DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
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. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 05/14/2026 has been entered.
Applicant’s amendment filed on 05/14/2026 was entered for the purpose of a compact prosecution even though the claim amendment does not comply with the requirements of 37 CFR 1.121(c) because the status identifier for claim 20 is incorrect. It should be “Currently amended” because claim 20 was not withdrawn from examination.
Amended claims 1-2, 4, 6, 8-9, 11-12, 17-22 and new claims 23-30 are pending in the present application.
Applicant elected previously without traverse of Group I, which is drawn to a nucleic acid molecule encoding a circRNA that is covalently closed comprising the elements a)-d) as recited independent claim 1, an AAV capsid or particle, and a composition comprising the same nucleic acid molecule.
Applicant also elected previously without traverse the following species: (a) HIPK3 intronic elements of SEQ ID Nos. 15 and 16; and (b) encephalomyocarditis virus IRES element.
Claims 9, 11-12 and 17-18 were also withdrawn previously from further consideration because they are directed to a non-elected invention. Claims 19 and 21-22 were also withdrawn previously from further consideration because they are drawn to non-elected species.
Accordingly, amended claims 1-2, 4, 6, 8, 20 and 23-30 are examined on the merits herein with the above elected species.
Terminal Disclaimer
The terminal disclaimer filed on 05/14/2026 disclaiming the terminal portion of any patent granted on this application which would extend beyond the expiration date of US Patent No. 11,718,862 and any patent granted on Application No. 18/334,530 has been reviewed and is accepted. The terminal disclaimer has been recorded.
Response to Amendment
1. All nonstatutory double patenting rejections over claims 1-25 of U.S. Patent No. 11,718,862 were withdrawn in light of the Terminal Disclaimer filed on 05/14/2026.
2. All provisional nonstatutory double patenting rejections as being unpatentable over claims 19-43 of copending Application No. 18/334,530 (reference application) were withdrawn in light of the Terminal Disclaimer filed on 05/14/2026.
3. The provisional nonstatutory double patenting rejection as being unpatentable over claims 20-33 of copending Application No. 17/796,874 (reference application) in view of Sena-Esteves et al (WO 2016/172155; IDS), Wang et al (RNA 21:172-179, 2015; IDS) and Liang et al (Genes & Development 28:2233-2247, 2014; IDS) with evidence from pIRES2-EGFP sequence from lifescience market (1 page, 2023) was withdrawn upon further consideration; and particular the copending Application No. 17/796,874 has a filing date of April 01, 2021, which is later than the filing date of the present application (November 7, 2018).
Claim Rejections - 35 USC § 112 (Lack of Written Description)
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.
Amended claims 1-2, 4, 6, 8, 20 and 23-30 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
MPEP 2163 - 35 U.S.C. 112(a) and the first paragraph of pre-AIA 35 U.S.C. 112 require that the “specification shall contain a written description of the invention ....” This requirement is separate and distinct from the enablement requirement. Ariad Pharm., Inc. v. Eli Lilly & Co., 598 F.3d 1336, 1340, 94 USPQ2d 1161, 1167 (Fed. Cir. 2010) (en banc). Vas-Cath Inc. v. Mahurkar, 19USPQ2d 1111 (Fed. Cir. 1991), clearly states that “applicant must convey with reasonable clarity to those skilled in the art that, as of the filing date sought, he or she was in possession of the invention. The invention is, for purposes of the ‘written description’ inquiry, whatever is now claimed.” Vas-Cath Inc. v. Mahurkar, 19USPQ2d at 1117. The specification does not “clearly allow persons of ordinary skill in the art to recognize that [he or she] invented what is claimed. ”Vas-Cath Inc. v. Mahurkar, 19USPQ2d at 1116.
The instant claims encompass an adeno-associated virus (AAV) genome, comprising: a nucleic caid molecule encoding a circular RNA, wherein the nucleic acid molecule comprises from 5’ to 3’: a) a promoter; b) a first intronic element having any structure; c) a gene of interest; and d) a second intronic element having any structure; wherein the first intronic element and the second intronic element are shorter than their corresponding wild-type intronic elements; wherein the AAV genome is packaged in an AAV9 capsid or any derivative thereof; and wherein the first intronic element and the second intronic element facilitate expression of the circular RNA following transcription of the AAV genome in a cell or tissue of a subject; the same AAV genome, wherein the first intronic element or the second intronic element comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:13-24 and 29-32 that encompass the pairing of any first intronic element (SEQ ID NOs: 13, 15, 17, 19, 21, 23, 29, and 31) with any second intronic element (SEQ ID NOs: 14, 16, 18, 20, 22, 24, 30, and 32) as long as the first and second intronic elements are selected from the recited group of SEQ ID NOs. (claim 2); an AAV capsid or particle comprising the same AAV genome (claim 6); and a composition comprising the same AAV genome and a pharmaceutically acceptable carrier (claim 8).
The instant specification discloses an AAV genome comprising a nucleic acid construct containing an exemplary split GFP gene (a gene of interest) cassette flanked by first and second intronic elements derived from the human ZKSCAN1 or HIPK3 genes, wherein the GFP gene fragments (FP and G gene fragments) are separated by an IRES such that upon RNA circularization, full length GFP is expressed; donor and acceptor splice sites are represented by grey triangles and the dotted lines indicate the back-splice pattern; a promoter (e.g., CMV promoter) in the 5’ untranslated region and outside of the intronic elements that flank the gene of interest; and an exemplary SV40 polyadenylation sequence in the 3’ UTR and outside of the intronic elements that flank the gene of interest as schematically depicted in Fig. 1B that is reproduced below. Additionally, the as-filed specification also discloses the use of specific pairs of left and right intronic elements that flank a gene of interest in the 5’ to 3’ direction (e.g., SEQ ID Nos. 15-16 pair, SEQ ID Nos. 13-14 pair), wherein the left intronic element comprises an acceptor splice site while the right intronic element comprises a donor splice site that can be backspliced by a cellular splicing machinery to yield a circular RNA that is covalently closed (see at least Summary of the Invention; Fig. 1B and Examples 1-2).
