DETAILED ACTION
Applicant’s amendment and Arguments/Remarks received on 18 November 2025 have been entered. Claims 1-23, 25-28, 30, 37, 43, and 65 were previously pending in the application. Claims 3, 4, 10-12, and 17-22 have been cancelled, and new claims 66-72 have been added by Applicant. Claims 1-2, 5-9, 13-16, 23, 25-28, 30, 37, 43, and 65-75 are currently pending in the application. Claims 1, 37, 43, 65, and 66 are independent claims. The following election of species remains in effect in the instant application, updated to reflect language from the amended claims:
Intracellular factors: a. Protein: vii. MBNL1,
First introns (now downstream flanking introns): a. a truncated version of a naturally occurring intron,
Second introns (now upstream flanking introns): jj. From gene MBNL1,
Exons: (now Alternatively regulated exons): aa. An exon 5 of MBNL1,
Nucleic acid sequences: SEQ ID NOs: 1-18, 22-40, and 42-47 read on the elected species for 1)-4) above,
Claim 25-26 remain withdrawn and new claims 66-72 are newly withdrawn from consideration as being directed to nonelected species.
Claims 1-2, 5-9, 13-16, 23, 27-28, 30, 37, 43, and 65 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. PCTUS2020/057796, filed 28 October 2020, which claims priority to U.S. Provisional Application No. 62/927,087, filed 28 October 2019.
Thus, the earliest possible priority for the instant application is 28 October 2019.
Information Disclosure Statement
The information disclosure statement filed 18 November 2025 has been considered by the Examiner.
Claim Objections
The objection to amended, previously presented, and cancelled claims 1-9, 12-23, 27-28, 30, 37, 43, and 65 for:
Claims 1-9, 12-23, 27-28, 30, 37, 43, and 65 each reciting “rAAV” without first writing out the term for which “rAAV” is an abbreviation;
Claim 8 recites the abbreviations “MBNL, “SR”, “hnRNP”, “RbFox”, “CELF”, and “PTB” without first writing out the terms for which they are abbreviations;
Claim 9 recites the abbreviations “MBNL1, MBNL2, MBNL3, hnRNP A1, hnRNP A2B1, hnRNP C, hnRNP D, hnRNP DL, hnRNP F, hnRNP H, hnRNP K, hnRNP L, hnRNP M, hnRNP R, hnRNP U, FUS, TDP43, PABPN1, ATXN2, TAF15, EWSR1, MATR3, TIA1, FMRP, MTM1, KIF5A, ... C90RF72, HTT, DNM2, BIN1, RYR1, NEB, ACTA, TPM3, TPM2, TNNT2, CFL2, KBTBD13, KLHL40, KLHL41, LMOD3, MYPN, SEPN1, TTN, SPEG, MYH7, TK2, POLG1, GAA, AGL, PYGM, SLC22A5, OCTN2, ETF, ETFH, PNPLA2, … CLCN1, SCN4A, DMPK, CNBP, MYOT, … CAV3, DNAJB6, DES, TNPO3, HNRPDL, CAPN3, DYSF, … TCAP, TRIM32, FKRP, POMT1, FKTN, POMT2, POMGnT1, DAG1, ANO5, PLEC1, TRAPPC11, GMPPB, ISPD, LIMS2, POPDC1, TOR1AIP1, POGLUT2, LAMA2, COL6A1, POMT1, POMT2, DUX4, EMD, PAX7, PMP22, MPZ, MFN2, SMCHD1, or GJB1” without first writing out the terms for which they are abbreviations;
Claim 14 recites the abbreviations “NMD”, “SmB/B'”, “SMN”, “hnRBP A2B1”, “Tia1”, “Bin1”, “hnRNP D”, “FMRP”, “ST7”, “NEXN”, “NRAP”, “MTM1”, “CACNA1C”, “MBNL1”, “PABPN1”, “TDP43”, “FUS”, “hnRNP A1”, and”ATP2A1” without first writing out the terms for which they are abbreviations;
Claim 19 recites the abbreviation “RNAi” without first writing out the term for which “RNAi” is an abbreviation;
Claim 27 recites the abbreviations “NMD” “SmB/B'”, “SMN”, “hnRBP A2B1”, “Tia1”, “Bin1”, “hnRNP D”, “FMRP”, “ST7”, “NEXN”, “NRAP”, “MTM1”, “CACNA1C”, “MBNL1”, ”PABPN1”, “TDP43”, “FUS”, “hnRNP A1”, and “ATP2A1” without first writing out the terms for which they are abbreviations;
Claim 30 recites the abbreviations “MBNL1, MBNL2, MBNL3, hnRNP A1, hnRNP A2B1, hnRNP C, hnRNP D, hnRNP DL, hnRNP F, hnRNP H, hnRNP K, hnRNP L, hnRNP M, hnRNP R, hnRNP U, FUS, TDP43, PABPN1, ATXN2, TAF1S, EWSR1, MATR3, TIA1, FMRP, MTM1, KIF5A, …, C90RF72, HTT, DNM2, BIN1, RYR1, NEB, ACTA, TPM3, TPM2, TNNT2, CFL2, KBTBD13, KLHL40, KLHL41, LMOD3, MYPN, SEPN1, TTN, SPEG, MYH7, TK2, POLG1, GAA, AGL, PYGM, SLC22A5, OCTN2, ETF, ETFH, PNPLA2, …, CLCN1, SCN4A, DMPK, CNBP, MYOT, LMNA, CAV3, DNAJB6, DES, TNPO3, HNRPDL, CAPN3, DYSF, …, TCAP, TRIM32, FKRP, POMT1, FKTN, POMT2, POMGnT1, DAG1, ANOS, PLEC1, TRAPPC11, GMPPB, ISPD, LIMS2, POPDC1, TOR1A1P1, POGLUT2, LAMA2, COL6A1, POMT1, POMT2, DUX4, EMD, PAX7, PMP22, MPZ, MFN2, SMCHD1, and/or GJB1” without first writing out the terms for which they are abbreviations;
Claim 43 recites the abbreviation “MBNL” without first writing out the term for which “MBNL” is an abbreviation; and
claim 65 reciting an rAAV comprising a nucleic acid having the sequence of any one or more of SEQ ID NOs: 1, 2, 4-6, 8-11, 13-15, and/or 17-49, wherein SEQ ID NO: 47 is an amino acid sequence;
is withdrawn in view of the amendment to the claims writing out the term for which rAAV is an abbreviation in claims 1, 37, 43, and 65, and removing SEQ ID NO: 47 from claim 65.
Claim Rejections - 35 USC § 112(b)
The rejection of cancelled claim 4 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 reciting “wherein the splicing of the intron is regulated by the encoded intracellular factor or wherein the splicing of the intron is regulated by an intracellular factor that is not encoded by the RNA”, is withdrawn.
The rejection of amended claims 9 and 30 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 reciting the proteins/genes POMT1 and POMT2 twice within the list in each claim is withdrawn in view of Applicant’s amendments to the claims such that the second recitations of POMT1 and POMT2 have been deleted.
The rejection of amended claims 14 and 27 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 claim 14 reciting “wherein the first and/or second intron is or is derived from” in lines 1-2 and claim 27 reciting “wherein the exon is or is derived from” in lines 1-2, is withdrawn in view of Applicant’s amendments to the claims such that claims 14 and 27 no longer recite “or is derived”. Amended claim 14 and 27 now recite, “is from”, which has been interpreted to indicate that the intron (claim 14) or exon (claim 27) comprises any portion (up to full length) of any of the recited introns or exons.
The rejection of amended claim 23 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 reciting “wherein the exon is naturally occurring or wherein the exon is a recombinant exon”, is withdrawn in view of Applicant’s amendments to claim 23 to now recite “wherein the alternatively regulated exon is naturally or non-naturally occurring exon”.
**The following new rejection is necessitated by amendments to the claims.**
Amended, previously presented claims 1-2, 5-9, 13-16, 23, 27-28, 30, 37, and 65 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 5-9, 13-16, 23, 27-28, and 30 are included in this rejection due to their dependence on or encompassing of amended independent claim 1 and/or claim 2.
