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 .
Applicant’s amendment filed on 09/24/2025 has been entered.
Amended claims 1-4, 6, 8-9, 11-13 and 15-22 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Claims 9, 11-13 and 15-18 were also withdrawn previously from further consideration because they are directed to a non-elected invention. Claims 19 and 21-22 were also withdrawn previously from further consideration because they are drawn to non-elected species.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 1-4, 6, 8 and 20 are examined on the merits herein with the above elected species.
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 1-4, 6, 8 and 20 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) and Wang et al (RNA 21:172-179, 2015; IDS). This is a modified rejection necessitated by Applicant’s amendment.
The instant claims are directed to an AAV genome comprising a nucleic acid molecule encoding a circular RNA (circRNA) that is covalently closed, wherein the nucleic acid molecule comprises the elements a)-d) recited in independent claim 1, wherein the circRNA is expressed in a cell or a tissue of a subject following administration of the AAV genome to the 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 that is covalently closed, 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, preferably the truncated intronic elements comprise the nucleotide sequence of any of SEQ ID NOs: 15-16; an IRES (e.g., the elected encephalomyocarditis virus IRES) driving translation of the translatable mRNA transcribed from the gene of interest; a promoter region outside of the truncated intronic elements that flank the gene of interest; and wherein the nucleic acid molecule is flanked by AAV inverted terminal repeats; the same AAV genome wherein the nucleic acid molecule further comprising a polyA sequence outside of the truncated intronic elements that flank the gene of interest; 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).
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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).
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 AAV vector/virion/genome comprising the following elements in the 5’ to 3’ order: (i) a first AAV ITR, (ii) a first endogenous truncated human HIPK3 intron containing a 3’ splice site such as SEQ ID NO: 15, (iii) a nucleotide sequence encoding a non-immunogenic circular target RNA encoding a therapeutic polypeptide, (iv) a second endogenous truncated human HIPK3 intron containing a 5’ splice site such as SEQ ID NO: 16, and (v) a second AAV ITR, wherein the produced target RNA transcript is circularized by backsplicing of the endogenous truncated/miniature human introns, the same recombinant AAV vector/virion/genome further comprising a promoter located between the first AAV ITR and the first endogenous truncated/miniature human HIPK3 intron containing a 3’ splice site, and a 3’-UTR including a polyadenylation sequence that is located immediately 5’ of the second AAV ITR and outside of the endogenous truncated/minature introns, in light of the teachings of Sena-Esteves et al, Wang et al and Liang 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 also taught at least an exemplary rAAV vector comprising two inverted terminal repeats (ITRs) flanking an expression vector on each end along with 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; and (iii) Wang et al already 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). 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 and Wang et al; coupled with a high level of skill for an ordinary skilled artisan in the relevant art.
The modified rAAV vector/virion/genome resulting from the combined teachings of Chang et al, Liang et al, Sena-Esteves et al and Wang et al as set forth above is indistinguishable and encompassed by the presently claimed invention. With respect to the “functional wherein” clause of “wherein the circRNA is expressed in a cell or a tissue of a subject following administration of the AAV genome to the subject”, the functional wherein clause does not provide further structural details but merely explains how the AAV genome functions when placed in the requisite environment. 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.
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 to the above modified 103 rejection in the Amendment filed on 09/24/2025 (pages 6-12) 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. Once again, 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 Wang, as a whole, generally discloses 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, and 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 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 and Wang to arrive at the AAV genome in currently amended claim 1.
