DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application is being examined under the pre-AIA first to invent provisions.
Applicant’s amendment filed on 08/04/2025 has been entered.
Amended claims 49, 51-59 and new claim 60 are pending in the present application, and they are examined on the merits herein.
Claim Rejections - 35 USC § 112
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.
New claim 60 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. This is a new ground of rejection necessitated by Applicant’s amendment.
The term “approximately” in claim 60 is a relative term which renders the claim indefinite. The term “approximately” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. For example, which of the following 20%, 21%, 22%, 23%, 24%, 26%, 27%, 28%, 29% and 30% would or would not be considered to be approximately 25%? Clarification is requested because the metes and bounds of the claims are not clearly determined.
Claim Rejections - 35 USC § 103
The following is a quotation of pre-AIA 35 U.S.C. 103(a) which forms the basis for all obviousness rejections set forth in this Office action:
(a) A patent may not be obtained though the invention is not identically disclosed or described as set forth in section 102, if the differences between the subject matter sought to be patented and the prior art are such that the subject matter as a whole would have been obvious at the time the invention was made to a person having ordinary skill in the art to which said subject matter pertains. Patentability shall not be negatived by the manner in which the invention was made.
Amended claims 49, 51-53, 56-58 and 60 are rejected under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Kariko et al (WO 2007/024708; IDS). This is a modified rejection necessitated by Applicant’s amendment.
The instant claims are directed to a method of decreasing binding of a polyribonucleotide to retinoic acid-inducible gene I (RIG-1), comprising: (a) providing a polyribonucleotide comprising a sequence which encodes a protein or protein fragment, wherein the polyribonucleotide contains a combination of unmodified and modified nucleotides, wherein 5 to 50%, preferably 5 to 30%, of uridine nucleotides in the polyribonucleotide are 2-thiouridine, and 5 to 50%, preferably 5 to 30%, of cytidine nucleotides in the polyribonucleotide are 5-methylcytidine, wherein the remainder of the adenosine triphosphate, cytidine triphosphate, guanosine triphosphate and uridine triphosphate in the polyribonucleotide does not include a modified nucleoside, and wherein the modified nucleotides and their percentages are selected to minimize binding of the polyribonucleotide to RIG-1 relative to a control polyribonucleotide that does not comprise the modified nucleotides; (b) providing the polyribonucleotide so produced; and (c) administering the polyribonucleotide to a subject or cell, wherein the protein or protein fragment encoded by the polyribonucleotide is expressed in the subject or cell, and wherein the polyribonucleotide has decreased binding to RIG-1 relative to a control polyribonucleotide that does not comprise the modified nucleotides; the same method further comprising encapsulating said polyribonucleotide in a nanoparticle or a cationic lipid.
Kariko et al already disclosed at least an in vitro transcribed RNA molecule (e.g., mRNA) comprising pseudouridine and/OR a modified nucleoside, wherein the modified nucleoside is m5C (5-methylcytidine), m5U, m6A, s2U (2-thiouridine), pseudouridine (ψ) or 2’-O-methyl-U, including a modified RNA molecule encoding Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) or EPO for treating cystic fibrosis and anemia in a subject, respectively; wherein the modified RNA molecule has reduced immunogenicity and/or enhancing translation efficiency (Abstract; Summary of the Invention; particularly paragraphs 5-7, 10-11, 17, 