DETAILED ACTION
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Applicant’s Request for Continued Examination, Amendment and Arguments/Remarks received on 08 June 2026 have been entered. Claims 1-2, 5-9, 13-16, 23, 25-28, 30, 37, 43, and 65-75 were previously pending in the application. Claims 2, 5-9, 14, 27, 28, 30, 43, and 66-72 have been newly cancelled by Applicant. Claims 1, 13, 15-16, 23, 25-26, 37, and 65 are currently pending in the application. Claims 1, 37, and 65 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,
Downstream flanking introns: a. a truncated version of a naturally occurring intron,
Upstream flanking introns: jj. From gene MBNL1,
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.
Regarding the election of species 1)-3), note that independent claim 1 now recites either the elected species or a species which is narrower than, and falls within, the elected species. However, independent claim 65 has not been amended to recite the specific species which are now recited within independent claim 1, and as such the election of species remains as elected for claim 65.
Claim 25-26 remain withdrawn from consideration as being directed to nonelected species.
Claims 1, 13, 15-16, 23, 37, 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.
Claim Objections
Amended claim 13 is newly objected to because of the following informalities: amended claim 13 now recites, “wherein the upstream and/or downstream flanking intron are truncated”, wherein “intron are” is grammatically mismatched between a singular “intron” and plural “are”. Appropriate correction is required.
Claim Rejections - 35 USC § 112(b)
The rejection of amended, previously presented, and cancelled claims 1-2, 5-9, 13-16, 23, 27-28, 30, 37, and 65 under 35 U.S.C. 112(b) as failing to particularly point out and distinctly claim the subject matter which the inventor(s) regards as the invention for multiple issues of indefiniteness is withdrawn in view of Applicant’s amendments to the claims.
Amended and previously presented claims 1, 13, 15-16, 23, and 37 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 15-16, 23, and 37 are included in this rejection due to their dependence on and/or encompassing of independent claim 1.
Amended independent claim 1 newly recites, “the MBNL1 transgene” in lines 21-22. There is insufficient antecedent basis for this limitation in the claim. Independent claim 1 has prior recitation of “a transgene” in line 12 and “the transgene encodes a functional MBNL1 protein” in line 14. However, claim 1 has no prior recitation of “an MBNL1 transgene”. Additionally, recitation of “the nucleic acid encoding the MBNL1 transgene” is further indefinite because transgenes are nucleic acid sequences which encode RNA and/or protein sequences, but which themselves are not encoded. As such, the metes and bounds of the claim cannot be determined.
Amended claim 13 newly recites the limitation "the upstream and/or downstream MBNL1 flanking intron" in lines 1-2. There is insufficient antecedent basis for this limitation in the claim. Claim 13 is dependent on claim 1. Claim 1 recites “an upstream flanking MBNL1 intron” in line 6, “a downstream flanking MBNL1 intron” in line 9, “the upstream flanking MBNL1 intron” in lines 17, 18-19, and “the downstream flanking MBNL1 intron” in lines 19-20, but has not prior recitation of any upstream or downstream “MBNL1 flanking” introns. As such, the metes and bounds of the claim cannot be determined.
Claim Rejections - 35 USC § 112(a)- New Matter
The rejection of amended, previously presented, and cancelled claims 1-2, 5-9, 13-16, 23, 27-28, 30, are 37 under 35 U.S.C. 112(a) for reciting new matter in the recitation of ““(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; is withdrawn in view of Applicant’s amendments to the claims such that amended independent claim 1 no longer recites, “having a first portion of”, “that comprises a second portion of the start codon”, nor “forming a complete start codon in”.
Claim Rejections - 35 USC § 112(a)- Scope of Enablement
The rejection of amended, previously presented, and cancelled claims 1-2, 5-9, 13-16, 23, 27-28, 30, and 37 under 35 U.S.C. 112(a) for 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;
is withdrawn in view of Applicant’s amendments to the claims such that the claims are now in alignment with the identified enabled scope.