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Apart from the above disclosed AAV genome having specific elements that are arranged in specific 5’ to 3’ orientation with respect to each other, the instant specification fails to provide sufficient description for any other AAV genome comprising a nucleic acid molecule with any first intronic element and any second intronic element of any structure, including those that do not necessarily possess a donor splice site and/or an acceptor splice site, that facilitate expression of a circular RNA following transcription of the AAV genome in a cell or tissue of a subject as encompassed broadly by the instant claims. Moreover, there is also no evidence of record indicating or remotely suggesting at least that the first intronic element of SEQ ID NO: 15 (elected HIPK3 left intronic element) is capable of backsplicing with the second intronic element of SEQ ID NO: 20 (ZKSCAN1 right intronic element), SEQ ID NO: 22 (Laccase 2 right intronic element), or SEQ ID NO: 24 (EPHB4 right intronic element) to generate a circular RNA following transcription of the AAV genome in a cell or tissue of a subject. Accordingly, as an example which additional or critical elements that the first intronic element of SEQ ID NO: 15 needs to possess in order for it to pair and/or backsplice with any one of SEQ ID NOs: 20, 22 and 24 as a second intronic element for the generation of a circular RNA following transcription of the AAV genome in a cell or tissue of a subject? Furthermore, the instant specification also fails to provide sufficient written description for any derivative of an AAV9 capsid to package the AAV genome of the present application for expression of a circular RNA in a cell or tissue of a subject. For example, at least which specific modifications (substitution(s), insertion(s), deletion(s), or a combination thereof) in the AAV9 capsid should be made such that the AAV9 capsid is still functional for packaging the AAV genome of the present application for expression of a circular RNA in a cell or tissue of a subject, let alone a derivation of an AAV9 capsid that may not share any common core structure of an AAV9 capsid?
Since the prior art before the effective filing date of the present application (11/07/2017) did not provide any guidance regarding the issues discussed above as evidenced at least by the teachings of Chang et al (US 2019/0345503 with an effective filing date of 06/20/2016; IDS), Liang et al (Genes & Development 28:2233-2247, 2014; IDS), Wang et al (RNA 21:172-179, 2015; IDS), Sena-Esteves et al (WO 2016/172155; IDS) and Kruse (WO 2014/186334; IDS); it is incumbent upon the present application to do so. The instant specification also fails to provide at least a sufficient number of a representative number of species for a broad genus of an AAV genome comprising a nucleic acid molecule having the recited elements (a)-(d) as claimed broadly.
The claimed invention as a whole is not adequately described if the claims require essential or critical elements which are not adequately described in the specification and which are not conventional in the art as of Applicants’ filing date. Possession may be shown by actual reduction to practice, clear depiction of the invention in a detailed drawing, or by describing the invention with sufficient relevant identifying characteristics such that a person skilled in the art would recognize that the inventor had possession of the claimed invention. Pfaff v. Wells Electronics, Inc., 48 USPQ2d 1641, 1646 (1998). The skilled artisan cannot envision the detailed structure at least of a representative number of species for a broad genus of an AAV genome comprising a nucleic acid molecule having the recited elements (a)-(d) as claimed broadly, and therefore conception is not achieved until reduction to practice has occurred, regardless of the complexity or simplicity of the method. Adequate written description requires more than a mere statement that it is part of the invention and reference to a method of isolating it. See Fiers v. Revel, 25 USPQ2d 1601, 1606 (Fed. Cir. 1993) and Amgen Inc. v. Chugai Pharmaceutical Co. Ltd., 18 USPQ2d 1016 (Fed. Cir. 1991). One cannot describe what one has not conceived. See Fiddes v. Baird, 30 USPQ2d 1481, 1483.
Applicant is reminded that Vas-Cath makes clear that the written description provision of 35 U.S.C. §112 is severable from its enablement provision (see page 1115).
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claim 6 is 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.
It is unclear what is encompassed by the limitation “An AAV capsid or particle comprising the AAV genome of claim 1”. This is because the AAV genome of claim 1 already recites the limitation “wherein the AAV genome is packaged in an AAV9 capsid or derivative thereof”. Thus, what exactly does Applicant intend to claim in claim 6? Clarification is requested because the metes and bounds of the claim are not clearly determined.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Amended claims 1-2, 4, 6, 8, 20 and 23-30 are rejected under 35 U.S.C. 103 as being unpatentable over Chang et al (US 2019/0345503 with an effective filing date of 06/20/2016; IDS) in view of Liang et al (Genes & Development 28:2233-2247, 2014; IDS), Sena-Esteves et al (WO 2016/172155; IDS), Wang et al (RNA 21:172-179, 2015; IDS) and Zincarelli et al (Molecular Therapy 16:1073-1080, 2008; IDS).