First, please refer to the above modified 103 rejection for details. Since the above rejection was made under 35 U.S.C. 103 none of the cited references 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 and Wang 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 AAV vector/virion/genome comprising the following elements in the 5’ to 3’ order: (i) a first AAV ITR, (ii) a first endogenous truncated human ZKSCAN1 intron containing a 3’ splice site such as SEQ ID NO: 15, (iii) a nucleotide sequence encoding a non-immunogenic circular target RNA encoding a therapeutic polypeptide, (iv) a second endogenous truncated human ZKSCAN1 intron containing a 5’ splice site such as SEQ ID NO: 16, and (v) a second AAV ITR, wherein the produced target RNA transcript is circularized by backsplicing of the endogenous truncated/miniature human introns, the same recombinant AAV vector/virion/genome further comprising a promoter located between the first AAV ITR and the first endogenous truncated/miniature human ZKSCAN1 intron containing a 3’ splice site, and a 3’-UTR including a polyadenylation sequence that is located immediately 5’ of the second AAV ITR and outside of the endogenous truncated/minature introns, 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 also taught at least an exemplary rAAV vector comprising two inverted terminal repeats (ITRs) flanking an expression vector on each end along with 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; and (iii) Wang et al already 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). 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, with respect to the new limitation “wherein the circRNA is expressed in a cell or a tissue of a subject following administration of the AAV genome to the subject”, it is noted that the functional wherein clause does not provide further structural details but merely explains how the AAV genome functions when placed in the requisite environment. 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. Applicant argued that even if there was a motivation to combine the cited references, which there was not, obviousness requires clear and convincing evidence of a reasonable expectation of success. 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. 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. 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; 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 cells. Instead, the Office 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; particularly in light of the teachings of Chang, Liang, Sena-Esteves and Wang as set forth above. 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).
Second, please note that the patentability of composition claims depends on the claimed structure; and the modified rAAV vector/virion/genome resulting from the combined teachings of Chang et al, Liang et al, Sena-Esteves et al and Wang et al as set forth in the above 103 rejection is indistinguishable from the claimed compositions. As such, the modified rAAV vector/virion/genome would exhibit the same properties when they are placed in the requisite environment (e.g., displaying a robust in vivo expression and translation of circular RNAs in comparison with the endogenous expression of circular RNAs and/or a recombinant adenoviral genome with truncated HIPK3 intronic elements flanking a gene of interest expresses only in astrocytes in brain tissue). Moreover, please also note that discrepancy in the in vivo expression and translation of circular RNAs mediated by AAV-circRNA cassettes comprising truncated intronic elements and the endogenous expression profile of circular RNAs can be attributed at least to the use of different promoters for expression. Additionally, a modified recombinant adeno-associated viral genome 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 and Wang et al would exhibit higher expression in eye tissue compared to the modified recombinant adeno-associated viral genome with truncated ZKSCAN1 intronic elements flanking a gene of interest.
Third, please note 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 truncated intronic elements of (b) comprise the nucleotide sequence of any 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 with respect to the new limitation “wherein the circRNA is expressed in a cell or a tissue of a subject following administration of the AAV genome to the subject”, it is noted that the functional wherein clause does not provide further structural details but merely explains how the AAV genome functions when placed in the requisite environment. 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.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/process/file/efs/guidance/eTD-info-I.jsp.
Amended claims 1-3, 6 and 20 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-25 of U.S. Patent No. 11,718,862.
