qy); see alignment below:
Query Match 99.4%; Score 2179; DB 1; Length 465;
Best Local Similarity 99.5%;
Matches 388; Conservative 0; Mismatches 2; Indels 0; Gaps 0;
Qy 1 KGQEMNATGGDDPRNAAGGCEREPQSLTRVDLSSSVLPGDSVGLAVILHTDSRKDSPPAG 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 76 KGQEMNATGGDDPRNAAGGCEREPQSLTRVDLSSSVLPGDSVGLAVILHTDSRKDSPPAG 135
Qy 61 SPAGRSIYNSAYVYCKGPCQRVQPGKLRVQCSTCRQATLTLTQGPSCWDDVLIPNRMSGE 12
|||||||||| |||||||||||||||||||||||||||||||||||||||||||||||||
Db 136 SPAGRSIYNSFYVYCKGPCQRVQPGKLRVQCSTCRQATLTLTQGPSCWDDVLIPNRMSGE 195
Qy 121 CQSPHCPGTSAEFFFKCGAHPTSDKETSVALHLIATNSRNITCITCTDVRSPVLVFQCNS 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 196 CQSPHCPGTSAEFFFKCGAHPTSDKETSVALHLIATNSRNITCITCTDVRSPVLVFQCNS 255
Qy 181 RHVICLDCFHLYCVTRLNDRQFVHDPQLGYSLPCVAGCPNSLIKELHHFRILGEEQYNRY 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 256 RHVICLDCFHLYCVTRLNDRQFVHDPQLGYSLPCVAGCPNSLIKELHHFRILGEEQYNRY 315
Qy 241 QQYGAEECVLQMGGVLCPRPGCGAGLLPEPDQRKVTCEGGNGLGCGFAFCRECKEAYHEG 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 316 QQYGAEECVLQMGGVLCPRPGCGAGLLPEPDQRKVTCEGGNGLGCGFAFCRECKEAYHEG 375
Qy 301 ECSAVFEASGTTTQAYRVDERAAEQARAEAASKETIKKTTKPCPRCHVPVEKNGGCMHMK 360
||||||||||||||||||||||||||| ||||||||||||||||||||||||||||||||
Db 376 ECSAVFEASGTTTQAYRVDERAAEQARWEAASKETIKKTTKPCPRCHVPVEKNGGCMHMK 435
Qy 361 CPQPQCRLEWCWNCGCEWNRVCMGDHWFDV 390
||||||||||||||||||||||||||||||
Db 436 CPQPQCRLEWCWNCGCEWNRVCMGDHWFDV 465
The two mismatches in the alignment above correspond to missense mutations leading to amino acid substitutions at positions 146 and 403 of the wild-type human Parkin protein : F146A and W403A.
Yi teaches that impaired mitophagy from naturally occurring Parkin variants that are pathogenic, could be rescued with structure-guided designer hyperactive Parkin variants comprising p.W403A or p.F146A (see abstract and Figure 5; see page 2819, “Structure-guided designer hyperactive Parkin mutants can rescue mitophagy in pathogenic variants” section).
Yi also teaches in Figure 3 that Ubl domain is comprised within residues 1-76 of Parkin protein (see Figure 3A).
Therefore, it would have been obvious to one with ordinary skills in the art before the effective filing date of the claimed invention to have combined the teachings of Zheng, NM_004562.3 and Yi and modified the Parkin fragment within the recombinant fusion protein taught by Zheng and introduced amino acid substitutions as taught by Yi. One with ordinary skills in the art motivated in making a recombinant parkin protein having the benefit of reversing the effect of pathogenic Parkin variant and enhancing mitophagy of dysfunctional mitochondria, could have substituted the ΔParkin protein of Zheng with a ΔParkin comprising two known mutations W403A and F146A, giving rise to a hyperactive chimeric Parkin protein that can be used as therapeutics. These modifications consist in routine optimization of a recombinant protein based on known elements in the art. Therefore, one with ordinary skills in the art could have performed these modifications with a reasonable expectation of success and arrived at the claimed invention.