The instant claims are directed to an AAV genome, comprising: a nucleic acid molecule encoding a circular RNA, wherein the nucleic acid molecule comprises from 5’ to 3’ the elements a)-d) recited in independent claim 1, wherein the first and the second intronic element are shorter than their corresponding elements; wherein the AAV genome is packaged in an AAV9 capsid or derivative thereof; and wherein the first intronic element and the second intronic element facilitate expression of the circRNA following transcription of the AAV genome in a cell or a tissue of a subject; an AAV capsid or particle and a composition comprising the same AAV genome. It is noted that the term “rAAV genome” in this specification refers to an AAV genome (e.g., vDNA) that comprises one or more heterologous nucleic acid sequences; and rAAV vectors generally require only the terminal repeat(s) in cis to generate virus and all other viral sequences are dispensible and may be supplied in trans (page 12, lines 31-34). Additionally, the specification explicitly teaches that the term “vector” may be used to refer to the vector genome/vDNA alone (page 12, lines 26-27).
With respect to the elected species, Chang et al already taught a recombinant nucleic acid encoding an immunogenic circular target RNA encoding an immunogenic polypeptide derived from a bacterium, a virus or a parasite, wherein the recombinant nucleic acid comprises in a 5’ to 3’ order: (i) a 3’ portion of an exogenous intron comprising a 3’ splice site (an acceptor site), (ii) a nucleic acid sequence encoding the target RNA, and (iii) a 5’ portion of an exogenous intron comprising a 5’ splice site (a donor site), wherein the produced target RNA transcript is circularized by backsplicing or splicing of the exogenous introns, the recombinant nucleic acid comprises a viral vector such as an adenovirus, a retrovirus, and adeno-associated virus and others, and the recombinant nucleic acid further comprises a nucleic acid sequence encoding an IRES (e.g., IRES derived from encephalomyocarditis virus (EMCV) UTR) operably linked to the nucleic acid encoding the immunogenic polypeptide (Abstract; Summary; particularly paragraphs [005]-[0008], [0018], [0021]-[0022], [0050], [0096], Z[0099]-[0101], [0106], [0116]; and Example 1). Chang et al also taught a recombinant nucleic acid encoding a non-immunogenic circular target RNA encoding a therapeutic polypeptide (e.g., an enzyme, hormone, neurotransmitter, cytokine and others), wherein the recombinant nucleic acid comprises in a 5’ to 3’ order: (i) a 3’ portion of an endogenous intron comprising a 3’ splice site (an acceptor site), (ii) a nucleic acid sequence encoding the target RNA, and (iii) a 5’ portion of an endogenous intron comprising a 5’ splice site (a donor site), wherein the produced target RNA transcript is circularized by backsplicing or splicing of the endogenous introns, the recombinant nucleic acid comprises a viral vector such as an adenovirus, a retrovirus, and adeno-associated virus and others, and the recombinant nucleic acid further comprises a nucleic acid sequence encoding an IRES (e.g., IRES derived from encephalomyocarditis virus (EMCV) UTR) operably linked to the nucleic acid encoding the therapeutic polypeptide (paragraphs [0026]-[0027], [0051], [0104]-[0105], [0106], [0116]; and Example 1). Chang et al also taught that the circular RNA is produced by transcription in vivo or in vitro under transcriptional control of a promoter (e.g., SV40 early promoter or a CMV promoter for mammalian cell expression) in the recombinant nucleic acid (paragraphs [0110]-[0112]). Fig. 1A below depicts a schematic of circRNA synthesis by in vitro transcription from a permuted intron-exon template (5’ half placed at the 3’ position and vice versa) via self-splicing of Group I intron of phage T4 thymidylate synthase (td) gene, and circularization brings the IRES upstream of GFP sequence that allows protein translation.
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In example 1 (paragraphs [0202]-[0205] and Fig. 5), Chang et al also disclosed the GFP-IRES circRNA exon and endogenous human ZKSCAN1 introns described in Liang et al (Genes Dev. 28:2233-2247, 2014) which do not have autocatalytic-splicing properties, but complementary Alu repeats that are present in these flanking introns enable human ZKSCAN1 to splice GFP-IRES into a circRNA without innate immune gene induction in human HeLa cells unlike the DNA construct that expresses the phage self-splicing IRES-GFP circRNA containing exogenous td introns as depicted in Fig. 5A below. Chang et al further taught a pharmaceutical composition comprising circular RNAs or recombinant nucleic acids encoding circular RNAs and a pharmaceutically acceptable carrier (paragraph [0151]).
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Chang et al did not teach explicitly at least an AAV genome comprising a nucleic acid molecule encoding a circRNA, wherein the nucleic acid molecule comprising: the elected truncated HIPK3 intronic elements that flank a gene of interest which can be transcribed into a translatable mRNA or a non-coding RNA, preferably the truncated first and second intronic elements comprise the nucleotide sequence of SEQ ID NO: 15 and 16, respectively; a promoter that is 5’ of a first intronic element, a gene of interest and a second intronic element; a first inverted terminal repeat and a second inverted terminal repeats, preferable they are derived derived from AAV serotype; the AAV genome is packaged in an AAV9 capsid or derivative thereof; the same AAV genome wherein the nucleic acid molecule further comprising a polyA sequence 3’ of the second intronic element; and an AAV capsid or virus comprising the same AAV genome.