Although the claims at issue are not identical, they are not patentably distinct from each other because an AAV genome encoding a circular RNA, wherein the AAV genome comprises, from 5’ to 3’: (a) a first inverted terminal repeat; (b) a first intronic element; (c) a nucleotide sequence of interest (e.g., encoding a non-coding RNA or a translatable mRNA; dependent claims 4-5, respectively); (d) a second intronic element; (c) a second inverted terminal repeat; wherein (c) is directly coupled to (b) and (d), respectively; and wherein the first intronic element and the second intronic element are at least one pair selected from the Markush group recited in independent claim 1 (capable of generating a covalently closed circular RNA; dependent claim 2); the same AAV genome further comprises an internal IRES (e.g., a viral IRES or a cellular IRES) capable of driving translation of a translatable mRNA (dependent claims 7-9); a promoter located between the first inverted terminal repeat and the first intronic element (outside of the first and second intronic elements that flank a gene of interest); dependent claims 16-17) and a 3’UTR comprising a translation regulation region (e.g., a polyadenylation sequence and/or a structural element that stabilizes the circRNA) located 5’ to the second inverted terminal repeat (dependent claims 19-21); and an AAV capsid or particle comprising the same AAV genome (claims 23-25) in claims 1-25 of U.S. Patent No. 11,718,862 anticipate/encompass the claimed genus in the application being examined and, therefore, a patent to the genus would, necessarily, extend the rights of the species or sub- should the genus issue as a patent after the species of sub-genus. Please note that SEQ ID NOs. 15-16 are truncated HIPK3 first and second intronic elements, respectively. With respect to the “functional wherein” clause of “wherein the circRNA is expressed in a cell or a tissue of a subject following administration of the AAV genome to the subject”, the functional wherein clause does not provide further structural details but merely explains how the AAV genome functions when placed in the requisite environment. 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.
Amended claims 1, 4 and 8 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-25 of U.S. Patent No. 11,718,862 in view of Sena-Esteves et al (WO 2016/172155; IDS) and Wang et al (RNA 21:172-179, 2015; IDS).
Claims 1-25 of U.S. Patent No. 11,718,862 are directed to an AAV genome encoding a circular RNA, wherein the AAV genome comprises, from 5’ to 3’: (a) a first inverted terminal repeat; (b) a first intronic element; (c) a nucleotide sequence of interest (e.g., encoding a non-coding RNA or a translatable mRNA; dependent claims 4-5, respectively); (d) a second intronic element; (c) a second inverted terminal repeat; wherein (c) is directly coupled to (b) and (d), respectively; and wherein the first intronic element and the second intronic element are at least one pair selected from the Markush group recited in independent claim 1 (capable of generating a covalently closed circular RNA; dependent claim 2); the same AAV genome further comprises an internal IRES (e.g., a viral IRES or a cellular IRES) capable of driving translation of a translatable mRNA (dependent claims 7-9); a promoter located between the first inverted terminal repeat and the first intronic element (outside of the first and second intronic elements that flank a gene of interest); dependent claims 16-17) and a 3’UTR comprising a translation regulation region (e.g., a polyadenylation sequence and/or a structural element that stabilizes the circRNA) located 5’ to the second inverted terminal repeat (dependent claims 19-21); and an AAV capsid or particle comprising the same AAV genome (claims 23-25).
The claims of the present application differ from claims 1-25 of U.S. Patent No. 11,718,862 in reciting specifically that “a polyadenylation (polyA) sequence outside of the intronic elements that flank the gene of interest”; “a composition comprising the nucleic acid molecule of claim 1 in a pharmaceutically acceptable carrier”; and “wherein the circRNA is expressed in a cell or a tissue of a subject following administration of the AAV genome to the subject”.
Before the effective filing date of the present application (11/07/2017), 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). Sena-Esteves et al further taught a composition comprising the rAAVs in a pharmaceutical acceptable carrier (e.g., sterile saline, lactose, sucrose and water) (page 35, second paragraph).
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Additionally, 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).
Accordingly, it would have been obvious for an ordinary skilled artisan before the effective filing date of the present application to modify the AAV genome encoding a circular RNA in claims 1-25 of U.S. Patent No. 11,718,862 by also locating a polyadenylation (polyA) sequence immediately 5’ to the second inverted terminal repeat and outside of the intronic elements that flank a gene of interest; as well as formulating the AAV genome in a pharmaceutically acceptable carrier, in light of the teachings of Sena-Esteves et al and Wang et al as presented above.
An ordinary skilled artisan would have been motivated to carry out the above modifications because Sena-Esteves et al already taught at least an exemplary rAAV vector comprising two inverted terminal repeats (ITRs) flanking an expression vector on each end along with 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; and a composition comprising the rAAV vector in a pharmaceutical acceptable carrier (e.g., sterile saline, lactose, sucrose and water). Moreover, Wang et al already 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).