Claims 20 and 28 are rejected under 35 U.S.C. §103 as being unpatentable over Zheng (Zheng, X. et al. "Parkin mitochondrial translocation is achieved through a novel catalytic activity coupled mechanism." Cell Research, Vol. 23 (2013), pp: 886-897), in view of Hauser (Hauser, D.N. et al. "The effects of variants in the PARK2 (parkin), PINK1, and PARK7 (DJ-1) genes along with evidence for their pathogenicity". Curr. Protein. Pept. Sci. Vol. 18, No. 7 (2017), pp: 702-714.), NM_032409.3 (Homo sapiens PTEN induced kinase 1 (PINK1), mRNA pdf, NCBI reference sequence: NM_032409.3 version updated and published November 22, 2018; cited previously), and Yamano (Yamano, K. et al. “PINK1 is degraded through the N-end rule pathway”. Autophagy, Vol. 9, No. 11 (2013), pp: 1758-1769), as applied to claim 1 above, and in view of NM_004562.3 (Homo sapiens parkin RBR E3 ubiquitin protein ligase (PRKN), transcript variant 1, mRNA pdf, NCBI reference sequence NM_004562.3 version updated and published November 23, 2018; cited previously), Okatsu (Okatsu, K. et al. Journal of Cell Science, Vol. 128 (2015), pp: 964-978; cited previously), Yi ( Yi, W. et al. “The landscape of Parkin variants reveals pathogenic mechanisms and therapeutic targets in Parkinson’s disease”. Human Molecular Genetics, Vol. 28, No. 17 (2019), pp: 2811-2825; cited previously), Trempe (Trempe, J.-F. et al. "Structure of Parkin reveals mechanisms for ubiquitin Ligase activation." Science, Vol. 340 (2013), pp: 1451-1455) and Liu (Liu, Y. et al. “The ubiquitination of PINK1 is restricted to its mature 52-KDa form”. Cell Reports, Vol. 20 (2017), pp: 30-39; cited previously).
The rejection of claim 1 is described above. It is noted that the elements of claim 1 are anticipated by Zheng as evidenced by Hauser, NM_032409.3 and Yamano.
Therefore, the combination of references does render elements of claim 1 obvious.
However, the combination of Zheng, Hauser, NM_032409.3 and Yamano does
not render obvious elements of claims 20 and 28, i.e., a polynucleotide encoding a fusion protein comprising a polypeptide sequence at least 95% identical to SEQ ID NO: 75 or a polypeptide at least 95% identical to SEQ ID NO: 97, comprising two or more amino acid substitutions selected from F104M, W403A and F463A, wherein F104M is relative to SEQ ID NO: 64 and W403A and F463A are relative to SEQ ID NO: 1.
Regarding claim 20, Zheng teaches a polynucleotide encoding for a fusion protein comprising fragments of PINK1 (N-terminus region: amino acids 1 to 156) and Parkin (C-terminus region: amino acids 216 to 465) (see page 890, left column, second paragraph; and see Figure 2 of Hauser).
Hauser teaches that the MTS-TMD fragments are contained withing amino acids 1 to 110 of PINK1 protein (see page 23, Figure 2B).
As evidenced by NM_032409.3 pdf, and the alignment below, the claimed sequence in SEQ ID NO: 75 (Qy) is 100% identical to the residues 1-110 of human PINK1 protein of NM_032409.3 (Db):
Query Match 21.0%; Score 581; DB 1; Length 110;
Best Local Similarity 100.0%;
Matches 110; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MAVRQALGRGLQLGRALLLRFTGKPGRAYGLGRPGPAAGCVRGERPGWAAGPGAEPRRVG 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MAVRQALGRGLQLGRALLLRFTGKPGRAYGLGRPGPAAGCVRGERPGWAAGPGAEPRRVG 60
Qy 61 LGLPNRLRFFRQSVAGLAARLQRQFVVRAWGCAGPCGRAVFLAFGLGLGL 110
||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 LGLPNRLRFFRQSVAGLAARLQRQFVVRAWGCAGPCGRAVFLAFGLGLGL 110
However, the combination of reference does not teach a limited region of PINK1 from 1 to 110 amino acids in the fusion protein.