Amended and previously presented claims 1, 13, 15-16, 23, 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; and
a downstream flanking MBNL1 intron,
wherein the downstream flanking MBNL1 intron 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;
wherein splicing of the RNA is regulated by the MBNL1 protein binding to the MBNL1 protein binding sites within the upstream flanking MBNL1 intron, wherein binding of the MBNL1 protein to the MBNL1 protein binding sites within the upstream flanking MBNL1 intron promotes exclusion of the alternatively regulated exon of MBNL1 within a spliced RNA molecule; 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 downstream flanking MBNL1 intron, thereby attaching the start codon in-frame to the transgene encoding the functional MBNL1 protein;
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:
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; and
a downstream flanking MBNL1 intron,
wherein the downstream flanking MBNL1 intron 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;
wherein splicing of the RNA is regulated by the MBNL1 protein binding to the MBNL1 protein binding sites within the upstream flanking MBNL1 intron in the absence of promoting exon exclusion; 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 downstream flanking MBNL1 intron, thereby attaching the start codon in-frame to the nucleic acid sequence encoding the MBNL1 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 practice the invention commensurate in scope with these claims.
This rejection addressed the issue of a lack of an enabling disclosure for MBNL1-regulated splicing of the RNA by the binding of MBNL1 protein to the MBNL1 protein binding sites within the upstream flanking MBNL1 intron to effect an outcome other than inclusion of the alternatively regulated exon within the spliced RNA molecule.
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.
The specification does not provide an enabling disclosure for MBNL1-regulated splicing of the RNA by the binding of MBNL1 protein to the MBNL1 protein binding sites within the upstream flanking MBNL1 intron to effect an outcome other than inclusion of the alternatively regulated exon within the spliced RNA molecule.
Independent claim 1 recites an rAAV comprising a nucleic acid comprising an alternatively regulated exon; 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; and a downstream flanking MBNL1 intron, wherein the downstream flanking MBNL1 intron is a natural or truncated intron selected from the group consisting of MBNL1 intron 2 and MBNL1 intron 6; and a transgene encoding a functional MBNL1 protein capable of binding to the MBNL1 protein binding sites, 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 downstream flanking MBNL1 intron, thereby attaching the start codon in-frame to the nucleic acid sequence encoding the MBNL1 transgene. Claims 13, 15-16, 23, and 37 depend on and/or encompass claim 1.
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.
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], wherein binding of MBNL1 protein to the MBNL1 binding sites results in exclusion of exon 5 from the spliced transcript [column 3 ¶ 1, column 4 ¶ 2; Figure 1].
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, cited in a prior action, abstract, column 3 ¶ 2, Figure 1-2]. Konieczny further teaches that MBNL1 binding to introns flanking exon 1 (e1) bridges them and initiates spliceosome-dependent formation of exon 1 circular RNA, thereby excluding exon 1 from the spliced transcript [column 9 ¶ 3].
Neither the specification nor the art at the time of filing teaches that MBNL1 can regulate alternative splicing of an exon by binding to an upstream intron to facilitate alternative splicing other than exon inclusion. Gates teaches that “when YGCY motifs are located upstream of the exon, MBNL1 binding generally leads to exon exclusion; when YGCY motifs are located downstream of an exon, MBNL1 binding generally leads to exon inclusion” [column 3 ¶ 2]. Thus, in view of the known mechanisms for MBNL1 regulation of alternatively regulated exons, wherein binding of the MBNL1 to intronic sequences upstream or downstream of the exon facilitates differential consequences to the alternatively regulated exon, the lack of teaching in the specification for MBNL1 binding in the upstream flanking intron to promote an outcome other than exon exclusion, and the breadth of the claims, the skilled artisan would have considered MBNL1-regulated splicing, wherein MBNL1 protein binds to MBNL1 protein binding sites within an upstream flanking intron to effect an outcome other than inclusion of the alternatively regulated exon within the spliced RNA molecule 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 independent 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: 1 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 186 nucleotides of SEQ ID NO: 1 of the instant application with 100% sequence identity. Applicant amended claim 65 to no longer recite SEQ ID NO: 1. However, SEQ ID NO: 1 of Thornton also comprises the full 171 nucleotides of instant SEQ ID NO: 18, still recited in amended claim 65, as shown in the alignment below.