An ordinary skilled artisan would have a reasonable expectation of success in light of the AAV genome in claims 1-25 of U.S. Patent No. 11,718,862 along with the teachings of Sena-Esteves et al and Wang et al, coupled with a high level of skill for an ordinary skilled artisan in the relevant art.
The modified AAV genome encoding a circular RNA resulting from claims 1-25 of U.S. Patent No. 11,718,862 along with the teachings of Sena-Esteves et al and Wang et al is indistinguishable and encompassed by the presently claimed invention. Once again, with respect to the “functional wherein” clause of “wherein the circRNA is expressed in a cell or a tissue of a subject following administration of the AAV genome to the subject”, the functional wherein clause does not provide further structural details but merely explains how the AAV genome functions when placed in the requisite environment. 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.
Amended claims 1-3, 6 and 20 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 19-43 of copending Application No. 18/334,530 (reference application).
Although the claims at issue are not identical, they are not patentably distinct from each other because an AAV genome comprising a nucleic acid molecule encoding a circular RNA, wherein the AAV genome comprises, from 5’ to 3’ the elements (a)-(e) as recited in independent claim 19; and an AAV capsid or particle comprising the same AAV genome in claims 19-43 of copending Application No. 18/334,530 anticipate/encompass the claimed genus in the application being examined and, therefore, a patent to the genus would, necessarily, extend the rights of the species or sub- should the genus issue as a patent after the species of sub-genus. Please note that SEQ ID NOs. 15-16 are truncated HIPK3 first and second intronic elements, respectively. With respect to the “functional wherein” clause of “wherein the circRNA is expressed in a cell or a tissue of a subject following administration of the AAV genome to the subject”, the functional wherein clause does not provide further structural details but merely explains how the AAV genome functions when placed in the requisite environment. 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.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Amended claims 1, 4 and 8 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 19-43 of copending Application No. 18/334,530 (reference application) in view of Sena-Esteves et al (WO 2016/172155; IDS) and Wang et al (RNA 21:172-179, 2015; IDS).
The claims of the present application differ from in claims 19-43 of copending Application No. 18/334,530 in reciting specifically that “a polyadenylation (polyA) sequence outside of the intronic elements that flank the gene of interest”; “a composition comprising the nucleic acid molecule of claim 1 in a pharmaceutically acceptable carrier”; and “wherein the circRNA is expressed in a cell or a tissue of a subject following administration of the AAV genome to the subject”.
Before the effective filing date of the present application (11/07/2017), 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). Sena-Esteves et al further taught a composition comprising the rAAVs in a pharmaceutical acceptable carrier (e.g., sterile saline, lactose, sucrose and water) (page 35, second paragraph).
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Additionally, 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).
Accordingly, it would have been obvious for an ordinary skilled artisan before the effective filing date of the present application to modify the AAV genome encoding a circular RNA in claims 19-43 of copending Application No. 18/334,530 by also locating a polyadenylation (polyA) sequence immediately 5’ to the second inverted terminal repeat and outside of the intronic elements that flank a gene of interest; as well as formulating the AAV genome in a pharmaceutically acceptable carrier, in light of the teachings of Sena-Esteves et al and Wang et al as presented above.
An ordinary skilled artisan would have been motivated to carry out the above modifications because Sena-Esteves et al already taught at least an exemplary rAAV vector comprising two inverted terminal repeats (ITRs) flanking an expression vector on each end along with 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; and a composition comprising the rAAV vector in a pharmaceutical acceptable carrier (e.g., sterile saline, lactose, sucrose and water). Moreover, Wang et al already 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).
An ordinary skilled artisan would have a reasonable expectation of success in light of the AAV genome in claims 19-43 of copending Application No. 18/334,530 along with the teachings of Sena-Esteves et al and Wang et al, coupled with a high level of skill for an ordinary skilled artisan in the relevant art.