21-22, 43, 47-48, 56, 62, 68-69, 71, 74, 76, 87-88, 100, 102, 114, 143, 207-208, 212, 214, 218, 275-276 and Examples 2, 7, 10-15 and 23). Kariko et al stated explicitly “In another embodiment, an RNA, oligoribonucleotide, or polyribonucleotide molecule of methods and compositions of the invention comprises a combination of 2 or more of the above modifications. In another embodiment, the RNA molecule or oligoribonucleotide molecule comprises a combination of 3 or more of the above modifications” (paragraph 71); and “In another embodiment, 0.1% of the residues of a given nucleotide (uridine, cytidine, guanosine, or adenine) are modified. In another embodiment, the fraction of the nucleotide is 0.2%....In another embodiment, the fraction is 5%. In another embodiment, the fraction is 6%. In another embodiment, the fraction is 8%. In another embodiment, the fraction is 10%....In another embodiment, the fraction is 20%. In another embodiment, the fraction is 25%. In another embodiment, the fraction is 30%. In another embodiment, the fraction is 35%. In another embodiment, the fraction is 40%. In another embodiment, the fraction is 45%. In another embodiment, the fraction is 50%” (paragraph 74). Kariko et al also taught that the modified RNA molecule is encapsulated in a nanoparticle, and methods for nanoparticle packaging are well known in the art (paragraph 118 and claim 7); and/or the modified RNA molecule is mixed with a cationic lipid transfection reagent (paragraphs 94-95). Kariko et al also disclosed a method for inducing a mammalian cell to produce a recombinant protein comprising contacting the mammalian cell with the above in vitro-synthesized modified RNA; and a treatment method for a subject comprising contacting a cell of the subject with the above in vitro-synthesized modified RNA encoding a therapeutic protein such as CFTR and EPO (paragraphs 10-11, 17, 104 and 114). In an exemplification, Kariko et al disclosed in vitro transcription reactions being performed in which 1 or 2 of the 4 nucleotide triphosphates (NTP) were substituted with a corresponding nucleoside-modified NTP (Example 2; paragraphs 187 and 194); and Fig. 2B below shows that nucleoside modifications such as m6A, m5C, m5U, s2U and pseudouridine, alone and in combination, reduce immunogenicity of RNA as mediated by TLR3, TLR7 and TLR8 signaling in comparison to unmodified, in vitro transcribed RNA..
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In another exemplification, Kariko et al determined the minimal frequency of particular modified nucleosides (1%, 10%, 50%, 90%, 99% and 100% of m6A, pseudouridine, or m5C) that is sufficient to reduce immunogenicity, and Figure 5 below demonstrated that pseudouridine and modified nucleosides reduce the immunogenicity of RNA molecules, even when present as a small fraction of the residues (Example 7; and paragraphs 212-214).
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Kariko et al further demonstrated that exemplary pseudouridine- and m5C-modified RNAs were translated almost 10-times and 4 times more efficiently, respectively, than unmodified mRNA in cultured cells (Example 11 and Figure 10); and in vivo (Example 12; paragraphs 231-233; Figure 11; and Examples 13-15, Figures 13-15).
Kariko et al did not teach explicitly a method using a polyribonucleotide encoding a protein or protein fragment, that contains a combination of 5 to 50% of uridine nucleotides as 2-thiouridine nucleotides and 5 to 50% of cytidine nucleotides as 5-methylcytidine nucleotides, wherein the modified nucleotides and their percentages are selected to minimize binding of the polyribonucleotide to RIG-1 relative to a control polyribonucleotide that does not comprise the modified nucleotides, and wherein the polyribonucleotide has decreased binding to RIG-1 relative to a control polyribonucleotide that does not comprise the modified nucleotides.