Alignment of instant SEQ ID NO: 18 (Qy) and Thornton SEQ ID NO: 1 (Db):
PNG
media_image1.png
331
642
media_image1.png
Greyscale
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: 1 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: 18.
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 cancelled claim 43 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 in view of Applicant’s cancellation of claim 43.
The rejection of amended, previously presented, and cancelled claims 1-2, 5-9, 13-16, 23, 27-28, 30, 37, 43, and 65 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]; is withdrawn in view of Applicant’s claims which now recite “an alternatively regulated exon of MBNL1 comprising a start codon”.
Amended and previously presented claims 1, 13, 15-16, 23, 37, and 65 are newly rejected under 35 U.S.C. 103 as being unpatentable over Gates et al. [2011, The Journal of Biological Chemistry, 286(39), 34224-34233, cited in a prior action]; in view of 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]; Konieczny et al. [2017, Nucleic Acids Research, 45(4), 1760-1775, IDS]; Fu et al. [2007, Nature Biotechnology, 25(3), 353-357]; Hayes et al. [2002, Cancer Gene Therapy, 9, 133-141]; Riedmayr et al. [2018, Bio-protocol, 8(5), 1-14, cited in a prior action];and Thornton et al. [US20100190689A1, published 29 July 2010, cited in a prior action].
Regarding independent claim 1, Gates teaches a nucleic acid molecule (e.g., mini-gene) encoding an RNA which includes or excludes the alternatively regulated MBNL1 exon 5 from the mature MBNL1 transcript, wherein the mini-gene RNA comprises:
a first sequence comprising:
an alternatively regulated exon 5 of MBNL1,
an upstream flanking natural MBNL1 intron 4 comprising MBNL1 protein binding sites, and
a downstream flanking natural MBNL1 intron 5 [Figure 1]; and
a second RNA which comprises:
a second sequence comprising a transgene having a first exon,
wherein the transgene encodes a functional MBNL1 protein capable of binding to the MBNL1 protein binding sites;
wherein splicing of the RNA is regulated by the MBNL1 protein binding to the MBNL1 protein binding sites within the upstream flanking intron 4, 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 downstream flanking MBNL1 intron [title, abstract, column 2 ¶ 2, column 4 ¶ 2, column 6 ¶ 4, column 8 ¶ 4, Figure 1].
Note that Gates refers to the introns which naturally flank exon 5 as introns 4 and 5 [Figure 1], which correspond to introns 5 and 6, respectively, of the instant application [see instant Figure 1B].
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]. Gates teaches that the MBNL1 binding sites in intron 4 are required for MBNL1 autoregulated splicing [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].
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.
Gates does not teach the following limitations:
the nucleic acid encoding the RNA comprising the first sequence and the second sequence is comprised within an rAAV;
that the alternatively regulated exon of MBNL1 comprises a start codon;
that the first exon of the transgene lacks a start codon; nor
that splicing of the RNA to exclude the downstream flanking MBNL1 intron attaches the start codon in-frame to the nucleic acid sequence encoding the MBNL1 transgene.
Regarding limitation A), as discussed above, Gates teaches the co-delivery and co-expression of both the MBNL1 minigene (expressing a portion of the MBNL1 protein) and a nucleic acid encoding the MBNL1 protein.
Gates further teaches that MBNL1 protein is 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]. Gates further teaches that in DM, the lack of properly localized MBNL1 leads to the missplicing of many pre-mRNAs, including the aberrant inclusion of exon 5 within the MBNL1 pre-mRNA [abstract].