Before the effective filing date of the present application (11/07/2017), Liang et al already demonstrated at least that miniature introns containing the splice sites along with short (about 30- to 40- nucleotide) inverted repeats, such as Alu elements, derived from human ZKSCAN1 and HIPK3 genes are sufficient to allow the intervening exons to circularize in cells; and the intronic repeats must base-pair to one another, thereby bringing the splice sites into close proximity to each other (Abstract; sections titled “Short repeat sequences are sufficient for ZKSCAN1 circular RNA production” on pages 2236-2238 and “Short repeats are sufficient for production of the HIPK3 circular RNA” on pages 2238 and 2240-2241; Figs. 2 and 4). Liang et al selected and cloned a 2803-nt region of the HIPK3 premRNA into pcDNA3.1 as depicted in reproduced Fig. 4A, 4D and 4E below and confirmed that this expression vector efficiently generates a circular RNA when transfected into HeLa cells, then demonstrated that a 32-nt region of the upstream AluSz element (nucleotides 300-331) and a 32-nt region of the downstream AluSq2 element (nucleotides 2607-2638) are sufficient to support HIPK3 circularization.
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The pcDNA3.1(+) HIPK3 300-2703∆2450-2609 (used in Figure 4E showing formation of a circular RNA) in Supplemental Methods comprises the first 391-nucleotide sequence that is 100% identical to the first intronic element of SEQ ID NO: 15 of the present application and the last 754-nucleotide sequence that is 100% identical to SEQ ID NO: 16 of the present application (see attached sequence below on page 34 of this office action; it is a typo error that the first G appears in front of the phrase “Used in Figure 4E” on the line above the main body of the listed sequence). Additionally, each of the pcDNA3.1(+) HIPK3 300-2703, pcDNA3.1(+) HIPK3 300-2703∆2450-2574, pcDNA3.1(+) HIPK3 300-2703∆2450-2599 and pcDNA3.1(+) HIPK3 300-2703∆2450-2609 constructs contain truncated flanking introns (relative to the wild-type 2803-nucleotide region) and are capable of generating circular RNA as shown in Figs. 4D-E above.
Additionally, Sena-Esteves et al already disclosed recombinant adeno-associated viruses/virions (rAAVs) comprising artificial genetic regulatory elements that modulate transgene expression to provide therapeutic amounts of transgene levels without the induction of adverse events for the treatment of lysosomal storage disorders, and that AAV vectors have emerged as an effective platform for in vivo gene therapy (Abstract; Summary of Invention; page 22, lines 22-25; page 23, lines 11-26). Sena-Esteves et al taught that the rAAV comprising a capsid (e.g., AAV9, AAV10 and others) containing a nucleic acid comprising a hybrid promoter operably linked to a transgene encoding a lysosomal storage disorder-associated protein, and the rAAV comprises two ITRs wherein the hybrid promoter and transgene are located between two ITRs, AAV-ITR sequences may be from any known AAV and the rAAV has the serotype AAV9 (page 3, second paragraph; page 29, lines 8-9). Fig. 1 depicts schematically an exemplary rAAV vector comprising two inverted terminal repeats (ITRs) flank an expression vector on each end as shown below. Sena-Esteves et al also taught the use of a polyadenylation sequence (e.g., SV40 polyA and BGH polyA) being inserted following the transgene sequences and immediately before the 3’ AAV-TR sequence (page 27, lines 4-5; and Fig. 1). Sene-Esteves et al also taught that a transgene may encode an interfering RNA such as dsRNA, siRNA, shRNA, miRNA or amiRNA (page 5, lines 3-8).
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Moreover, Wang et al also disclosed at least the pCircGFP vector comprising a minigene with split GFP in a reverse order which transcription is driven by the CMV promoter located 5’ of the minigene and terminated by the SV40 polyadenylation signal located 3’ of the minigene, wherein minigene is flanked immediately with canonical introns (intron 12 of IGF2BP1) which can be joined into a circular RNA through backsplicing to generate an intact open reading frame of GFP, and the minigene also comprises an internal ribosome entry site (IRES) being inserted upstream of the start codon of GFP, which can drive cap-independent synthesis (page 173, right column, first full paragraph; page 177, right column, last full paragraph; and Figure 1A below).
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Wang et al also investigated whether the poly(A) sequence has similar activities in stimulating translation from circular mRNA as translation from linear mRNA by inserting a 40-nt poly(A) fragment after the stop codon of GFP; and surprisingly they found that rather than enhancing protein translation, poly(A) sequence actually reduced the protein production as compared with the circular mRNA without poly(A), but the level of circular mRNA did not change by inserting poly(A) sequences (page 176, left column, second full paragraph; and Fig. 3E).
Furthermore, Zincarelli et al already investigated the tropism and kinetics of expression for nine different serotypes of AAV (AAV 1-9 in the form a recombinant vector with each having flanking AAV2 ITRs), packaging the same transgene, produced and purified in the same manner, and injected in mice through the same systemic route (tail vein injection) (Abstract and Figure 1). They found that AAV9 had the best viral genome distribution and highest protein levels, with rapid-onset transgene expression and AAV9-mediated luciferase enzyme activity was found mainly at least in the liver, in the heart, as well as in other tissues such as the lung, the hamstring and the brain (Figure 4). Zincarelli et al stated clearly “AAV8 and AAV9 transduce tissues more ubiquitously than the other serotypes do, with AAV9 having the most robust tissue expression” (page 1078, right column, middle of last full paragraph).