The modified AAV genome encoding a circular RNA resulting from claims 19-43 of copending Application No. 18/334,530 along with the teachings of Sena-Esteves et al and Wang et al is indistinguishable and encompassed by the presently claimed invention. Once again, with respect to the “functional wherein” clause of “wherein the circRNA is expressed in a cell or a tissue of a subject following administration of the AAV genome to the subject”, the functional wherein clause does not provide further structural details but merely explains how the AAV genome functions when placed in the requisite environment. 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.
This is a provisional nonstatutory double patenting rejection.
Amended claims 1-4, 6, 8 and 20 are provisionally rejected on the ground of nonstatutory double patenting 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).
The claims of the present application differ from claims 20-33 of copending Application No. 17/796,874 in reciting specifically at least “a promoter region outside of the intronic elements that flank the gene of interest”, “a polyadenylation (polyA) sequence outside of the intronic elements that flank the gene of interest”, “wherein the truncated intronic elements of (b) comprise the nucleotide sequence of any of SEQ ID Nos:13-24 and 29-32”, and “wherein the circRNA is expressed in a cell or a tissue of a subject following administration of the AAV genome to the subject”.
Before the effective filing date of the present application (11/07/2017), 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). Sena-Esteves et al further taught a composition comprising the rAAVs in a pharmaceutical acceptable carrier (e.g., sterile saline, lactose, sucrose and water) (page 35, second paragraph).
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Additionally, 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 disclosed that the IRES fragment was amplified from pIRES2-EGFP vector (page 177, right column, last full paragraph), and accordingly the IRES fragment is an encehalomyocarditis virus (ECMV) IRES as evidenced by the pIRES2-EGFP sequence information from lifescience market. 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).
Moreover, 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.
Accordingly, it would have been obvious for an ordinary skilled artisan before the effective filing date of the present application to modify a nucleic acid molecule (e.g., in the form of an AAV vector/particle; see dependent claims 31-33 of the co-pending application) comprising a nucleic acid sequence encoding at least two circular RNAs comprising a first circRNA and a second circRNA in claims 20-33 of copending Application No. 17/796,874 by also locating at least one promoter region between the first AAV ITR and a left backsplicing intronic element, including the sequence of SEQ ID NO: 15, that flanks the first circRNA-encoding sequence, as well as locating a polyadenylation (polyA) sequence immediately 5’ to the second inverted terminal repeat and outside of the intronic elements that flank a gene of interest, in light of the teachings of Sena-Esteves et al, Wang et al and Liang et al as presented above.
An ordinary skilled artisan would have been motivated to carry out the above modifications because Sena-Esteves et al already taught at least an exemplary rAAV vector comprising two inverted terminal repeats (ITRs) flanking an expression vector on each end along with 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; and a composition comprising the rAAV vector in a pharmaceutical acceptable carrier (e.g., sterile saline, lactose, sucrose and water). Moreover, Wang et al already 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). Furthermore, 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 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. Please note that dependent claim 27 of the copending Application already recites the limitation “wherein the left backsplicing intronic element and the right backsplicing intronic element comprises a deletion of about 750 nucleotides”, and therefore the use of truncated intronic elements.
An ordinary skilled artisan would have a reasonable expectation of success in light of claims 20-33 of copending Application No. 17/796,874 along with the teachings of Sena-Esteves et al, Wang et al and Liang et al; coupled with a high level of skill for an ordinary skilled artisan in the relevant art.
The modified nucleic acid molecule (e.g., in the form of an AAV vector/particle) resulting from claims 20-33 of copending Application No. 17/796,874 along with the teachings of Sena-Esteves et al, Wang et al and Liang et al is indistinguishable and encompassed by the presently claimed invention. With respect to the “functional wherein” clause of “wherein the circRNA is expressed in a cell or a tissue of a subject following administration of the AAV genome to the subject”, the functional wherein clause does not provide further structural details but merely explains how the AAV genome functions when placed in the requisite environment. 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.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
In the Amendment dated 09/24/2025 (pages 12-13), Applicant simply requested the above non-statutory double patenting rejections be held in abeyance.