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].
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 to promote native autoregulated distribution of MBNL1 within the cell and the accompanying native functions.
Additionally, 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 type 1 or type 2to rescue disease-associated muscle hyperexcitability or myotonia, thereby treating the DM in the subject.
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 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 an rAAV encoding 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.
Regarding limitations B)-D), Gates teaches that the protein levels of MBNL1 play a significant role in the regulation of MBNL1 exon 5 [column 8 ¶ 4].
Further, Konieczny teaches that, in addition to autoregulating alternative splicing to control alternative subcellular localization, MBNL1 autoregulates its own protein levels through alternative splicing which results in altered translational activity and protein stability, such that upon expression of MBNL1, there is a shift towards isoforms which result in downregulation of endogenous MBNL1 levels [pg. 1761 col. 1 ¶ 2, pg. 1768 col. 1 ¶ 1, col. 2 ¶ 2-3, pg. 1769 col. 1 ¶ 1]. Konieczny also teaches that the knowledge concerning MBNL1 expression level fluctuations might offer a possible therapeutic strategy against DM aiming at the autoregulatory overexpression of MBNL1 [pg. 1770 col. 2. ¶ 2].
Therefore, in expressing an MBNL1 protein for gene therapy, an ordinarily skilled artisan at the time of filing the instant application would be motivated to include an autoregulated exon which controls the translation and/or stability of the MBNL1 protein to recapitulate the autoregulation of protein levels to avoid either too much or too little MBNL1 protein from a gene therapy vector administering MBNL1 for the treatment of DM.
Fu teaches an alternative splicing construct for producing sex-lethal phenotypes in insects, wherein an upstream exon comprises a start codon and a downstream sequence comprises a transgene comprising a first exon which lacks a start codon, such that alternative splicing alternatively produces an mRNA wherein the start codon is fused in frame with the downstream transgene to allow expression of the transgene or an mRNA which comprises an alternatively regulated exon comprising a premature stop codon which prevents expression of the transgene [Figure 1, 2]. Fu additionally teaches that their results demonstrate the potential of controlling gene expression through alternative splicing and that their approach may have wide applicability for regulating gene expression in other organisms [pg. 356 col. 1 ¶ 2, abstract]. Therefore, an ordinarily skilled artisan would have been motivated to employ alternative splicing as a mechanism for controlling expression of a transgene, wherein an upstream exon comprises a start codon and is alternatively spliced to a sequence comprising a transgene having a first exon lacking a start codon, thereby allowing the expression of the transgene only under conditions which promote the inclusion of the upstream exon and exclusion of the downstream intron.
Hayes teaches the use of alternative splicing as a means of regulating the expression of therapeutic genes, wherein a series of episomal plasmid-based “splice-activated gene expression” (pSAGE) vectors were generated which contain minigene cassettes composed of various combinations of the three alternatively spliced exons present in the differentially expressed adhesion protein CD44R1 (v8, v9, and v10) with or without their corresponding intronic sequences, positioned in-frame between the CD44 leader sequence and a “leaderless” human ALP cDNA, wherein the expression and enzymatic activity of ALP are dependent up on the accurate removal of intronic sequences from the pre-mRNA transcripts [abstract]. Hayes further teaches that the constructs exhibited cell-type specific expression of ALP only in CD44R1+ cells [abstract]. Hayes also teaches that whereas splice-activated gene expression can clearly be used to target expression of essentially any therapeutic gene, and that there are numerous other genes that are differentially spliced such that incorporation of such alternatively spliced exons into minigene constructs may further expand the potential usefulness of alternative splicing as s novel means of targeting gene expression in vivo [pg. 140 col. 1 ¶ 2]. Therefore, given the teachings of Hayes to target cancer cells by using a tumor-cell specific alternatively spliced minigene for controlling the expression of a therapeutic gene, an ordinarily skilled artisan at the time of filing the instant application would be motivated to use an alternatively spliced minigene construct to control the expression of a therapeutic gene for administering to a human subject to treat a human disease.