Accordingly, it would have been obvious for an ordinary skilled artisan before the effective filing date of the present application to modify the teachings of Chang et al by also preparing at least a recombinant AAV9 vector/virion/genome comprising the following elements in the 5’ to 3’ order: (i) a first AAV ITR, (ii) a promoter, (iii) a first endogenous truncated human HIPK3 intron containing a 3’ splice site such as SEQ ID NO: 15, (iv) a nucleotide sequence encoding a non-immunogenic circular target RNA encoding a therapeutic polypeptide or a non-coding RNA such as siRNA, shRNA or miRNA, (v) a second endogenous truncated human HIPK3 intron containing a 5’ splice site such as SEQ ID NO: 16, and (vi) a second AAV ITR, wherein the produced target RNA transcript is circularized by backsplicing of the endogenous truncated human introns, the same recombinant AAV9 vector/virion/genome further comprising a polyadenylation sequence that is 3’ of the second truncated human HIPK3 intron, in light of the teachings of Liang et al, Sena-Esteves et al, Wang et al and Zincarelli et al as presented above.
An ordinary skilled artisan would have been motivated to carry out the above modifications because: (i) Liang et al already demonstrated successfully that miniature introns containing the splice sites along with short (about 30- to 40- nucleotide) inverted repeats, such as Alu elements, derived from human ZKSCAN1 and HIPK3 genes are sufficient to allow the intervening exons to circularize in cells; and at least the exemplary pCDNA3.1(+) HIPK3 300-2703∆2450-2609 (used in Figure 4E showing formation of a circular RNA) in Supplemental Methods comprises the first 391-nucleotide sequence that is 100% identical to the first truncated intronic element of SEQ ID NO: 15 of the present application and the last 754-nucleotide sequence that is 100% identical to the second truncated intronic element of SEQ ID NO: 16 of the present application; (ii) Sena-Esteves et al already taught at least a rAAV comprising an AAV9 capsid containing a nucleic acid comprising a hybrid promoter operably linked to a transgene (e.g. a transgene encoding a therapeutic lysosomal storage disorder-associated protein or an interfering RNA such as dsRNA, siRNA, shRNA, miRNA or amiRNA); the rAAV comprises two ITRs wherein the hybrid promoter and transgene are located between two ITRs; AAV ITR sequences may be derived from any known AAV such as AAV2 ITRs; as well as the use of a polyadenylation sequence such as SV40 polyA signal being inserted following the transgene sequences and before the 3’ AAV-TR sequence; (iii) Wang et al also taught that rather than enhancing protein translation, poly(A) sequence inserted after the stop codon of GFP gene (within the splicing intronic sequences) in a circular RNA reporter construct actually reduced the protein production as compared with the circular mRNA without poly(A); and (iv) Zincarelli et al taught that AAV9 containing AAV2 ITRs had the best viral genome distribution and highest protein levels among tested AAV serotypes 1-9, with rapid-onset transgene expression and AAV9-mediated luciferase enzyme activity was found mainly at least in the liver, in the heart, as well as in other tissues such as the lung, the hamstring and the brain. Please also noting that the primary Chang reference already taught the circular RNA is produced by transcription in vivo or in vitro under transcriptional control of a promoter and the recombinant nucleic acid comprises a viral vector such an adeno-associated virus, as well as the use of a 3’ portion of an endogenous or exogenous intron comprising a 3’ splice site (a truncated intron) together with a 5’ portion of an endogenous or exogenous intron comprising a 5’ splice site (a truncated intron) for back-splicing.
An ordinary skilled artisan would have a reasonable expectation of success in light of the teachings of Chang et al, Liang et al, Sena-Esteves et al, Wang et al and Zincarelli et al; coupled with a high level of skill for an ordinary skilled artisan in the relevant art.
The modified rAAV9 vector/virion/genome resulting from the combined teachings of Chang et al, Liang et al, Sena-Esteves et al, Wang et al and Zincarelli et al as set forth above is indistinguishable and encompassed by the presently claimed invention.
Therefore, the claimed invention as a whole was prima facie obvious in the absence of evidence to the contrary.
Response to Arguments
Applicant’s arguments related in part to the above modified 103 rejection in the Amendment filed on 05/14/2026 (pages 8-12) along with the 1.132 Declaration of Dr. Aravind Asokan filed on 11/08/2024 have been fully considered, but they are respectfully not found persuasive for the reasons discussed below.
A. Motivation to combine Chang, Liang, Wang and/or Sena-Esteves.
Applicant argued basically that an ordinary skill would not have had reason or motivation to combine the cited references to arrive at the presently claimed an AAV genome. This is because Applicant argued that Chang, as a whole, discloses circular RNAs in vitro and delivering the circular RNAs to target cells using liposomes in the working examples; and Chang suggests that “[n]aked circRNA or liposome-encapsulated cirRNA are active in vivo” (paragraph [0212]). Similarly, Applicant argued that both Liang and Wang, as a whole, generally disclose non-viral plasmids containing circular RNA minigenes that are delivered to cells in vitro using liposome-mediated transfection techniques. Applicant argued that given the effectiveness of the in vitro production of circular RNAs and the liposome-mediated delivery methods described in Chang, Liang, and Wang, there is no reason or motivation for one of ordinary skill in the art to modify the circRNA production and delivery methods of the cited references to arrive at the claimed AAV genome; particularly it was unknown at the time of filing whether circular RNA could be expressed in vivo using the subject’s cellular splicing machinery. Applicant further argued that the only relevant disclosure in Chang is a generic statement about viral delivery with a laundry list of bacterial or viral vectors, none of which were described in further detail or actually tested in the working examples; and the Office attempts to construct a prima facie case of obviousness by selecting the adeno-associated viral vector from Chang and picking and choosing other claim elements from the cited references while using the Applicant’s specification as a template. Such an approach is impermissible because it uses hindsight. Applicant also argued that the Office has not provided any motivation from Sena-Esteves, and Sena-Esteves merely discloses the modulation of AAV vectors for transgene expression and it does not disclose an AAV genome encoding a circular RNA. Accordingly, an ordinary skill in the art would not have had a reason or motivation to combine the generic AAV vectors of Sena-Esteves with the circular RNAs taught in Chang, Liang and Wang to arrive at the AAV genome in currently amended claim 1, particular an AAV genome packaged in an AAV9 capsid capable of expressing a circRNA in a subject’s cell or tissue as presently claimed.