Amended independent claim 1 has multiple issues of indefiniteness.
The term “downstream” in amended independent claim 1 is a relative term which renders the claim indefinite. The term “downstream” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. It is unclear what the downstream flanking intron is downstream of. As such, the metes and bounds of the claim cannot be determined.
Amended independent claim 1 newly recites, “wherein splicing of the RNA by the intracellular factor”, which is indefinite because it is unclear what is encompassed by the intracellular factor. The phrase says that the RNA is spliced by the intracellular factor; however, the elected intracellular factor is MBNL1, which is a protein which regulates splicing but does not perform the splicing reaction. Additionally, dependent claims 5-9 recite that the intracellular factor is selected from a variety of proteins, including RNA binding proteins. Given that splicing is performed by RNA-protein complexes, wherein the RNA is the catalytic molecule performing the splicing reaction, it is unclear how the recited (and elected) proteins are performing the splicing of the RNA of amended claim 1. As such, the metes and bounds of the claim cannot be determined.
The term “upstream” in amended claim 2 is a relative term which renders the claim indefinite. The term “upstream” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. It is unclear what the upstream flanking intron is upstream of. As such, the metes and bounds of the claim cannot be determined.
Independent amended claim 37 newly recites the limitations “the intracellular factor of regulated splicing” in line 3, "the first truncated intron" in line 3, and “the second intron” in line 4. There is insufficient antecedent basis for this limitation in the claim. Claim 37 depends on independent claim 1. Neither claim 37 nor claim 1 have any prior recitations of an intracellular factor of regulated splicing, a first truncated intron, or a second intron. As such, the metes and bounds of the claim cannot be determined.
Independent amended claim 65 newly recites, “any one or more of SEQ ID NOs: 1, 2, 4, 6, 8-11, 13-15, and/or 17-46, 48, and 49”, which is indefinite because the recitation of both “and/or” and “and” makes it unclear which SEQ ID NOs: are required to be included. Specifically it is unclear whether “17-46, 48, and 49” are grouped together as a single option of the list. As such, the metes and bounds of the claim cannot be determined.
Claim Rejections - 35 USC § 112(a)- New Matter
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
**The following new rejection is necessitated by amendments to the claims.**
Amended and previously presented claims 1-2, 5-9, 13-16, 23, 27-28, 30, are 37 are newly rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention. This is a new matter rejection.
The applicant is reminded that an amendment to the claims or the addition of a new claim must be supported by the description of the invention in the application as filed. In re Wright, 866 F.2d 422, 9 USPQ2d 1649 (Fed. Cir. 1989). New or amended claims which introduce elements or limitations which are not supported by the as-filed disclosure violate the written description requirement. See, e.g., In re Lukach, 442 F.2d 967, 169 USPQ 795 (CCPA 1971); In re Smith, 458 F.2d 1389, 1395, 173 USPQ 679, 683 (CCPA 1972).
Amended independent claim 1 newly recites, “(i) an alternatively regulated exon having a first portion of a start codon” in line 3, “a transgene having a first exon that comprises a second portion of the start codon” in line 4-5, and “forming a complete start codon in the nucleic acid sequence” in lines 7-8. However, the instant disclosure does not provide any teachings, explicitly or implicitly, to support his new limitation.
The specification recites:
“In some embodiments, a recombinant nucleic acid for which splicing is regulated is a synthetic construct configured to regulate expression of a protein by including a 5’ exon comprising an amino terminal amino acid encoding sequence (e.g., an ATG or part of the ATG)…, wherein the 5’ exon is separated from subsequent exon(s) by an intron for which splicing is regulated. In some embodiment, if the intron is spliced out of the RNA transcript, the recombinant 5’ exon is spliced in frame to the subsequent exon(s) and the resulting spliced transcript encodes a protein that is expressed. In some embodiments, if the intron is not spliced out of the RNA transcript, the recombinant 5’ exon is not spliced to the subsequent exon(s) and as a result a protein is not expressed from the transcript” [page 24 ¶ 1].
As such, the specification teaches a partial stop codon in a 5’ exon (e.g., the beginning A or AT). However, the specification does not teach a partial stop codon in a regulated exon. Additionally, the specification does not teach that the subsequent exon has a partial stop codon (e.g., the ending TG or G), such that splicing of a regulated exon which results in retention of the exon and removal of the intron downstream of the regulated exon thereby forms a complete start codon (e.g., ATG).
The drawings present various schematics for splicing [e.g., Figure 1, 8, 10], including a construct in which MBNL1 exon 5 comprises a 3’ terminal ATG start codon and the subsequent exon has its ATG removed from the natural location of a coding sequence [Figure 14B], but none teach a partial/split start codon which is spliced together by bringing together an alternatively regulated exon with a subsequent exon to form a complete start codon. None of the working examples discussed in the specification utilize a split start codon, and none of the data presented in the drawings are generated from a split start codon system. As such, none of the data in the drawings support the limitations of “(i) an alternatively regulated exon having a first portion of a start codon”, “a transgene having a first exon that comprises a second portion of the start codon”, and “forming a complete start codon in the nucleic acid sequence” as claimed.
Therefore, the disclosure does not provide support for the limitations newly added to amended independent claim 1 of “(i) an alternatively regulated exon having a first portion of a start codon”, “a transgene having a first exon that comprises a second portion of the start codon”, and “forming a complete start codon in the nucleic acid sequence”, and the limitations represent new matter.
Claim Rejections - 35 USC § 112(a)- Scope of Enablement
The rejection of amended claim 37 under 35 U.S.C. 112(a) for
while being enabling for:
a method of treating myotonic dystrophy (DM) type 1 or DM type 2 in a subject having DM, comprising administering an rAAV comprising:
a) an MBNL1 protein coding sequence,
b) at least one first regulated intron of the MBNL1 gene, and
c) at least one second intron of the MBNL1 gene,
wherein splicing of the at least one first intron is regulated by the intracellular protein MBNL1, and wherein the at least one first regulated intron of the MBNL1 gene is a natural or truncated intron selected from the group consisting of a) MBNL1 intron 1c and b) MBNL1 intron 5, and wherein the at least one second intron of the MBNL1 gene is a natural or truncated intron selected from the group consisting of a) MBNL1 intron 2 and b) MBNL1 intron 6;
to the subject;
does not reasonably provide enablement for:
a method of treating any disease or any condition in any subject comprising administering any rAAV comprising a nucleic acid encoding any RNA, wherein the RNA comprises any first intron and wherein splicing of the any first intron is regulated by any intracellular factor, to the any subject. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to practice the invention commensurate in scope with these claims;
is withdrawn in view of Applicant’s amendments to the claims such that amended claim 37 now recites the limitations identified as the enabled scope in the prior action.
**The following new rejection is necessitated by amendments to the claims.**
Amended and previously presented claims 1-2, 5-9, 13-16, 23, 27-28, 30, and 37 are newly rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for:
A recombinant adeno-associated virus (rAAV) comprising a nucleic acid molecule encoding an RNA, wherein the RNA comprises:
a first sequence comprising
an alternatively regulated exon of MBNL1 comprising a start codon,
an upstream flanking MBNL1 intron comprising MBNL1 protein binding sites,
wherein the upstream flanking MBNL1 intron is a natural or truncated intron selected from the group consisting of MBNL1 intron 1c and MBNL1 intron 5,
a downstream flanking MBNL1 intron,
wherein the downstream flanking is a natural or truncated intron selected from the group consisting of MBNL1 intron 2 and MBNL1 intron 6; and
a second sequence comprising a transgene having a first exon and lacking a start codon;
wherein the transgene encodes a functional MBNL1 protein capable of binding to the MBNL1 protein binding sites and regulating alternative splicing of the alternatively regulated exon of MBNL1;
wherein splicing of the RNA is regulated by the MBNL1 protein binding to the MBNL1 protein binding sites within the upstream flanking MBNL1 intron; and
wherein in the absence of MBNL1 binding to the MBNL1 protein binding sites within the upstream flanking MBNL1 intron, splicing of the RNA excludes the flanking intron, thereby attaching the start codon in-frame to the nucleic acid sequence encoding the MBNL1 transgene;
does not reasonably provide enablement for:
A recombinant adeno-associated virus (rAAV) comprising a nucleic acid molecule encoding an RNA, wherein the RNA comprises:
a first sequence comprising
any alternatively regulated exon having a first portion of a start codon,
any or no upstream flanking intron, and
any downstream flanking intron, and
a second sequence comprising a transgene having any first exon that comprises a second portion of the start codon, wherein the transgene encodes any intracellular factor, wherein splicing of the RNA by the intracellular factor excludes the flanking intron thereby forming a complete start codon in the nucleic acid sequence encoding the transgene.