Regarding claim 13, Gates, Kanadia, Cheng, Wang, Konieczny, Fu, and Hayes teach the limitations of claim 1. Gates, Kanadia, Cheng, Wang, Konieczny, and Fu teach do not teach wherein the upstream and/or downstream MBNL1 flanking introns are truncated.
Additionally, Riedmayr teaches a protocol for the design and cloning of minigenes into recombinant adeno-associated virus (rAAV) vectors for gene delivery and mRNA splicing in a native context [abstract]. Reidmayr also teaches that most native genes exceed the limited packaging capacity of AAVs, and so they designed minigenes lacking large intronic parts which usually do not contain information required for correct mRNA splicing [pg. 2 ¶ 3]. Reidmayr also 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 [pg. 2 ¶ 3]. Therefore, an ordinarily skilled artisan at the time of filing the instant application would have been motivated to truncate an intronic region to allow the gene comprising the intron to be packaged in an rAAV vector.
Regarding claims 15-16, 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, Gates teaches wherein the alternatively regulated exon 5 of MBNL1 is a naturally occurring exon [Figure 1].
Regarding claim 37, as discussed above, Gates, Kanadia, Cheng, Wang, Konieczny, Fu, and Hayes teach the limitations of claim 1, including teachings of the role of MBNL1 in causing the adverse phenotypes associated with DM, teachings to administer MBNL1 to patients with DM to treat DM type 1 and/or 2, and teachings of the importance of autoregulated alternative splicing for controlling the appropriate activity and levels of MBNL1 protein within a cell. Therefore, Gates, Kanadia, Cheng, Wang, Konieczny, Fu, And Hayes provide the motivation for treating DM type 1 or type 2 in a subject comprising administering the rAAV of claim 1 to the subject.
Regarding claim 65, as discussed above, Gates, Kanadia, Cheng, Wang, Konieczny, Fu, and Hayes teach the limitations 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: 18 with 100% identity [0158, 0202, 0206].
Alignment of instant SEQ ID NO: 18 (Qy) and Thornton SEQ ID NO: 1 (Db):
PNG
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331
642
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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.
Given the motivation taught by Gates to include a sequence of MBNL1 intron 4 (i.e., instant MBNL1 intron 5) as an upstream flanking intron along with the alternatively regulated MBNL1 exon 5 (i.e., instant MBNL1 intron 6) 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 type 1 or type 2to rescue disease-associated muscle hyperexcitability or myotonia, thereby treating the DM in the subject; the teachings of Cheng and Wang that the proper balance of MBNL1 splicing and cytoplasmic/nuclear localization is important for proper functioning of cells such that an imbalance between the two in either direction can be problematic for cells; the motivation taught by Gates, Kanadia, Cheng, and Wang 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; the motivation taught by Konieczny to include an autoregulated exon which controls the translation and/or stability of the MBNL1 protein to recapitulate the autoregulation of protein levels to avoid either too much or too little MBNL1 protein expression from a gene therapy vector administering MBNL1 for the treatment of DM; the motivation taught by Fu to employ alternative splicing as a mechanism for controlling expression of a transgene, wherein an upstream exon comprises a start codon and is alternatively spliced to a sequence comprising a transgene having a first exon lacking a start codon, thereby allowing the expression of the transgene only under conditions which promote the inclusion of the upstream exon and exclusion of the downstream intron; the motivation taught by Hayes to use an alternatively spliced minigene construct to control the expression of a therapeutic gene for administering to a human subject to treat a human disease; the motivation taught by Riedmayr to truncate an intronic region to allow the gene comprising the intron to be packaged in an rAAV vector; and the motivation taught by Riedmayr 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 RNA of Gates to combine the minigene construct with the MBNL1 transgene construct within an rAAV vector to administer to a subject having DM as a gene therapy to treat the DM within the subject, such that the construct comprises an autoregulated exon comprising a start codon and the transgene comprises a first exon lacking a start codon, wherein in the absence of MBNL1 binding to the upstream flanking intron, splicing removes the downstream flanking intron and the ATG comprised within the alternatively regulated exon is attached in-frame to the nucleic acid sequence encoding the MBNL1 transgene, thereby providing for autoregulated expression of the MBNL1 protein, with a reasonable expectation of success.