First, please refer to the above modified 103 rejection for details. The Zincarelli reference was additionally cited to supplement the combined teachings of Chang et al, Liang et al, Sena-Esteves et al and Wang et al for providing a specific motivation why an ordinary skill in the art would select AAV9 capsid to package an AAV genome. Since the above rejection was made under 35 U.S.C. 103 none of the cited references individually has to teach every limitation of the claims. For example, neither the Sena-Esteves reference nor the Wang reference have to teach an AAV genome encoding a circular RNA. It is also apparent that Applicant considered each of the cited references in total isolation one from the others, without taking into account of the specific combination of Chang et al, Liang et al, Sena-Esteves et al, Wang et al and Zincarelli et al.
Second, the primary Chang reference already teaches explicitly at least a recombinant nucleic acid encoding a non-immunogenic circular target RNA encoding a therapeutic polypeptide (e.g., an enzyme, hormone, neurotransmitter, cytokine and others), wherein the recombinant nucleic acid comprises in a 5’ to 3’ order: (i) a 3’ portion of an endogenous intron comprising a 3’ splice site (an acceptor site), (ii) a nucleic acid sequence encoding the target RNA, and (iii) a 5’ portion of an endogenous intron comprising a 5’ splice site (a donor site), wherein the produced target RNA transcript is circularized by backsplicing or splicing of the endogenous introns, the recombinant nucleic acid comprises a viral vector such as an adenovirus, a retrovirus, and adeno-associated virus and others (see at least paragraphs [0027], [0111], [0114] and [0116]), and the recombinant nucleic acid further comprises a nucleic acid sequence encoding an IRES (e.g., IRES derived from encephalomyocarditis virus (EMCV) UTR) operably linked to the nucleic acid encoding the therapeutic polypeptide. The Chan reference stated clearly and explicitly “In certain embodiments, the recombinant nucleic acid encoding the circular RNA comprises a vector….Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated vectors, retroviral vectors, lentiviral vectors, and the like” (paragraph [0111]); and “There are a number of ways in which nucleic acids (e.g., circular RNAs or recombinant nucleic acids encoding them) may be introduced into cells. In certain embodiments, a virus or engineered construct derived from a viral genome is used for delivery of a circular RNA to a cell. A number of viral based systems have been developed for transfer of nucleic acids into mammalian cells. These include adenoviruses, retroviruses (γ-retroviruses and lentiviruses), poxviruses, adeno-associated viruses, baculoviruses, and herpes simplex viruses” (paragraph [0114]). Thus, the teachings of the primary Chang reference are not necessarily limited only to the use of naked circRNA or liposome-encapsulated circRNA. Nor do the teachings of the primary Chang reference are limited only to the working examples. Moreover, before the effective filing date of the present application Sena-Esteves et al already stated “AAV vectors have emerged as an effective platform for in vivo gene transfer” (page 1, lines 20-21), and disclosed recombinant AAV vectors that provide therapeutic amounts of transgene without the induction of adverse events (Summary of Invention). Please also note that the Chang reference already teaches clearly using a truncated endogenous intron comprising a 3’ splice site (a 3’ portion of an endogenous intron) and a truncated endogenous intron comprising a 5’ splice site (a 5’ portion of an endogenous intron) for back-splicing.
Third, as set forth in the above modified 103 rejection an ordinary skilled artisan would have been motivated to modify the teachings of Chang et al by also preparing at least a recombinant AAV9 vector/virion/genome comprising the following elements in the 5’ to 3’ order: (i) a first AAV ITR, (ii) a promoter, (iii) a first endogenous truncated human HIPK3 intron containing a 3’ splice site such as SEQ ID NO: 15, (iv) a nucleotide sequence encoding a non-immunogenic circular target RNA encoding a therapeutic polypeptide or a non-coding RNA such as siRNA, shRNA or miRNA, (v) a second endogenous truncated human HIPK3 intron containing a 5’ splice site such as SEQ ID NO: 16, and (vi) a second AAV ITR, wherein the produced target RNA transcript is circularized by backsplicing of the endogenous truncated human introns, the same recombinant AAV9 vector/virion/genome further comprising a polyadenylation sequence that is 3’ of the second truncated human HIPK3 intron, because: (i) Liang et al already demonstrated successfully that miniature introns containing the splice sites along with short (about 30- to 40- nucleotide) inverted repeats, such as Alu elements, derived from human ZKSCAN1 and HIPK3 genes are sufficient to allow the intervening exons to circularize in cells; and at least the exemplary pCDNA3.1(+) HIPK3 300-2703∆2450-2609 (used in Figure 4E showing formation of a circular RNA) in Supplemental Methods comprises the first 391-nucleotide sequence that is 100% identical to the first truncated intronic element of SEQ ID NO: 15 of the present application and the last 754-nucleotide sequence that is 100% identical to the second truncated intronic element of SEQ ID NO: 16 of the present application; (ii) Sena-Esteves et al already taught at least a rAAV comprising an AAV9 capsid containing a nucleic acid comprising a hybrid promoter operably linked to a transgene (e.g. a transgene encoding a therapeutic lysosomal storage disorder-associated protein or an interfering RNA such as dsRNA, siRNA, shRNA, miRNA or amiRNA); the rAAV comprises two ITRs wherein the hybrid promoter and transgene are located between two ITRs; AAV ITR sequences may be derived from any known AAV such as AAV2 ITRs; as well as the use of a polyadenylation sequence such as SV40 polyA signal being inserted following the transgene sequences and before the 3’ AAV-TR sequence; (iii) Wang et al also taught that rather than enhancing protein translation, poly(A) sequence inserted after the stop codon of GFP gene (within the splicing intronic sequences) in a circular RNA reporter construct actually reduced the protein production as compared with the circular mRNA without poly(A); and (iv) Zincarelli et al taught that AAV9 containing AAV2 ITRs had the best viral genome distribution and highest protein levels among tested AAV serotypes 1-9, with rapid-onset transgene expression and AAV9-mediated luciferase enzyme activity was found mainly at least in the liver, in the heart, as well as in other tissues such as the lung, the hamstring and the brain. Please also noting that the primary Chang reference already taught the circular RNA is produced by transcription in vivo or in vitro under transcriptional control of a promoter and the recombinant nucleic acid comprises a viral vector such an adeno-associated virus, as well as the use of a 3’ portion of an endogenous or exogenous intron comprising a 3’ splice site (a truncated intron) together with a 5’ portion of an endogenous or exogenous intron comprising a 5’ splice site (a truncated intron) for back-splicing.