The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the invention commensurate in scope with these claims.
This rejection comprises three (3) separate issues: 1) the absence of an enabling disclosure for an rAAV comprising a nucleic acid encoding an RNA, wherein the RNA comprises a) a first sequence comprising any alternatively regulated exon other than an alternatively regulated exon selected from the group consisting of MBNL1 exon 1 and MBNL1 exon 5, any or no upstream flanking intron other than a natural or truncated intron selected from the group consisting of MBNL1 intron 1c and MBNL1 intron 5, and any downstream flanking intron other than a natural or truncated intron selected from the group consisting of MBNL1 intron 2 and MBNL1 intron 6, and b) a second sequence comprising a transgene having a first exon that comprises a second portion of the start codon, wherein the transgene encodes any intracellular factor other than a functional MBNL1 protein capable of binding to the MBNL1 protein binding sites and regulating alternative splicing of the alternatively regulated exon; 2) the absence of an enabling disclosure for an rAAV comprising a nucleic acid encoding an RNA comprising a regulated exon having a first portion of a start codon, a downstream flanking intron, and a first exon of a transgene comprising a second portion of the start codon such that splicing of the RNA excludes the flanking intron thereby forming a complete start codon in the nucleic acid sequence encoding the transgene; and 3) the absence of an enabling disclosure for splicing of the RNA by any intracellular factor other than regulation of splicing by MBNL1.
These issues were identified by the Office after analysis of the disclosure provided by the specification. The Office has analyzed the specification in direct accordance to the factors outlined in In re Wands, namely 1) the nature of the invention, 2) the state of the prior art, 3) the predictability of the art, 4) the amount of direction or guidance present, and 5) the presence or absence of working examples, and presented detailed scientific reasons supported by publications from the prior art for the finding of a lack of enablement for the scope of the instant methods. The Wands analysis and supporting specific evidence are presented below for each of the identified issues.
As a first issue (1), the specification does not provide an enabling disclosure for an rAAV comprising a nucleic acid encoding an RNA, wherein the RNA comprises a) a first sequence comprising any alternatively regulated exon other than an alternatively regulated exon selected from the group consisting of MBNL1 exon 1 and MBNL1 exon 5, any or no upstream flanking intron other than a natural or truncated intron selected from the group consisting of MBNL1 intron 1c and MBNL1 intron 5, and any downstream flanking intron other than a natural or truncated intron selected from the group consisting of MBNL1 intron 2 and MBNL1 intron 6, and b) a second sequence comprising a transgene having a first exon that comprises a second portion of the start codon, wherein the transgene encodes any intracellular factor other than a functional MBNL1 protein capable of binding to the MBNL1 protein binding sites and regulating alternative splicing of the alternatively regulated exon.
The broadest independent claim, claim 1, recites an rAAV comprising a nucleic acid encoding an RNA, wherein the RNA comprises an alternatively regulated exon, a downstream flanking intron, and a transgene, wherein the transgene encodes an intracellular factor, wherein splicing of the RNA to remove the flanking intron forms a start codon of the transgene coding sequence. Claims 2, 5-9, 13-16, 23, 27-28, and 30 depend on claim 1. Independent claim 37 recites a method of treating DM type 1 or DM type 2 by administering the rAAV of claim 1 to a subject, wherein the intracellular factor is MBNL1, the first truncated intron is MBNL1 intron 1c or MBNL1 intron 5, and the second intron is MBNL1 intron 2 or MBNL1 intron 6.
The specification discloses rAAV constructs comprising nucleic acids encoding RNAs comprising MBNL1 regulated exons 1 or 5 flanked by (truncated) introns 1c and 2 or 5 and 6, respectively, with exon and introns numbered as illustrated in instant Figure 1B [Example 1-4, 6-8, Figure 1B-C, 2, 3, 4, 5, 6, 7, 8, 9, 14B-C, 15, 16]. The specification discloses AR constructs comprising autoregulated MBNL1 exon 5 constructs as minigenes (e.g., miniAR5.1-5.4, Figure 2), within the coding sequence of MBNL1 (e.g., AR5.1-5.5, Example 2, 4, Figure 3), and also as a construct in which exon 5 has been placed upstream of the MBNL1 coding sequence and altered to end with a start codon ATG (e.g., repurposed AR5.5, Example 6, Figure 14B-C). The specification also discloses AR constructs comprising autoregulated MBNL1 exon 1 (e.g., AR1.1-AR1.5, Example 3, 4, Figure 6, 7, 9), and constructs comprising both autoregulated MBNL1 exon 1 and autoregulated MBNL1 exon 5 (e.g., combined AR1.2 + AR5.5, Example 4, Figure 9), wherein exon 1 comprises the start codon for the MBNL1 transgene.
Example 5 of the disclosure illustrates discusses proposed constructs for PABPN1, TDP43, Fus, and hnRNPA1 [Figure 10], with demonstrated splicing effects of autoregulated constructs for hnRNPA1 and TDP43 [Figure 11-12]. However, the hnRPPA1 and TDP43 constructs only include introns within the body of the hnRPPA1 or TDP43 coding sequence, and do not have regulated exon sequence upstream of the start codon of the transgene coding sequence. Figure 13B also presents a proposed gene therapy construct comprising an MBNL1-dependent regulated exon of ATP2A1, but this construct has the regulated exon within the ATP2A1 coding sequence and has no accompanying data using the construct.
Example 8 of the disclosure discusses an overall process for developing autoregulated AAV cargos, wherein MBNL1 autoregulated exons 1 and 5 are the examples [page 69].
The art at the time of filing teaches that a variety of mutations can affect splicing, including intronic and exonic mutations, that mRNA occurs in a highly cell type-specific manner, and that mRNA splicing should be analyzed on the native gene and in the native tissue, with the accompanying flanking introns [Riedmayr 2018, Bio-protocol, 8(5), 1-14, cited in a prior action, abstract, page 2 ¶ 1, page 3 ¶ 1-7, page 6 ¶ 14, page 12 ¶ 2]. Additionally, Stoilov teaches that alternative splicing is monitored for alternatively regulated exons along with nucleotides from each of the native flanking intronic sequences [Stoilov & Black, US20100233685A1, published 16 September 2010, ¶ 0100, 0131, 0150]. Additionally, Gates teaches that the upstream flanking intron to exon 5 (referred to in Gates as intron 4, and in the instant application as intron 5, see instant Figure 1) comprises MBNL1 binding sites which are required for MBNL1 autoregulated splicing [Gates et al. 2011, The Journal of Biological Chemistry, 286(39), 34224-34233, cited in a prior action, column 3 ¶ 2- column 4 ¶ 2, Figure 1]. Gates also teaches that the MBNL1 binding sites are sufficient for regulation by MBNL1 in an exon not normally regulated by MBNL1 [column 1 ¶ 4].
Further, Konieczny teaches that MBNL1 binding within exon 1 precludes exon 1 retention, thereby facilitating autoregulated splicing [Konieczny 2018, RNA Biology, 15(1), 1-8, IDS, abstract, column 3 ¶ 2, Figure 1-2]. Konieczny further teaches that MBNL1 binding to intron flanking exon 1 (e1) bridges them and initiates spliceosome-dependent formation of exon 1 circular RNA [column 9 ¶ 3]. Konieczny also teaches that the length of the intron preceding exon 1 also contributes to determining exon 1 retention [column 5 ¶ 2].