However, Okatsu teaches using fragments of PINK1 corresponding to amino acids 1 to 110 in constructs comprising GFP as fusion proteins (see Figure 6Am and page 971, left column, “The second mitochondrial localization signal allows PINK1 to localize on OMM when the typical MTS is inhibited” section, line 6).
Therefore, it would have been obvious to one with ordinary skills in the art, before the effective filing date of the claimed invention, to have substituted the N-terminal fragment of PINK1 taught by Zheng, with a region consisting of amino acid residues 1 to 110 of PINK1 since Okatsu verified it contains all the elements required for mitochondrial localization. One with ordinary skills in the art, motivated in obtaining optimum mitochondrial translocation of the recombinant fusion PINK1-Parkin protein could have made this modification with a reasonable expectation of success and arrived at the claimed invention.
The combination of references, Zheng, Hauser, NM_032409.3, Yamano and Okatsu does not teach SEQ ID NO: 75 specifically.
However, as taught by NM_004562.3, the remainder of the fusion protein’s sequence is a mutated Parkin fragment; the alignment below between SEQ ID NO: 75 (Qy) and NM_004562.3, i.e. human Parkin, (Db) shows that residues 111 to 500 of SEQ ID NO: 75 are 99.5% identical to a wild-type human Parkin protein from amino acids 76 to 465:
Query Match 78.6%; Score 2179; DB 1; Length 465;
Best Local Similarity 99.5%;
Matches 388; Conservative 0; Mismatches 2; Indels 0; Gaps 0;
Qy 111 KGQEMNATGGDDPRNAAGGCEREPQSLTRVDLSSSVLPGDSVGLAVILHTDSRKDSPPAG 170
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 76 KGQEMNATGGDDPRNAAGGCEREPQSLTRVDLSSSVLPGDSVGLAVILHTDSRKDSPPAG 135
Qy 171 SPAGRSIYNSAYVYCKGPCQRVQPGKLRVQCSTCRQATLTLTQGPSCWDDVLIPNRMSGE 230
|||||||||| |||||||||||||||||||||||||||||||||||||||||||||||||
Db 136 SPAGRSIYNSFYVYCKGPCQRVQPGKLRVQCSTCRQATLTLTQGPSCWDDVLIPNRMSGE 195
Qy 231 CQSPHCPGTSAEFFFKCGAHPTSDKETSVALHLIATNSRNITCITCTDVRSPVLVFQCNS 290
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 196 CQSPHCPGTSAEFFFKCGAHPTSDKETSVALHLIATNSRNITCITCTDVRSPVLVFQCNS 255
Qy 291 RHVICLDCFHLYCVTRLNDRQFVHDPQLGYSLPCVAGCPNSLIKELHHFRILGEEQYNRY 350
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 256 RHVICLDCFHLYCVTRLNDRQFVHDPQLGYSLPCVAGCPNSLIKELHHFRILGEEQYNRY 315
Qy 351 QQYGAEECVLQMGGVLCPRPGCGAGLLPEPDQRKVTCEGGNGLGCGFAFCRECKEAYHEG 410
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 316 QQYGAEECVLQMGGVLCPRPGCGAGLLPEPDQRKVTCEGGNGLGCGFAFCRECKEAYHEG 375
Qy 411 ECSAVFEASGTTTQAYRVDERAAEQARAEAASKETIKKTTKPCPRCHVPVEKNGGCMHMK 470
||||||||||||||||||||||||||| ||||||||||||||||||||||||||||||||
Db 376 ECSAVFEASGTTTQAYRVDERAAEQARWEAASKETIKKTTKPCPRCHVPVEKNGGCMHMK 435
Qy 471 CPQPQCRLEWCWNCGCEWNRVCMGDHWFDV 500
||||||||||||||||||||||||||||||
Db 436 CPQPQCRLEWCWNCGCEWNRVCMGDHWFDV 465
The two mismatches in the alignment above correspond to missense mutations leading to amino acid substitutions at positions 146 and 403 of the wild-type human Parkin protein : F146A and W403A.