Conclusions
No claim is allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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
cctcagcctctcaaagtgctaggattacagggatctatacttttcttttgagggaaaatgttggcaccgtttctagggcatattggccatttcagcttctcagtaaatatttgttaagtaattaaatgcacttgattctttattcttagccttttaacgcaatactcagaatagctgaagcaccaattaactgaaatggagatattataaagatagttatcttctccaagggaaaaaatcatcttcatggaaattaattacttttttacaaattgtgaatttgacccttaagagttttcttcctgatatttaaaattgaaaaaaaaattgttgacattaatatttcttctttccttttttttcttttcctttttttttttttttttgcaggtatggcctcacaagtcttggtctacccaccatatgtttatcaaactcagtcaagtgccttttgtagtgtgaagaaactcaaagtagagccaagcagttgtgtattccaggaaagaaactatccacggacctatgtgaatggtagaaactttggaaattctcatcctcccactaagggtagtgcttttcagacaaagataccatttaatagacctcgaggacacaacttttcattgcagacaagtgctgttgttttgaaaaacactgcaggtgctacaaaggtcatagcagctcaggcacagcaagctcacgtgcaggcacctcagattggggcgtggcgaaacagattgcatttcctagaaggcccccagcgatgtggattgaagcgcaagagtgaggagttggataatcatagcagcgcaatgcagattgtcgatgaattgtccatacttcctgcaatgttgcaaaccaacatgggaaatccagtgacagttgtgacagctaccacaggatcaaaacagaattgtaccactggagaaggtgactatcagttagtacagcatgaagtcttatgctccatgaaaaatacttacgaagtccttgattttcttggtcgaggcacgtttggccaggtagttaaatgctggaaaagagggacaaatgaaattgtagcaatcaaaattttgaagaatcatccttcttatgcccgtcaaggtcaaatagaagtgagcatattagcaaggctcagtactgaaaatgctgatgaatataactttgtacgagcttatgaatgctttcagcaccgtaaccatacttgtttagtctttgagatgctggaacaaaacttgtatgactttctgaaacaaaataaatttagtcccctgccactaaaagtgattcggcccattcttcaacaagtggccactgcactgaaaaaattgaaaagtcttggtttaattcatgctgatctcaagccagagaatattatgttggtggatcctgttcggcagccttacagggttaaagtaatagactttgggtcggccagtcatgtatcaaagactgtttgttcaacatatctacaatctcggtactacaggtaggtaacaactccatactttttggttgtttattaatgtgaaatttctgctaaatgaaatacttttgtgtgtgtttgtggtagaagagaccacttcagttaaataaggaaatcaagagaggatcaatttaggttcgttttaaagagattaaaaaaaatcaagacataaaatctacccaagcaggatagaaatctccactgcaaagttccatgccaaagacatctggttatttttatttttaatggaagacttgaaggaatgataggtgattaataatgatcaaacagaagtctttaaatgttggaaagtatttacattaatctttgtatatatcattgggcattttagcacttgagagaaatagtttattaaagatataatcaatcatatgtaactgaacatttagaaaaattatatacaggtttgagtagcccttatctgaaacttttggggccagaagtgttttggattccagatttttccggattttggaatatttgcactgccaactagttaagcacccccaaatttgaaaattcgtttcctttgagtgtcatgtcaatgcccaaaaagtttcagatatttggatttgagatgctcaacctgtataaggattcagaaagttattctgattaatgattttaagattcagatatacaataatcccagcaacttgggaggctgaggcaggagaatcacttgaacccaggagatggaggttgcagtgagccgagatcatgccattgcactcca