Neither the specification nor the art at the time of filing teaches that any alternatively regulated exon combined with any downstream flanking intron will allow for splicing by any intracellular factor that excludes the flanking intron to form a start codon of the transgene coding sequence. Thus, in view of the varied alternatively regulated exon and intron sequences, the criticality of sequences and length in both introns and exons for regulating alternative splicing, the requirement for the upstream flanking intron MBNL1 binding sites for MBNL1 exon 5 autoregulated splicing, and the requirement for intra-exon MBNL1 binding sites as well as intra-intronic MBNL1 binding sites for MBNL1-regulated splicing of MBNL1 exon 1, and the breadth of the claims, the skilled artisan would have considered combining any alternatively regulated exon with any downstream flanking intron with or without any upstream flanking intron to achieve alternatively regulated splicing as highly unpredictable. As such, it would have required undue experimentation to practice the scope of applicant’s invention as claimed.
As a second issue (2), the specification does not provide an enabling disclosure for an rAAV comprising a nucleic acid encoding an RNA comprising a regulated exon having a first portion of a start codon, a downstream flanking intron, and a first exon of a transgene comprising a second portion of the start codon such that splicing of the RNA excludes the flanking intron thereby forming a complete start codon in the nucleic acid sequence encoding the transgene.
The broadest independent claim, claim 1, recites an rAAV comprising a nucleic acid encoding an RNA comprising (a) a first sequence comprising an alternatively regulated exon having a first portion of a start codon and a downstream flanking intron, and (b) a second sequence comprising a transgene having a first exon that comprises a second portion of the start codon, wherein splicing of the RNA excludes the flanking intron thereby forming a complete start codon in the nucleic acid encoding the transgene. Claims 2, 5-9, 13-16, 23, 27-28, and 30 depend on claim 1. Independent claim 37 recites a method of treating DM type 1 or DM type 2 by administering the rAAV of claim 1 to a subject, wherein the intracellular factor is MBNL1, the first truncated intron is MBNL1 intron 1c or MBNL1 intron 5, and the second intron is MBNL1 intron 2 or MBNL1 intron 6.
The specification discloses:
“In some embodiments, a recombinant nucleic acid for which splicing is regulated is a synthetic construct configured to regulate expression of a protein by including a 5’ exon comprising an amino terminal amino acid encoding sequence (e.g., an ATG or part of the ATG)…, wherein the 5’ exon is separated from subsequent exon(s) by an intron for which splicing is regulated. In some embodiment, if the intron is spliced out of the RNA transcript, the recombinant 5’ exon is spliced in frame to the subsequent exon(s) and the resulting spliced transcript encodes a protein that is expressed. In some embodiments, if the intron is not spliced out of the RNA transcript, the recombinant 5’ exon is not spliced to the subsequent exon(s) and as a result a protein is not expressed from the transcript” [page 24 ¶ 1].
As such, the specification teaches a partial stop codon in a 5’ exon (e.g., the beginning A or AT). However, the specification does not teach a partial stop codon in a regulated exon. Additionally, the specification does not teach that the subsequent exon has a partial stop codon (e.g., the ending TG or G), such that splicing of a regulated exon which results in retention of the exon and removal of the intron downstream of the regulated exon thereby forms a complete start codon (e.g., ATG) when joined with the subsequent exon.
The drawings present various schematics for splicing [e.g., Figure 1, 8, 10], including a construct in which MBNL1 exon 5 comprises a 3’ terminal ATG start codon and the subsequent exon has its ATG removed from the natural location of a coding sequence [Figure 14B], but none teach a partial/split start codon which is spliced together by bringing together an alternatively regulated exon with a subsequent exon to form a complete start codon. None of the working examples discussed in the specification utilize a split start codon, and none of the data presented in the drawings are generated from a split start codon system. As such, none of the data in the drawings support the limitations of “(i) an alternatively regulated exon having a first portion of a start codon”, “a transgene having a first exon that comprises a second portion of the start codon”, and “forming a complete start codon in the nucleic acid sequence” as claimed.
The art at the time of filing teaches a viral vector comprising a nucleic acid molecule encoding an RNA [Stoilov 0106-0108, 0113-0114], wherein the RNA comprises (a) a first sequence comprising (i) and alternatively regulated exon having a first portion (e.g., A) of a start codon and (ii) a downstream flanking intron, and (b) a second sequence comprising a GFP transgene having a first exon that comprises a second portion of the start codon (e.g., TG), wherein the transgene encodes a protein (e.g., GFP), wherein splicing of the RNA excludes the flanking intron thereby forming a complete start codon in the nucleic acid sequence encoding the GFP transgene [0054, 0131, 0138-0139, 0147, Figure 1c]. Stoilov teaches that an mRNA encoding a GFP transgene is divided into a first exon ending in a “G” nucleotide, a second exon beginning at the “TG” nucleotides of the GFP start codon and comprises the remainder of the GFP sequence [0147]. Stoilov further teaches that inclusion of an alternative exon that has an “A” at its 3’ end produces a start codon and results in GFP expression, whereas GFP is not expressed when the alternative exon is spliced out [0147]. Therefore, Stoilov teaches that to achieve differential expression to monitor the alternative splicing, the exon upstream of the alternative exon should not end in an A so that it cannot reconstitute a start codon for the expression of the GFP transgene. Stoilov also teaches the inclusion of an upstream flanking intron adjacent to the alternatively regulated exon [Figure 1C].
Therefore, although the art teaches a split start codon which can form a complete start codon upon removal of a downstream flanking intron adjacent to the alternatively regulated exon, such that retention of the alternatively regulated exon is necessary for expression of a transgene coding sequence initiated by the formed start codon, the art also teaches that the utility of the system is for producing alternative expression of the transgene to reflect the alternatively regulated splicing, thereby requiring a lack of forming the start codon when either the downstream flanking intron is retained or when the alternatively regulated exon is not retained following splicing.
Neither the specification nor the art at the time of filing teaches to include a first portion of a start codon in an alternatively regulated exon wherein the upstream exon and flanking introns are any sequences such that a lack of splicing and/or lack of retention of the alternatively regulated exon could also form a complete start codon for expression of the transgene.
Thus, in view of the complete lack of teachings in the disclosure for an alternatively regulated exon comprising a first portion of a start codon paired with a downstream flanking intron and a second sequence comprising a transgene having a first exon comprising a second portion of the start codon, wherein splicing excludes the flanking intron to form a complete start codon in the nucleic acid encoding the transgene, the teachings in the art that a split start codon system as claimed requires a lack of a first portion of a start codon at the 3’ ends of both the exon immediately upstream of the alternatively regulated exon and the downstream flanking intron, and the breadth of the claims, the skilled artisan would have considered an rAAV comprising a nucleic acid encoding an RNA comprising (a) any first sequence comprising (i) any regulated exon comprising a first portion of a start codon and (ii) any downstream flanking intron, and (b) any second sequence comprising any transgene having any first exon that comprises a second portion of the start codon, wherein splicing of the RNA excludes the flanking intron thereby forming a complete start codon in the nucleic acid sequence encoding the transgene as highly unpredictable for producing alternative expression of the transgene dependent on the alternative splicing of the alternatively regulated exon. As such it would have required undue experimentation to practice the scope of applicant’s invention as claimed.
As a third issue (3), the specification does not provide an enabling disclosure for splicing of the RNA by any intracellular factor other than regulation of splicing by MBNL1.
The broadest independent claim, claim 1, recites an rAAV comprising a nucleic acid encoding an RNA comprising (a) a first sequence comprising an alternatively regulated exon having a first portion of a start codon and a downstream flanking intron, and (b) a second sequence comprising a transgene encoding an intracellular factor having a first exon that comprises a second portion of the start codon, wherein splicing of the RNA by the intracellular factor excludes the flanking intron thereby forming a complete start codon in the nucleic acid encoding the transgene. Claims 2, 5-9, 13-16, 23, 27-28, and 30 depend on claim 1. Independent claim 37 recites a method of treating DM type 1 or DM type 2 by administering the rAAV of claim 1 to a subject, wherein the intracellular factor is MBNL1, the first truncated intron is MBNL1 intron 1c or MBNL1 intron 5, and the second intron is MBNL1 intron 2 or MBNL1 intron 6.