Yi teaches that impaired mitophagy from naturally occurring Parkin variants that are pathogenic, could be rescued with structure-guided designer hyperactive Parkin variants comprising p.W403A or p.F146A (see abstract and Figure 5; see page 2819, “Structure-guided designer hyperactive Parkin mutants can rescue mitophagy in pathogenic variants” section).
Yi also teaches in Figure 3 that Ubl domain is comprised within residues 1-76 of Parkin protein (see Figure 3A).
Trempe teaches recombinant Parkin proteins devoid of fragments of different lengths in the N-terminal of Parkin protein. Trempe teaches that the Ubl domain (1-76) can be removed from Parkin protein without affecting the its activity, i.e., ΔUBL1 (77-465) (see Figures 2G and 2H).
Trempe also teaches that point mutations in RING0 (Phe146>Ala) or RING2 (Phe463>Ala) that have been predicted to disrupt the RING0-RNIG2 interface both increased Parkin activity (see page 1453, right column).
Trempe also teaches other mutations capable of interfering with the integrity of the C-terminal helix in RING2, are also able to activate Parkin, such as Cys457 (see page 1453, right column) and F463 (see Figure 2F), and stability and mitochondrial uptake, such as W403 (Figures 3 and 4).
Therefore, it would have been obvious to one with ordinary skills in the art before the effective filing date of the claimed invention, to have combined the teachings of Zheng, Hauser, NM_032409.3, Yamano and Okatsu, with the teachings of NM_004562.3, Trempe and Yi, and modified the Parkin fragment within the recombinant fusion protein taught by Zheng modified by Okatsu. One with ordinary skills in the art motivated in making a recombinant parkin protein that is more active, capable of reversing the effect of pathogenic Parkin variant and enhancing mitophagy of dysfunctional mitochondria, could have deleted the Ubl domain of Parkin protein as taught by Trempe, and substituted the Ubl domain of Parkin with residues 1-110 of PINK1 for mitochondrial outer membrane localization, and introduced the two known mutations given rise to a hyperactive Parkin recombinant protein that can be used as therapeutics. These modifications consist in routine optimization of a recombinant protein based on known elements in the art. Therefore, one with ordinary skills in the art could have performed these modifications with a reasonable expectation of success and arrived at the claimed invention.
Regarding claim 28, the combination of references Zheng as evidenced by Hauser/NM_32409.3/Yamano, with Okatsu, NM_004562.3, Trempe and Yi, renders sequence SEQ ID NO: 75 obvious. SEQ ID NO: 75 is the sequence of the recombinant protein consisting of residues 1-110 of PINK1 protein and residues 76-465 of Parkin protein, comprising two amino acid substitutions at p.W403A and p.F146A.