Fourth, with respect to Applicant’s argument on impermissible hindsight reconstruction Examiner would like to recite a paragraph from in re Oetiker, 977, F.2d 1443, 1448 (Fed. Cir. 1992).
"[T]here must be some teaching, reason, suggestion, or motivation found "in the prior art" or "in the prior art references" to make a combination to render an invention obvious within the meaning of 35 U.S.C. 103 (1998). Similar language appears in a number of opinions and if taken literally would mean that an invention cannot be held to have been obvious unless something specific in a prior art reference would lead an inventor to combine the teachings therein with another piece of prior art. This restrictive understanding of the concept of obviousness is clearly wrong…. While there must be some teaching, reason, suggestion, or motivation to combine existing elements to produce the claimed device, it is not necessary that the cited references or prior art specifically suggest making the combination…. In sum, it is off the mark for litigants to argue, as many do, that an invention cannot be held to have been obvious unless a suggestion to combine the prior art teachings is found in a specific reference."
Although the cited artisans do not specifically point out a motivation to in their disclosure, an ordinarily skilled artisan would have been able to identify the need for the combination of the teachings without the disclosure of the instant application. 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). Please see the above modified 103 rejection for details along with the provided motivations, as well as Examiner’s responses in the preceding paragraphs.
Fifth, please note that the patentability of composition claims depends on the claimed structure, not on the use or the purpose of the structure; and stating an intended use is not sufficient to structurally distinguish from the prior art.
B. Reasonable expectation of success.
Applicant argued that even if there was a motivation to combine the cited references, obviousness requires clear and convincing evidence of a reasonable expectation of success, particularly none of the previously cited references teach or suggest the presently claimed AAV9-packaged genome that expresses circular RNA in a cell or tissue of a subject following its adminnstration. In support of this, Applicant submitted the 1.132 Declaration describing that it was not known by Chang or others at the time of filing whether rAAV containing truncated backsplicing introns would be capable of expressing circular RNAs in vivo. Instead, Applicant demonstrates for the first time in the as-filed specification that AAV can deliver transgene cassettes that express circular RNAs in a variety of tissue and cell types in vivo (e.g., Example 1, FIGs. 3A-3F and 4A-4E; and Example 2, FIGs. 12A-12F). The findings were not obvious for the following reasons:
(1) The in vivo expression profile of AAV-circRNA cassettes comprising truncated intronic elements differed from the endogenous expression profile of circular RNAs. For example, endogenous circular ZKSCAN1 and circular HIPK3 are expressed at low levels in the heart, whereas surprisingly robust expression and translation of circular GFP RNA produced from the backsplicing of either truncated ZKSCAN1 introns or truncated HIPK3 introns in heart tissue (Figs. 3A-C, E of the application). Additionally, in contrast to endogenous circular ZKSCAN1 and circular HIPK3 are highly expressed in the brain, Applicant’s constructs were expressed at low levels in brain tissue (Fig. 4A of the application). There were even cell-type specificities within brain tissue that could not have been predicted based on general knowledge of the prior art since ZKSCAN1 and HIPK3 GFP vectors showed expression only in astrocytes and not neurons, which were known to express circular RNAs that function in neuronal processes; and
(2) Applicant observed differences in expression between the truncated ZKSCAN1 and HIPK3 GFP constructs themselves that was not expected. The truncated ZKSCAN1 GFP construct was more highly expressed in heart and brain tissue, whereas the truncated HIPK3 GFP construct was more highly expressed in eye tissue (Figs. 3A-C, E and 4A, C-D of the present application); even though both vectors had the same promoter and open reading frame, and therefore, should have been transcribed and translated with equal efficiencies. The differences in expression were also not due to dosing because all animals had similar numbers of vector genomes per cell.
Instead, the Office has relied on conclusive statements while dismissing Applicant’s objective evidence of record provided in the as-filed application and the Asokan Declaration.