The instant disclosure teaches the expression of transgenes which are intracellular factors which regulate the splicing of their own pre-mRNA constructs by binding to the pre-mRNA and affecting the inclusion or exclusion of alternatively spliced regions, including MBNL1, MBNL2, PABPN1, TDP43, Fus, and hnRNPA1 [page 2 ¶ 1, 4, Figure 1, 10]. The instant disclosure also teaches the expression of MBNL1, hnRNPA1, or TDP43 from constructs comprising alternatively regulated exons, wherein only MBNL1 is expressed from a coding sequence in which at least a portion of the start codon was comprised in an alternatively regulated exon [Figures 2-6, 8-9, 11-12, 14-16]. However, the instant disclosure does not teach any intracellular factors which perform the splicing.
The art at the time of filing teaches that intracellular factors which regulate alternative splicing by binding to pre-mRNAs to affect the inclusion or exclusion of alternatively spliced exons or introns, such as MBNL1, can hinder formation of a functional spliceosome, cause the spliceosome to omit a sequence, or initiate spliceosome-dependent formation of circular RNA [Konieczny column 3 ¶ 2, column 5 ¶ 3, column 9 ¶ 3], thereby teaching that the spliceosome, and not MBNL1 or similar alternative splicing regulating factors, is the factor performing splicing.
Neither the specification nor the art at the time of filing teaches that any intracellular factor performs splicing of RNA. Additionally, neither the specification nor the art at the time of filing teaches that the intracellular factors disclosed in the specification and taught to be encoded by the transgenes of the instantly claimed rAAVs, such as MBNL1, are capable of splicing an RNA.
Thus, in view of the well-known role of the spliceosome in performing splicing reactions, the teachings in the art and the instant disclosure that the disclosed factors are splicing regulating proteins and not splicing factors which splice RNA, and the breadth of the claims, the ordinarily skilled artisan at the time of filing the instant application would have considered splicing of the RNA by any intracellular factor as highly unpredictable. As such it would have required undue experimentation to practice the scope of applicant’s invention as claimed.
Claim Rejections - 35 USC § 102
The rejection of amended claim 65 under 35 U.S.C. 102(a)(1) as being anticipated by Thornton et al. (US20100190689A1, published 29 July 2010), is maintained. 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.
Claim 65 was rejected for being anticipated by Thornton for teaching a recombinant adeno-associated virus (rAAV) comprising a nucleic acid (e.g., SEQ ID NO: 1) having the sequence of SEQ ID NO: 5 of the instant application [0055, 0113, 0122-0125, 0158, 0252, 0257, SEQ ID NO: 1]. SEQ ID NO: 1 of Thornton is 4207 nucleic acids long and comprises the full 54 nucleotides of SEQ ID NO: 5 of the instant application with 100% sequence identity. Applicant amended claim 65 to no longer recite SEQ ID NO: 5. However, SEQ ID NO: 1 of Thornton also comprises the full 186 nucleotides of instant SEQ ID NO: 1, still recited in amended claim 65.
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As such, by teaching all of the limitations of amended claim 65 as recited, Thornton still anticipates the invention as claimed, and Applicant’s amendments do not overcome a finding of anticipation under 35 USC 102(a)(1).
Applicant argues that the removal of SEQ ID NO: 5 obviates the rejection. However, this is not agreed. As discussed above, Thornton still anticipates the claim by teaching an AAV vector comprising the sequence according to instant SEQ ID NO: 1.
Therefore, Applicant’s arguments do not overcome a finding of anticipation under 35 USC 102(a)(1), and the rejection is maintained.
Claim Rejections - 35 USC § 103
The rejection of amended, previously presented, and cancelled claims 1-9, 12-23, 27-28, 30, 37, 43, and 65 under 35 U.S.C. 103 as being unpatentable over Gates et al. (2011, The Journal of Biological Chemistry, 286(39), 34224-34233) in view of Riedmayr et al. (2018, bio-protocol, 8(5), 1-14); Kino et al. (2015, Human Molecular Genetics, 24(3), 740-756, IDS); Kanadia et al. (2006, Proceedings of the National Academy of Science, 103(31), 11748-11753, IDS); Cheng et al. (2014, Blood, 124(4), 598-610); and Wang et al. (2018, Cell Reports, 22, 2294-2306), is withdrawn over amended, previously presented and cancelled claims 1-9, 12-23, 27-28, 30, 37, and 65 and maintained over amended claim 43 in view of Applicant’s claims which now recite an alternatively regulated exon having a first portion of a start codon and a first exon that comprises a second portion of the start codon, wherein splicing forms a complete start codon in the nucleic acid sequence encoding the transgene, and the removal of SEQ ID NO: 5 from claim 65. 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 amended claim 43 to recite “wherein the at least one truncated intron retains a sequence motif for binding to the MBNL protein. Gates was cited for teaching a nucleic acid (e.g., mini-gene) encoding an RNA for studying the process of alternative splicing within includes or excludes the MBNL1 exon 5 from the mature MBNL1 transcript, wherein the RNA comprises a) a first intron (e.g., MBNL1 intron 4), wherein the first intron is regulated by an intracellular factor (e.g., the autoregulatory RNA-binding protein MBNL1), b) an exon (e.g., MBNL1 exon 5), and c) a second intron (e.g., MBNL1 intron 5) [title, abstract, column 2 ¶ 2, column 4 ¶ 2, column 8 ¶ 4, Figure 1C]. As such, Gates teaches an intron which retains a sequence motif for binding to the MBNL protein.
Additionally, Riedmayr was cited for teaching a protocol for the design and cloning of minigenes into recombinant adeno-associated virus (rAAV) vectors for gene delivery and investigation of mRNA splicing in a native context [abstract], thereby teaching to retains a sequence motif for binding to the splicing regulator proteins. Riedmayr also teaches that most native genes exceed the limited packaging capacity of AAVs (approx. 4.7 kb), and so designing minigenes lacking large intronic parts, which usually do not contain information required for correct mRNA splicing, allows for the packaging on the gene into rAAV vectors [page 2 ¶ 3]. Also, Riedmayr teaches that for genes which do not contain large exon numbers or sizes, shortening of the intronic sections also allows for introducing the entire protein coding region into the rAAV vector-based minigenes [page 2 ¶ 3]. Riedmayr further teaches that large introns can be shortened to enable minigene-based analysis of mutation on mRNA splicing, such as for the minigene they created consisting of three native exons including two shortened flanking introns [page 6 ¶ 2, Figure 2]. Therefore, an ordinarily skilled artisan at the time of filing the instant application would have been motivated to shorten/truncate the natural intron(s) in a minigene (or other nucleic acid comprised in an rAAV construct) to retain native sequence motifs for binding to splicing regulatory factors while also allowing the minigene/ nucleic acid to fit within the packaging capacity of the rAAV vector and/or allowing for the inclusion of the entire protein coding region for the gene of interest (such as a gene with alternatively spliced exon(s)).
Therefore, Applicant’s amendment to claim 43 does not overcome a finding of obviousness under 35 USC 103.
Applicant argues that the combination of the cited references does not disclose or suggest the subject matter of the amended claims, specifically:
that Gates does not disclose a rAAV construct, let along an rAAV construct comprising a nucleic acid molecule encoding an RNA, wherein the RNA comprises (a) a first sequence comprising (i) an alternatively regulated exon having a first portion of a start codon and (ii) a downstream flanking intron, and (b) a second sequence comprising a transgene having a first exon that comprises a second portion of the start codon, wherein the transgene encodes an intracellular factor, wherein splicing of the RNA by the intracellular factor excludes the flanking intron thereby forming a complete start codon in the nucleic acid sequence encoding the transgene; and
Riedmayr merely focuses on generating PRPH2 minigenes by truncating PRPH2 introns, and that controlling splicing outcomes with rAAV-encoded minigenes is not predictable for all genes.
However, this is not agreed.