The alignment below between SEQ ID NO: 97 (Qy) and SEQ ID NO: 75 (Db) is below:
Query Match 99.0%; Score 2752; DB 1; Length 500;
Best Local Similarity 99.4%;
Matches 497; Conservative 0; Mismatches 3; Indels 0; Gaps 0;
Qy 1 MAVRQALGRGLQLGRALLLRFTGKPGRAYGLGRPGPAAGCVRGERPGWAAGPGAEPRRVG 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MAVRQALGRGLQLGRALLLRFTGKPGRAYGLGRPGPAAGCVRGERPGWAAGPGAEPRRVG 60
Qy 61 LGLPNRLRFFRQSVAGLAARLQRQFVVRAWGCAGPCGRAVFLAFGLGLGLKGQEMNATGG 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 LGLPNRLRFFRQSVAGLAARLQRQFVVRAWGCAGPCGRAVFLAFGLGLGLKGQEMNATGG 120
Qy 121 DDPRNAAGGCEREPQSLTRVDLSSSVLPGDSVGLAVILHTDSRKDSPPAGSPAGRSIYNS 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 DDPRNAAGGCEREPQSLTRVDLSSSVLPGDSVGLAVILHTDSRKDSPPAGSPAGRSIYNS 180
Qy 181 FYVYCKGPCQRVQPGKLRVQCSTCRQATLTLTQGPSCWDDVLIPNRMSGECQSPHCPGTS 240
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 AYVYCKGPCQRVQPGKLRVQCSTCRQATLTLTQGPSCWDDVLIPNRMSGECQSPHCPGTS 240
Qy 241 AEFFFKCGAHPTSDKETSVALHLIATNSRNITCITCTDVRSPVLVFQCNSRHVICLDCFH 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 AEFFFKCGAHPTSDKETSVALHLIATNSRNITCITCTDVRSPVLVFQCNSRHVICLDCFH 300
Qy 301 LYCVTRLNDRQFVHDPQLGYSLPCVAGCPNSLIKELHHFRILGEEQYNRYQQYGAEECVL 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 LYCVTRLNDRQFVHDPQLGYSLPCVAGCPNSLIKELHHFRILGEEQYNRYQQYGAEECVL 360
Qy 361 QMGGVLCPRPGCGAGLLPEPDQRKVTCEGGNGLGCGFAFCRECKEAYHEGECSAVFEASG 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 QMGGVLCPRPGCGAGLLPEPDQRKVTCEGGNGLGCGFAFCRECKEAYHEGECSAVFEASG 420
Qy 421 TTTQAYRVDERAAEQARWEAASKETIKKTTKPCPRCHVPVEKNGGCMHMKCPQPQCRLEW 480
||||||||||||||||| ||||||||||||||||||||||||||||||||||||||||||
Db 421 TTTQAYRVDERAAEQARAEAASKETIKKTTKPCPRCHVPVEKNGGCMHMKCPQPQCRLEW 480
Qy 481 CWNCGCEWNRVCMGDHWADV 500
||||||||||||||||| ||
Db 481 CWNCGCEWNRVCMGDHWFDV 500
The alignment shows that the modified recombinant fusion PINK1-Parkin protein is 99% identical to SEQ ID NO: 97. Therefore, the claim encompasses the species rendered obvious by the combination of references.
It is noted that there is a mutation at position F463, consisting in an amino acid substitution F463A, which also was taught by Trempe.
Also Okatsu teaches that the phenylalanine residue at position 104 of the N-terminus of PINK1 acts as a signal for N-end Rule pathway mediated degradation of PINK1 (see page 964, right column, third paragraph).
Yamano also teaches that the N-end mutation F104M in PINK1 is stabilizing, and that the mutant can recruit Parkin to depolarized mitochondria followed by mitophagy (see page 1761, left column, second paragraph).
Liu teaches inducing a mutation at this position F104 and making a recombinant protein PINK1 with an amino acid substitution F104M or F104A. Liu teaches that the half-life of the mutated PINK1 increased from about 2.3 hours to about 6.4 hour for PINK1 F104A, and to about 11.4 hours for PINK1 F104M. Therefore, mutating the PINK1 protein and making a F104M mutation leads to the most stable PINK1 variant (see page 36, left column, “Is 52-KDa PINK1 degraded via the N-end Rule pathway?” section).
Therefore, it would have been obvious to one with ordinary skills in the art before the effective filing date of the claimed invention to have added a supplemental modification/amino acid substitution at F104 as taught by Yamano and Liu to the fragment of PINK1 (1-110) within the recombinant fusion parkin protein taught by the combination of Zheng/Hauser, Yamano, Okatsu, NM_32409.3, NM_004562.3, Trempe and Yi. One with ordinary skills in the art motivated in stabilizing the recombinant fusion protein PINK1(1-110)-Parkin(76-465; p.W403A+ p.F146A) onto the outer mitochondrial membrane could have performed this modification with a reasonable expectation of success and arrived at the claimed invention.