First, there is no objective evidence of record indicating or even suggesting that before the effective filing date of the present application (11/07/2017) that an ordinary skill in the art would not have a “reasonable” expectation of success for constructing an AAV genome comprising a nucleic acid encoding a circular RNA of the present application that is packaged in an AAV9 capsid; particularly in light of the teachings of Chang, Liang, Sena-Esteves, Wang and Zincarelli as set forth in the above modified 103 rejection. Especially, Liang already demonstrated all of the pcDNA3.1(+) HIPK3 300-2703, pcDNA3.1(+) HIPK3 300-2703∆2450-2574, pcDNA3.1(+) HIPK3 300-2703∆2450-2599 and pcDNA3.1(+) HIPK3 300-2703∆2450-2609 constructs contain truncated flanking introns (relative to the wild-type 2803-nucleotide region) and they are capable of generating circular RNAs as shown in Figs. 4D-E. Additionally, the pcDNA3.1(+) HIPK3 300-2703∆2450-2609 (used in Figure 4E showing formation of a circular RNA) in Supplemental Methods comprises the first 391-nucleotide sequence that is 100% identical to the first intronic element of SEQ ID NO: 15 of the present application and the last 754-nucleotide sequence that is 100% identical to SEQ ID NO: 16 of the present application (see attached sequence below on page 34 of this office action; it is a typo error that the first G appears in front of the phrase “Used in Figure 4E” on the line above the main body of the listed sequence). The Zincarelli reference was additionally cited to supplement the combined teachings of Chang et al, Liang et al, Sena-Esteves et al and Wang et al for providing a specific motivation why an ordinary skill in the art would select AAV9 capsid to package an AAV genome.
Second, please note that the patentability of composition claims depends on the claimed structure; and the modified rAAV9 vector/virion/genome resulting from the combined teachings of Chang et al, Liang et al, Sena-Esteves et al, Wang et al and Zincarelli et al as set forth in the above modified 103 rejection is indistinguishable from the claimed compositions. As such, the modified rAAV9 vector/virion/genome would exhibit the same properties when they are placed in the requisite environment. Nevertheless, an ordinary skill in the art would reasonably expect a robust in vivo expression and translation of circular GFP RNAs mediated by the truncated ZKSCAN1 and HIPK3 GFP constructs in the form of a recombinant AAV9 vector in heart tissue in comparison with the low endogenous expression of circular ZKSCAN1 and HIPK3 in the heart because rAAV9 vector has been demonstrated to have the highest transgene expression in heart tissue by Zincarelli et al (see at least Figure 4 in the Zincarelli reference). Similarly, an ordinary skill in the art would reasonably expect the truncated ZKSCAN1 and HIPK3 GFP constructs in the form of a recombinant AAV9 vector were expressed at low levels in brain tissue in comparison with the high endogenous expression of circular ZKSCAN1 and HIPK3 in brain tissue because rAAV9 vector has also been demonstrated to have a relatively low transgene expression in brain tissue by Zincarelli et al (see at least Figure 4 in the Zincarelli reference). With respect to the issue that ZKSCAN1 and HIPK3 GFP vectors showed expression only in astrocytes and not neurons, an ordinary skill in the art would readily attribute such cell-type specificity to the selective host cell tropism of the AAV9 capsid. Additionally, since the modified recombinant adeno-associated viral genome packaged in an AAV9 capsid with truncated HIPK3 intronic elements flanking a gene of interest resulting from the combined teachings of Chang et al, Liang et al, Sena-Esteves et al, Wang et al and Zincarelli et al is indistinguishable from the rAAV9 vector of the present application, it would also exhibit higher expression in eye tissue relative to a corresponding recombinant adeno-associated viral genome with truncated ZKSCAN1 intronic elements flanking a gene of interest; and the difference in their expression levels may be attributed to the difference in backsplicing efficacy between truncated HIPK3 intronic elements and truncated ZKSCAN1 intronic elements in eye tissue.
Third, should there be any “surprising/unexpected” result it must be commensurate with the scope of the claims. None of the claims under rejection are directed specifically to a recombinant AAV construct/genome containing “truncated synthetic introns” having the specific pair of SEQ ID NOs of the present application (e.g., the elected pair of SEQ ID NOs. 15-16). Even dependent claim 2 simply recites “wherein the first intronic element or the second intronic element comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 13-24 and 29-32”, which limitation does not require the specific pair of SEQ ID NOs. 15-16 that flank a gene of interest. Nevertheless, please note that Liang already disclosed the exemplary pcDNA3.1(+) HIPK3 300-2703∆2450-2609 construct (used in Figure 4E showing formation of a circular RNA) in Supplemental Methods that comprises the first 391-nucleotide sequence that is 100% identical to the first intronic element of SEQ ID NO: 15 of the present application and the last 754-nucleotide sequence that is 100% identical to SEQ ID NO: 16 of the present application.
Fourth, please also note that the standard under 35 U.S.C. 103 is a “reasonable” expectation of success, and not certainty, let alone clear and convincing evidence as required by Applicant.
Fifth, once again please note that the patentability of composition claims depends on the claimed structure, not on the use or the purpose of the structure; and stating an intended use is not sufficient to structurally distinguish from the prior art.
Conclusions
No claim is allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Quang Nguyen, Ph.D., at (571) 272-0776.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s SPE, James Douglas (Doug) Schultz, Ph.D., may be reached at (571) 272-0763.
To aid in correlating any papers for this application, all further correspondence regarding this application should be directed to Group Art Unit 1631; Central Fax No. (571) 273-8300.
Any inquiry of a general nature or relating to the status of this application or proceeding should be directed to (571) 272-0547.
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/QUANG NGUYEN/Primary Examiner, Art Unit 1631
pcDNA3.1(+) HIPK3 300-2703 D2450-2609
g Used in Figure 4E
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