In response to applicant’s arguments against the references individually, 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). One cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). 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 arguing 1, note that claim 43 does not depend upon claim 1, and as such does not recite nor incorporate the recited limitations. As discussed above, Gates in view of Riedmayr, Kino, Kanadia, Cheng, and Wang teach all of the limitations of amended claim 43.
Regarding argument 2, that Riedmayr teaches that controlling splicing outcomes with rAAV-encoded minigenes is not predictable for all genes by teaching that “not all genes are suitable for an in vivo splice analysis using rAAV-mediated gene expression” [page 11] and “shortening of the native introns should be done only if necessary” [page 12].
Riedmayr teaches that “Not all genes are suitable for an in vivo splice analysis using rAAV-mediated gene expression. If the size of the expression cassette containing a promoter, the minigene, and a polyA signal exceeds 4.7 kb, lentiviral derived vector systems might be used.” [page 11]. Accordingly, Riedmayr is teaching that the suitability is dependent on the size if the minigene. Gates teaches a minigene without truncating the introns which is 2492 nucleotides long [Figure 1]. Gates also teaches a plasmid encoding amino acids 1-260 (e.g., 780 nucleotides) which is functional to block inclusion of exon 5 by binding to MBNL1 binding sites in intron 4 [column 4 ¶ 4, column 6 ¶ 4, Figure 1]. Gates also teaches that the MBNL1 binding sites in intron 4 are within about 200 nucleotides upstream of exon 4 [column 10 ¶ 5- column 12 ¶ 1, Figure 1]. Therefore, even without truncation of the introns 4 and 5, the total minigene + MBNL1 coding sequence amounts to 3272 nt, leaving an additional 1.5 kb for promoter and polyA sequences, which is more than enough for a CMV enhancer/promoter (at 584 nt) and a BGH poly A (at 225 nt). As such, Riedmayr does not teach away from incorporating MBNL1 minigenes into rAAV vectors. Additionally, given that the teaching of Riedmayr of suitability is merely a reference to the packaging capacity of rAAV, Riedmayr is not teaching unpredictability of minigene incorporation but mere nonuniversality.
Note that, as discussed above, Riedmayr also teaches the motivation for shortening of introns, indicating that the shortening can be necessary to fit the intron and desired exons into the packaging capacity of the rAAV [page 2 ¶ 7, page 6 ¶ 14].
Accordingly, Applicant’s arguments do not overcome a finding of obviousness over Gates in view of Riedmayr, Kino, Kanadia, Cheng, and Wang for claim 43 under 35 USC 103, and the rejection is maintained.
**The following new rejection is necessitated by Applicant’s amendments to the claims.
Amended and previously presented claims 1-2, 5-9, 13-16, 23, 27-28, 30, 37, 43, and 65 are newly rejected under 35 U.S.C. 103 as being unpatentable over Stoilov & Black [US20100233685A1, published 16 September 2010]; in view of Riedmayr et al. [2018, Bio-protocol, 8(5), 1-14, cited in a prior action]; Gates et al. [2011, The Journal of Biological Chemistry, 286(39), 34224-34233, cited in a prior action]; Kanadia et al. [2006, Proceedings of the National Academy of Science, 103(31), 11748-11753, IDS, cited in a prior action]; Cheng et al. [2014, Blood, 124(4), 598-610, cited in a prior action]; Wang et al. [2018, Cell Reports, 22, 2294-2306, cited in a prior action]; and Thornton et al. [US20100190689A1, published 29 July 2010, cited in a prior action].
Regarding claim 1, Stoilov teaches a viral vector comprising a nucleic acid molecule encoding an RNA [0106-0108, 0113-0114], wherein the RNA comprises (a) a first sequence comprising (i) and alternatively regulated exon having a first portion (e.g., A) of a start codon and (ii) a downstream flanking intron, and (b) a second sequence comprising a transgene having a first exon that comprises a second portion of the start codon (e.g., TG), wherein the transgene encodes a protein (e.g., GFP), wherein splicing of the RNA excludes the flanking intron thereby forming a complete start codon in the nucleic acid sequence encoding the transgene [0054, 0131, 0138-0139, 0147, Figure 1c].
Regarding the elected species of flanking intron, Stoilov teaches that the flanking intron is a truncated version of a naturally occurring intron (as elected), and that the constructs are useful for detecting alternative splicing of exons in a gene of interest, such as genes associated with disease states [0100, 0150].
Stoilov does not teach that the viral vector is an rAAV, that the transgene is an intracellular factor (e.g., the elected MBNL1 protein), wherein splicing of the RNA by the intracellular factor excludes the flanking intron, nor that the exon is an exon 5 of MBNL1 (as elected).
However, Riedmayr teaches a protocol for the design and cloning of minigenes into recombinant adeno-associated virus (rAAV) vectors for gene delivery and investigation of mRNA splicing in a native context [abstract]. Riedmayr also teaches that rAAVs allow stable and specific ectopic expression of minigenes, that rAAVs are capable of transducing a variety of different cell types in vivo, and that the design, cloning, and production and purification of rAAV vectors can be completed in a few weeks without elaborate technical equipment [page 2 ¶ 2]. Therefore, an ordinarily skilled artisan at the time of filing the instant application would have been motivation to package a nucleic acid encoding an alternative splicing construct into an rAAV vector for stable and specific ectopic expression of the alternative splicing construct, including for transduction of a variety of different cell types in vivo.
Additionally, Gates teaches a nucleic acid molecule (e.g., mini-gene) encoding an RNA for studying the process of alternative splicing which includes or excludes the MBNL1 exon 5 from the mature MBNL1 transcript, wherein the RNA comprises (a) a first sequence comprising (i) an alternatively regulated exon (e.g., MBNL1 exon 5, as elected) and (ii) a downstream flanking intron (e.g., MBNL1 intron 5), and (b) a second sequence comprising a transgene having a first exon, wherein the transgene encodes a coding sequence of an intracellular factor (e.g., MBNL1), wherein splicing of the RNA by the intracellular factor (e.g., MBNL1) excludes the flanking intron [title, abstract, column 2 ¶ 2, column 4 ¶ 2, column 8 ¶ 4, Figure 1C].
Gates also teaches the co-transfection of the mini-gene nucleic acid (comprising MBNL1 exon 4, intron 4, exon 5, intron 5, and exon 6) and a nucleic acid encoding the intracellular factor MBNL1 into HeLa cells to overexpress the MBNL1 protein and test the effect therefrom on the inclusion of the MBNL1 exon 5 in minigene transcripts [column 6 ¶ 4, column 8 ¶ 4], thereby teaching the delivery of both the MBNL1 minigene (expressing a portion of the MBNL1 protein) and a nucleic acid encoding the MBNL1 protein. Additionally, as discussed above, Riedmayr teaches that rAAVs allow stable and specific ectopic expression, that rAAVs are capable of transducing a variety of different cell types in vivo, and that the design, cloning, and production and purification of rAAV vectors can be completed in a few weeks without elaborate technical equipment [page 2 ¶ 2]. Riedmayr also teaches that for genes which do not contain large exon numbers or sizes, the entire protein coding region can be included in the rAAV vector-based minigenes [page 2 ¶ 3].
Gates further teaches that MBNL1 are involved in causing myotonic dystrophy (DM) types 1 and 2, in that MBNL1 sequestration by expanded CUG or CCUG repeats sequester MBNL1 protein into nuclear foci, leading to a loss of active MBNL1 protein, thereby leading to missplicing of developmentally regulated events [column 2 ¶ 2]. Gates further teaches that Exon 5 of the MBNL1 pre-mRNA are themselves mis-spliced in DM due to autoregulated splicing of the MBNL1 pre-mRNA by MBNL1 protein, and that inclusion of exon 5 causes MBNL1 to localize primarily to the nucleus, whereas exclusion of exon 5 results in MBNL1 proteins which localize to both the cytoplasm and nucleus [column 2 ¶ 3- column 3 ¶ 1, Figure 1].
As such, an ordinarily skilled artisan at the time of filing the instant application would have been motivated to use an MBNL1 exon 5, which truncated flanking introns 4 and 5, as the regulated exon of the alternative splicing construct, and further to include a sequence encoding the MBNL1 protein in the nucleic acid of the rAAV encoding an alternative splicing construct to assess the effects of overexpression of MBNL1 protein on the inclusion of the alternatively spliced exon 5.