Insofar as applicant’s arguments apply to this new grounds of rejection, Applicant argues that:
Riedmayr teaches away from the predictability of the claimed approach by expressly reporting that large intronic sequences “usually do not contain information required for correct mRNA splicing”, that controlling splicing outcomes with rAAV-encoded minigenes is “not [predictable] for all genes”, that shortening of native introns “should be done only if necessary”, and that avoiding undesirable splicing outcomes- including disease-associated alternative splicing- requires “exact knowledge of molecular mechanisms underpinning the disease-causing mutations” and that splicing outcomes are highly gene-dependent [Riedmayr pages 1, 11-12];
Gates does not disclose rAAV of any kind, let alone the specific rAAV architecture recited in claim 1;
a rejection that strings together without a particularized showing that each missing element is taught or suggested by a specific reference, and without a clear rationale for why a skilled artisan would have been motivated to combine all of them in the precise manner claimed does not satisfy the requirements of KSR Int’l Co. v. Teleflex Inc., 550 U.S. 398 (2007), and its progeny; and
the rejection should be withdrawn because it is the product of impermissible hindsight reconstruction in that the claimed invention requires the convergence of a highly specific set of elements: an rAAV delivery vehicle, a transgene encoding MBNL1, exons and introns specifically derived from MBNL1, an autoregulatory architecture in which flanking intronic sequences containing MBNL1 protein binding sites govern splicing outcomes, and an autoregulatory feedback loop in which the expressed MBNL1 protein controls its own production.
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 Applicant’s argument 1), note that Riedmayr was cited for teaching the motivation to truncate a natural gene when necessary to fit into the packaging capacity of an rAAV vector. Applicant’s claim that Riedmayr teaches away from the instant invention by teaching that intronic sequences “usually do not contain information required for correct mRNA splicing” is a gross mischaracterization of the teaching of Riedmayr. Riedmayr actually recites, “we designed PRPH2 minigenes lacking large intronic parts, which usually do not contain information required for correct mRNA splicing” [page 2 ¶ 3]. As such, the “large intronic parts” removed usually do not contain the required information for correct mRNA splicing and the intronic parts remaining are those with the information required for correct mRNA splicing. As such, Riedmayr teaches that large parts can be removed and paired down to smaller sequences to fit within the packaging capacity of the rAAV vector. Regarding Riedmayr’s teaching that avoiding undesirable outcomes requires exact knowledge of the molecular mechanisms involved with disease-associated alternative splicing, and that splicing outcomes is gene dependent, is likewise not teaching away from the predictability of the instant invention. As discussed above, Gates, Kanadia, Cheng, Wang, and Konieczny teach extensive details regarding the role of MBNL1 in autoregulation of splicing as well as regulation of splicing at other sites involved in DM types 1 and 2. As such, sufficient understanding of the molecular mechanisms underpinning the alternative splicing of MBNL1 and other genes affected in DM is known in the prior art to arrive at the instant invention as claimed.
Regarding Applicant’s argument 2), Gates was not relied on for teaching to package the construct into an rAAV. Kanadia was cited for teaching the motivation to deliver an rAAV comprising a nucleic acid encoding an MBNL1 therapeutic transgene to a subject having DM to rescue disease-associated muscle hyperexcitability or myotonia. Additionally, Gates was cited for teaching an MBNL1 minigene and MBNL1 transgene having most of the claimed architecture. As discussed above, Konieczny, Fu, and Hayes provide additional teachings and motivations to arrive at the instantly claimed architecture.