Claims 31-33 and 52-53 are rejected under 35 U.S.C. §103 as being unpatentable over Zheng (Zheng, X. et al. "Parkin mitochondrial translocation is achieved through a novel catalytic activity coupled mechanism." Cell Research, Vol. 23 (2013), pp: 886-897), in view of Hauser (Hauser, D.N. et al. "The effects of variants in the PARK2 (parkin), PINK1, and PARK7 (DJ-1) genes along with evidence for their pathogenicity". Curr. Protein. Pept. Sci. Vol. 18, No. 7 (2017), pp: 702-714.), NM_032409.3 (Homo sapiens PTEN induced kinase 1 (PINK1), mRNA pdf, NCBI reference sequence: NM_032409.3 version updated and published November 22, 2018; cited previously), and Yamano (Yamano, K. et al. “PINK1 is degraded through the N-end rule pathway”. Autophagy, Vol. 9, No. 11 (2013), pp: 1758-1769), as applied to claims 1 and 30 above, and in further view of Youle (Youle, R.J. et al. US 2012/0277286 A1, published November 1, 2012; cited previously), Cearley (Cearley, C.N. et al. “A single injection of an Adeno-Associated virus vector into nucleic with divergent connections results in widespread vector distribution in the brain and global correction of a neurogenetic disease”. The Journal of Neuroscience, Vol. 27, No. 37 (2007), pp: 9928-9940; cited previously), pdf-GENBANK accession # AAS99282.1 (NCBI-“Capsid protein VP1 [Adeno-associated virus]; GENBANK # AAS99282.1; published June 24, 2004) and Gao (Gao, G. et al. “Clades of Adeno-Associated viruses are widely disseminated in Human tissues”. Journal of Virology, Vol. 78, No. 12 (2004), pp: 6381-6388).
The rejections of claims 1 and 30 are described above. The combination of references Zheng/Hauser, Yamano, and NM_32409.3 renders the elements of claims 1 and 30 obvious.
Regarding claims 31-33 and 52-53, Zheng teaches that cDNAs for human Parkin and PINK1 were cloned from the MGC human cDNA library, mutations were generated using QuickChange® site-directed mutagenesis kit from STRATAGENE and expressed in pEGFP CLONTECH vectors and Flag-tagged versions were expressed in pCMV-3tag from AGILENT vectors (see page 895, left column, “Materials and Methods” section). Zheng teaches that recombinant fusion proteins comprising a fragment of Parkin and a fragment of PINK1 were constructed and expressed. Zheng teaches a new chimeric protein constructed by replacing the PINK1 kinase domain (PINK1, 157-581 aa) with the Parkin RBR domain (216-465 aa), and adding a flag tag sequence between them, i.e., PINK1Δkinase-Flag-RBR protein (see page 890, left column, second paragraph; and see Figure 2).
According to Hauser, in human PINK1, the MTS corresponds to amino acids 1 to 77 and the transmembrane domain corresponds to amino acids 94 to 110 (see Figure 2, and see below).
PNG
media_image1.png
232
768
media_image1.png
Greyscale
Therefore, the remainder of human PINK1 within the chimeric protein is aa 1-156, since only the kinase domain (157-581 aa) is removed. Parkin RBR domain corresponds to amino acids 216 to 465. Therefore, inherently, to express this chimeric protein, a polynucleotide comprising the sequence encoding a fusion protein comprising a MTS of PINK1 and a TMD of PINK1, fused to a fragment of Parkin protein, had to be cloned by Zheng. Zheng teaches transfecting HeLa cells with constructs comprising the recombinant chimeric protein (see page 891, Figure 2), therefore, Zheng teaches the polynucleotide as claimed.
Zheng also teaches vectors comprising the polynucleotide as claimed in claim 1 (see page 895, left column, “Materials and Methods” section).
The combination of references does not teach the elements of claims 31-33 and 52-53 obvious, i.e., a vector comprising an AAV9 capsid or functional variant sharing at least 98% identity to SEQ ID NO: 60 ( claims 31-33), nor a pharmaceutical composition comprising the vector and one or more pharmaceutically acceptable carriers, diluents or excipients, as well as a kit comprising the vector and instructions for use (claims 52-53).