Regarding claim 2, Stoilov teaches wherein the first sequence/sequencing comprising the alternatively regulated exon further comprises an upstream flanking intron [0100]. Additionally, Gates teaches wherein the sequence comprising the MBNL1 exon 5 further comprises an upstream flanking intron which is an intron 4 from gene MBNL1 (as elected) [Figure 1]. Gates also teaches that sequences in the MBNL1 intron 4 bind to MBNL1 protein to promote inclusion of the MBNL1 exon 5, thereby regulating the alternative splicing of the MBNL1 exon 5 [Figure 1]. Therefore, an ordinarily skilled artisan would have been motivated to include a sequence of MBNL1 intron 4 as an upstream flanking intron along with the alternatively regulated MBNL1 exon 5 to allow MBNL1 autoregulatory alternative splicing of exon 5 in its native context.
Regarding claims 5- 9, as discussed above, Stoilov, Gates, and Riedmayr provide the teachings and motivation for encoding an MBNL1 protein, which is an intracellular factor which autoregulates the alternative splicing of the MBNL1 exon 5 by binding to intron 4 of the MBNL1 RNA to detect alternative splicing of exons in a gene of interest (e.g., MBNL1 exon 5), such as genes associated with disease states (e.g., DM1 and DM2) [Gates column 2 ¶ 3- column 3 ¶ 1, Figure 1, Stoilov 0100, 0150].
Regarding claim 13, as discussed above, Stoilov teaches that the downstream flanking intron is a truncated version of a naturally occurring intron (as elected) [0100, 0150].
Regarding claim 14, as discussed above, Stoilov, Gates, and Riedmayr provide the teachings and motivation for the upstream flaking intron which is an intron 4 (exon 4-flanking and exon 5-flanking intron) from the MBNL1 gene.
Regarding claims 15, Stoilov teaches that the upstream and downstream flanking introns comprise 5’ splice donor sites and/or 3’ splice acceptor sites [0006, 0012, 0016, 0044, 0050, 0109, 0147, claim 1-2].
Regarding claim 16, as discussed above, Stoilov, Gates, and Riedmayr provide the teachings and motivation for claims 1 and 15. Stoilov does not teach wherein the 5’ splice donor site is a GU or an AU and/or the 3’ splice acceptor site is an AG or an AC. However, Gates teaches that MBNL1 introns 4 and 5 comprise 3’ splice sites (3’-ss) and 5’ splice site (5’-ss) which contribute to the alternative inclusion of exon 5, wherein the intron 4 3’-ss is an AG and the intron 4 5’-ss is a GU [column 8 ¶ 5, column 10 ¶ 2-4, Figure 1, 2].
Regarding claim 23, as discussed above, Gates teaches that the MBNL1 exon 5 is a naturally occurring exon [abstract, column 2 ¶ 3, Figure 1].
Regarding claim 27, as discussed above, Stoilov, Gates, and Riedmayr provide the teachings and motivation for claim 1, wherein the alternatively regulated exon is MBNL1 exon 5.
Regarding claim 28 and 30, as discussed above, Stoilov, Gates, and Riedmayr provide the teachings and motivation for claim 1, wherein the alternatively regulated exon is MBNL1 exon 5. Gates also teaches wherein both the upstream flanking intron and the downstream flanking intron are from MBNL1 [Figure 1].
Regarding claim 37, Stoilov, Gates, and Riedmayr provide the teachings and motivation for claim 1. As discussed above, Gates teaches that MBNL proteins associate with expanded CUG repeats located in the 3’ UTR region of the DMPK gene that have been shown to act as toxic RNA and contribute to causing myotonic dystrophy (DM) type 1 by sequestering MBNL proteins into nuclear foci, leading to loss of active MBNL1 protein [column 2 ¶ 2]. Gates further teaches that expanded CCUG repeats within the first intron of ZNF9 also sequester MBNL proteins, which is thought to be at least partially responsible for causing DM type 2 [column 2 ¶ 2]. The sequestration of MBNL proteins leads to missplicing of developmentally regulated events, which have been linked directly to symptoms in DM types 1 and 2, such as myotonia and heart defects [column 2 ¶ 2]. Gates also teaches that the inclusion of exon 5 causes MBNL1 to be localized primarily in the nucleus, whereas isoforms of MBNL1 lacking exon 5 are found in both the nucleus and cytoplasm [column 2 ¶ 3].
However, Stoilov, Gates, and Riedmayr do not teach a method of treating a disease or condition in a subject comprising administering the rAAV as described above to the subject.
Kanadia teaches that myotonic dystrophy (DM) is a multisystemic degenerative disease which includes skeletal muscle myotonia, weakness/wasting, heart conduction defects, particulate subcapsular cataracts, and insulin insensitivity [column 1 ¶ 1]. Kanadia also teaches that the genetic basis of DM is such that DM type 1 is caused by the expansion of a (CTG)n repeat in the 3’UTR of the DMPK gene and DM type 2 results from a (CCTG)n expansion in the first intron of ZNF9, such that the C/CTG repeats sequester MBNL1 to effectively reduce available levels of MBNL1 within the cell, resulting in missplicing of MBNL1 target genes [column 1 ¶ 2, column 2 ¶ 1, 3, column 3 ¶ 1]. Kanadia further teaches that overexpression of MBN1 in vivo mediated by transduction of skeletal muscle with an rAAV vector rescues disease-associated muscle hyperexcitability, or myotonia, in the HSALR poly(CUG) mouse model for DM [abstract]. Therefore, an ordinarily skilled artisan at the time of filing the instant application would have been motivated to deliver an rAAV comprising a nucleic acid encoding MBNL1 to a subject having DM to vector rescues disease-associated muscle hyperexcitability, or myotonia.
Cheng teaches that MBNL proteins are predominantly expressed in skeletal muscle, neuronal tissues, thymus, liver, and kidney and are important for terminal differentiation of myocyte and neurons [column 2 ¶ 1]. Cheng also teaches that MBNL1 transcripts themselves undergo extensive alternative splicing, generating a variety of protein isoforms, such that the inclusion of the highly conserved exon 5 during differentiation of heart and muscle tissues is important for nuclear localization and splicing activity of the MBNL1 protein [column 2 ¶ 1]. Cheng teaches that perturbation of MBNL1 is associated with myotonic dystrophy (DM), resulting in cataract formation, abnormal muscle relaxation, heart and nerve dysfunction, and other pathologies [column 2 ¶ 1]. Cheng also teaches that the inclusion of this exon was reported to enhance nuclear localization of the splicing activity of MBNL1 [column 7 ¶ 2]. Cheng teaches that different MBNL1 isoforms have different subcellular localizations and that the inclusion but not the exclusion isoform is mainly localized in the nucleus [column 8 ¶ 4]. Cheng teaches that the MBNL1 exclusion isoform displayed both nuclear and cytoplasmic localizations with an enrichment in the cytoplasm, whereas the MBNL1 inclusion isoform was mainly localized in the nucleus, consistent with previous observations [column 8 ¶ 4, Figure 3].
Further, Wang teaches that MBNL1 cytoplasmic, but not nuclear, isoform promotes neurite morphogenesis and reverses the morphological defects caused by expanded CUG RNA [abstract]. Wang also teaches that expanded CUG RNA induced the deubiquitination of cytoplasmic MBNL1, which resulted in nuclear translocation and morphological impairment that could be ameliorated by inhibiting K63-linked ubiquitin chain degradation [abstract]. Therefore, given the teachings of Cheng and Wang, it is apparent that the proper balance of MBNL1 splicing and cytoplasmic/nuclear localization is important for proper functioning of cells. As such, an imbalance between the two in either direction can be problematic for cells.