Regarding Applicant’s argument 3), the number of references needed to teach and motivate all the limitations of the claimed invention is not an indication of non-obviousness.
MPEP 2143.01 states: “Obviousness can 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. In re Kahn, 441 F.3d 977, 986, 78 USPQ2d 1329, 1335 (Fed. Cir. 2006) (discussing rationale underlying the motivation-suggestion-teaching test as a guard against using hindsight in an obviousness analysis). Axonics, Inc. v. Medtronic, Inc., 73 F.4th 950, 957-58, 2023 USPQ2d 795 (Fed. Cir. 2023) (the court found an erroneous framing of the motivation inquiry led to an incorrect conclusion of nonobviousness). A "motivation to combine may be found explicitly or implicitly in market forces; design incentives; the ‘interrelated teachings of multiple patents’; ‘any need or problem known in the field of endeavor at the time of invention and addressed by the patent’; and the background knowledge, creativity, and common sense of the person of ordinary skill." Zup v. Nash Mfg., 896 F.3d 1365, 1371, 127 USPQ2d 1423, 1427 (Fed. Cir. 2018) (quoting Plantronics, Inc. v. Aliph, Inc., 724 F.3d 1343, 1354 [107 USPQ2d 1706] (Fed. Cir. 2013) (citing Perfect Web Techs., Inc. v. InfoUSA, Inc., 587 F.3d 1324, 1328 [92 USPQ2d 1849] (Fed. Cir. 2009) (quoting KSR, 550 U.S. at 418-21)).” The MPEP does not put a limit on the number of references used to establish a prima facie case of obviousness.
As discussed in detail above, each modification to the base reference of Gates is clearly articulated with teachings and motivations taught by the prior art references, including teachings and motivations to alter the constructs of Gates such that:
the nucleic acid encoding the RNA comprising the first sequence and the second sequence is comprised within an rAAV;
that the alternatively regulated exon of MBNL1 comprises a start codon;
that the first exon of the transgene lacks a start codon; and
that splicing of the RNA to exclude the downstream flanking MBNL1 intron attaches the start codon in-frame to the nucleic acid sequence encoding the MBNL1 transgene.
For example, Fu and Hayes teach to use alternative splicing as a mechanism for controlling gene expression of a transgene administered to a subject, including therapeutic transgenes for the treatment of human diseases exhibiting altered alternative splicing, wherein splicing combines an upstream exon comprising a start codon with a downstream transgene sequence comprising a first exon and lacking a start codon to facilitate expression of the transgene only under the specific conditions which promote that particular splicing outcome.
Regarding Applicant’s argument 4), as discussed above, it must be recognized that any judgment on obviousness is in a sense necessarily a reconstruction based upon hindsight reasoning. But so long as it takes into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made, and does not include knowledge gleaned only from the applicant's disclosure, such a reconstruction is proper. See In re McLaughlin, 443 F.2d 1392, 170 USPQ 209 (CCPA 1971). The rejection of record, as presented above, relies upon publicly available references which provide the teachings and motivations for the ordinarily skilled artisan to modify the base reference of Gates to arrive at all of the features of the instant application as claimed with a reasonable expectation of success. As such, the rejection does not rely upon impermissible hindsight reasoning.
Therefore, Applicant’s arguments do not overcome a finding of obviousness under 35 U.S.C. 103.
Conclusion
No claim is allowed.
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DR. KATIE L. PENNINGTON
Examiner
Art Unit 1634
/KATIE L PENNINGTON/Examiner, Art Unit 1634
Dr. A.M.S. Wehbé
/ANNE MARIE S WEHBE/Primary Examiner, Art Unit 1634
Prosecution Insights