However, regarding claims 31-33, Youle teaches compositions and methods for the treatment or prevention of diseases associated with a mitochondrial defect (see title and abstract). Youle teaches transfection of recombinant parkin encoding polynucleotides or recombinant PINK1 protein encoding polynucleotides using different vectors (§ [0007]-[0009], [0093]-[0094]). Youle teaches that a vector can be a viral or retroviral vector, or adenoviral and adeno-associated viral vector (§ [0022], [0137]).
Regarding claims 52-53, Youle teaches a pharmaceutical composition comprising the vector, and also comprising acceptable carriers, diluents or excipients (§ [0167]-[0188]).
Youle also teaches kits comprising the vector encoding for a recombinant PINK1 or recombinant parkin proteins, and instructions (see § [0011], [0199]-[0200]).
Youle does not teach an AAV9 vector specifically.
However, Cearley teaches that adeno-associated virus vectors can be used for transfer of cDNA to correct metabolic defects at the site of an injection, but treating the whole brain requires widespread delivery. Cearley teaches that adeno-associated virus of serotype AAV9 has the particularity of being the most widely disseminated after injection in the ventral tegmental area (see title and abstract).
Gao, as shown in pdf-GENBANK accession # AAS99282.1 (Db), teaches the isolation of an AAV9 vector with 98.4% similarity to SEQ ID NO: 60 (Qy) as shown in the alignment below:
Query Match 98.4%; Score 3923; DB 1; Length 736;
Best Local Similarity 98.1%;
Matches 722; Conservative 8; Mismatches 6; Indels 0; Gaps 0;
Qy 1 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLD 60
||||||||||||| ||||||:|| |||| | || ::|:|::||||||||||||||||||
Db 1 MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPGNGLD 60
Qy 61 KGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQ 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 KGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQ 120
Qy 121 AKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTE 180
|||||||||||||||||||||||||||||||||||||||||||:|||||:||||||||||
Db 121 AKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGSQPAKKKLNFGQTGDTE 180
Qy 181 SVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVI 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 SVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVI 240
Qy 241 TTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQR 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 TTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQR 300
Qy 301 LINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAH 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 LINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAH 360
Qy 361 EGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENV 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 EGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENV 420
Qy 421 PFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIP 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 PFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIP 480
Qy 481 GPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGS 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 481 GPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGS 540
Qy 541 LIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQG 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 541 LIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQG 600
Qy 601 ILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPT 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 601 ILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPT 660
Qy 661 AFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGV 720
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 661 AFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGV 720
Qy 721 YSEPRPIGTRYLTRNL 736
||||||||||||||||
Db 721 YSEPRPIGTRYLTRNL 736
It is therefore obvious to one of ordinary skills in the art before the effective filing date of the claimed invention to have substituted the vector taught by Zheng with the AAV9 vector as taught by Cearley/Youle/ Gao in preparing a delivery method for the polynucleotide encoding the recombinant fusion protein PINK(1-110)-Parkin(76-465, p.W403A + p.F146A + pF104M) taught by Zheng/Hauser/Yamano, Okatsu, NM_32409.3, NM_004562.3, Liu, Trempe and Yi. One with ordinary skills in the art motivated in a single intracerebral injection leading in the most widespread dissemination of the therapeutic polynucleotide product as taught by Cearley could have performed this modification with a reasonable expectation of success and arrived at the claimed invention.
Response to Arguments
Applicant’s arguments with respect to claims 1, 3-5, 8-9, 12-13, 15, 18, 20, 28, 30-33, and 50-53 have been considered but are moot because the new ground of rejections does not rely on the same combination of references applied in the prior rejection of record for any teaching or matter specifically challenged in the argument.
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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/A.D./Examiner, Art Unit 1636
/NANCY J LEITH/Primary Examiner, Art Unit 1636
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