Accordingly, given the teachings of Gates, discussed above, of the role of introns in the autoregulation of MBNL1 alternative splicing, the teachings of Kanadia that expression of MTNB1 alleviates symptoms of DM1, the teachings of Cheng of the importance of the exon 5 inclusion isoform for regulating splicing, and the teachings of Wang that the cytoplasmic MBNL1 (which Cheng teaches is the exon 5 excluding isoform) is important for proper neuronal cell function such that cytoplasmic localization of MBNL1 ameliorates morphological impairment induced by expanded CUG RNA, an ordinarily skilled artisan at the time of filing the instant application would have been motivated, in administering MBNL1 for the treatment of DM, to utilize a nucleic acid encoding MBNL1 which comprises MBNL1 introns, at least the introns flanking exon 5, to ensure proper production of both the exon 5 inclusion (nuclear) and exon 5 exclusion (cytoplasmic and nuclear) isoforms to avoid the problems that arise from a deficiency of either pool.
Note that the instant specification teaches that the introns flanking MBNL1 exon 5 are introns 5 and 6 [Figure 1B]. As such, the teachings of Gates, Kanadia, Cheng, and Wang of the introns flanking exon 5, referred to above as introns 4 and 5, are the introns recited in instant claim 37 as introns 5 and 6.
Regarding claim 43, as discussed above, Stoilov, Gates, and Riedmayr provide the teachings and motivation for the rAAV of claim 1, wherein the rAAV comprises a recombinant MBNL1 gene comprising an MBNL1 protein coding sequence and at least one truncated intron of the MBNL1 gene, wherein the at least one truncated intron retains a sequence motif for binding to the MBNL1 protein (e.g., truncated intron 4), and wherein splicing of the truncated intron is regulated by the MBNL1 protein. Specifically, Riedmayr teaches to include the full coding sequence of gene in a minigene construct comprising truncated introns to allow the minigene to fit into the packaging size of the rAAV. Accordingly, as discussed above, an ordinarily skilled artisan would have been motivated to include the coding sequence for the MBNL1 protein in an rAAV comprising the MBNL1 minigene to assess the effects of overexpression of the MBNL1 protein on splicing regulation of the MBNL1 transcript.
Regarding claim 65, as discussed above, Stoilov, Gates, and Riedmayr provide the teachings and motivation for the rAAV of claim 1, including the inclusion of the MBNL1 protein coding sequence in the rAAV. Additionally, Thornton teaches a nucleotide sequence encoding an MBNL1 protein (SEQ ID NO: 1) which comprises the full-length sequence of instant SEQ ID NO: 1 with 100% identity [0158, 0202, 0206].
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Given the teaching of Thornton that the sequence of SEQ ID NO: 1 is a sequence of an MBNL1 coding sequence with encodes an MBNL1 protein, and ordinarily skilled artisan at the time of filing would have been motivated to use the sequence of Thornton SEQ ID NO: 1 as the sequence encoding MBNL1 in the rAAV.
Therefore, given the motivation taught by Riedmayr to package a nucleic acid encoding a minigene into an rAAV vector for stable and specific ectopic expression of the minigenes, including for transduction of a variety of different cell types in vivo; the motivation taught by Gates and Riedmayr to use an MBNL1 exon 5, which truncated flanking introns 4 and 5, as the regulated exon of the alternative splicing construct, and further to include a sequence encoding the MBNL1 protein in the nucleic acid of the rAAV encoding an alternative splicing construct to assess the effects of overexpression of MBNL1 protein on the inclusion of the alternatively spliced exon 5; the further motivation taught by Gates and Riedmayr to include a sequence of MBNL1 intron 4 as an upstream flanking intron along with the alternatively regulated MBNL1 exon 5 to allow MBNL1 autoregulatory alternative splicing of exon 5 in its native context; the motivation taught by Kanadia to deliver an rAAV comprising a nucleic acid encoding MBNL1 to a subject having DM to vector rescues disease-associated muscle hyperexcitability, or myotonia; the motivation taught by Gates, Kanadia, Cheng, and Wang to utilize a nucleic acid encoding MBNL1 in administering MBNL1 for the treatment of DM which comprises MBNL1 introns, at least the introns flanking exon 5, to ensure proper production of both the exon 5 inclusion (nuclear) and exon 5 exclusion (cytoplasmic and nuclear) isoforms to avoid the problems that arise from a deficiency of either pool; the motivation taught by Riedmayr to include the coding sequence for the MBNL1 protein in an rAAV comprising the MBNL1 minigene to assess the effects of overexpression of the MBNL1 protein on splicing regulation of the MBNL1 transcript; and the motivation taught by Thornton to use the sequence of Thornton SEQ ID NO: 1 as the sequence encoding MBNL1 in the rAAV; it would have been prima facie obvious to an ordinarily skilled artisan at the time of filing the instant application to modify the vector comprising the alternative splicing construct of Stoilov such that the alternatively regulated exon is MBNL1 exon 5 and the upstream and downstream flanking introns are truncated MBNL1 introns 4 and 5, which retain their MBNL1 binding sites, to include the coding sequence for MBNL1 protein as the downstream coding sequence, and to administer the rAAV to a subject to treat DM type 1 or type 2 with a reasonable expectation of success.
Insofar as applicant’s arguments apply to this new grounds of rejection, Applicant argues that the combination of the cited references does not disclose or suggest the subject matter of the amended claims, specifically:
that Gates does not disclose a rAAV construct, let along an rAAV construct comprising a nucleic acid molecule encoding an RNA, wherein the RNA comprises (a) a first sequence comprising (i) an alternatively regulated exon having a first portion of a start codon and (ii) a downstream flanking intron, and (b) a second sequence comprising a transgene having a first exon that comprises a second portion of the start codon, wherein the transgene encodes an intracellular factor, wherein splicing of the RNA by the intracellular factor excludes the flanking intron thereby forming a complete start codon in the nucleic acid sequence encoding the transgene; and
Riedmayr merely focuses on generating PRPH2 minigenes by truncating PRPH2 introns, and that controlling splicing outcomes with rAAV-encoded minigenes is not predictable for all genes.
However, this is not agreed.
In response to applicant’s arguments against the references individually, 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). One cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). 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, as discussed above, the combination of Stoilov, Gates, and Riedmayr provide the teachings and motivations for the rAAV of claim 1.
Regarding argument 2, that Riedmayr teaches that controlling splicing outcomes with rAAV-encoded minigenes is not predictable for all genes by teaching that “not all genes are suitable for an in vivo splice analysis using rAAV-mediated gene expression” [page 11] and “shortening of the native introns should be done only if necessary” [page 12].
Riedmayr teaches that “Not all genes are suitable for an in vivo splice analysis using rAAV-mediated gene expression. If the size of the expression cassette containing a promoter, the minigene, and a polyA signal exceeds 4.7 kb, lentiviral derived vector systems might be used.” [page 11]. Accordingly, Riedmayr is teaching that the suitability is dependent on the size if the minigene. Gates teaches a minigene without truncating the introns which is 2492 nucleotides long [Figure 1]. Gates also teaches a plasmid encoding amino acids 1-260 (e.g., 780 nucleotides) which is functional to block inclusion of exon 5 by binding to MBNL1 binding sites in intron 4 [column 4 ¶ 4, column 6 ¶ 4, Figure 1]. Gates also teaches that the MBNL1 binding sites in intron 4 are within about 200 nucleotides upstream of exon 4 [column 10 ¶ 5- column 12 ¶ 1, Figure 1]. Therefore, even without truncation of the introns 4 and 5, the total minigene + MBNL1 coding sequence amounts to 3272 nt, leaving an additional 1.5 kb for promoter and polyA sequences, which is more than enough for a CMV enhancer/promoter (at 584 nt) and a BGH poly A (at 225 nt). As such, Riedmayr does not teach away from incorporating MBNL1 minigenes into rAAV vectors. Additionally, given that the teaching of Riedmayr of suitability is merely a reference to the packaging capacity of rAAV, Riedmayr is not teaching unpredictability of minigene incorporation but mere nonuniversality.
Note that, as discussed above, Riedmayr also teaches the motivation for shortening of introns, indicating that the shortening can be necessary to fit the intron and desired exons into the packaging capacity of the rAAV [page 2 ¶ 7, page 6 ¶ 14].
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
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