However, since Kariko et al taught an in vitro transcribed RNA molecule (e.g., mRNA) comprising pseudouridine and/OR a modified nucleoside, wherein the modified nucleoside is m5C (5-methylcytidine), m5U, m6A, s2U (2-thiouridine), pseudouridine (ψ) or 2’-O-methyl-U, including a modified RNA molecule encoding Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) or EPO for treating cystic fibrosis and anemia in a subject, respectively; the RNA molecule comprises a combination of 2 or more of the above modifications, wherein the fraction of a given nucleotide (uridine, cytidine, guanosine or adenine) to be modified can be 5%, 10%, 25%, 30% or 50%; coupled with exemplary modified RNA molecules with 1%, 10%, 50%, 90%, 99% or 100% of m6A, pseudouridine or 5-methyl cytidine (m5C) have reduced immunogenicity even when modified nucleosides are present as a small fraction of the RNA molecules, and exemplary RNA molecules with m6A, m5C, m5U, s2U and pseudouridine nucleosides, alone or in combination such as the combination of m5C and pseudouridine modified nucleosides, reduce immunogenicity of RNA mediated by TLR3, TLR7 and TLR8 signaling relative to control unmodified RNA molecules; it would have been obvious to an ordinary skilled in the art that the teachings of Kariko et al would also encompass or modify the teachings of Kariko et al to prepare at least an in vitro transcribed RNA molecule coding for a therapeutically useful protein, which comprises a combination of 5 to 50% of uridine nucleotides as 2-thiouridine nucleotides and 5 to 50% of cytidine nucleotides as 5-methylcytidine nucleotides, while the remainder of the nucleotides in the RNA molecule contains no modified nucleosides; and the RNA molecule has reduced immunogenicity and/or enhanced translation efficiency. Additionally, since the modified RNA molecule containing a combination of 5 to 50% of uridine nucleotides as 2-thiouridine nucleotides and 5 to 50% of cytidine nucleotides as 5-methylcytidine nucleotides of Kariko et al is structurally indistinguishable from the polyribonucleotide used in the claimed method of the present application, such modified RNA polyribonucleotide would also possess a decreased binding to RIG-1 relative to a control unmodified polyribonucleotide and/or whether Kariko et al recognized or not that such modified RNA molecule is selected for minimizing binding of the polyribonucleotide to RIG-1 relative to an unmodified RNA molecule, particularly Kariko et al already demonstrated that exemplary RNA molecules with m6A, m5C, m5U, s2U and pseudouridine nucleosides, alone or in combination such as the combination of m5C and pseudouridine modified nucleosides, reduce immunogenicity of RNA mediated by TLR3, TLR7 and TLR8 signaling relative to control unmodified RNA molecules. Please, also note that where, as here, the claimed and prior art products are identical or substantially identical, or are produced by identical or substantially identical processes, the PTO can require an applicant to prove that the prior art products do not necessarily or inherently possess the characteristics of his claimed product. See In re Ludtke. Whether the rejection is based on "inherency" under 35 USC 102, or "prima facie obviousness" under 35 USC 103, jointly or alternatively, the burden of proof is the same, and its fairness is evidenced by the PTO's inability to manufacture products or to obtain and compare prior art products. In re Best, Bolton, and Shaw, 195 USPQ 430, 433 (CCPA 1977) citing In re Brown, 59 CCPA 1036, 459 F.2d 531, 173 USPQ 685 (1972). Accordingly, the method of administering a modified RNA polyribonucleotide containing a combination of 5 to 50% of uridine nucleotides as 2-thiouridine nucleotides and 5 to 50% of cytidine nucleotides as 5-methylcytidine nucleotides of Kariko et al to a mammalian cell in a subject is also indistinguishable from a method of decreasing binding of polyribonucleotide to RIG-1 as claimed.
Therefore, the claimed invention as a whole was prima facie obvious in the absence of evidence to the contrary.
Claims 54-55 and 59 are rejected under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Kariko et al (WO 2007/024708; IDS) as applied to claims 49, 51-53, 56-58 and 60 above, and further in view of Naldini et al (US 2010/0041737; IDS) and Adamis et al (WO 2005/014814).
The teachings of Kariko et al were presented above. However, Kariko et al did not teach or suggest that the modified polyribonucleotide further includes a micro-RNA binding site (claim 54), preferably the micro-RNA binding site is 3’ to the sequence encoding a protein or protein fragment and 5’ from a 3’ polyA tail (claim 55); and the method further comprising the step of introducing the polyribonucleotide into a delayed release matrix before the step of administration (claim 59).
At the effective filing date of the present application (07/31/2009), Naldini et al already taught vectors (e.g., DNA or RNA vectors, viral vectors such as lentiviral vectors as well as non-viral vectors) for transgene expression for gene transfer and therapy can be engineered with miRNAs target sequence in order to be recognized by endogenous miRNAs cell type specific, thus regulating transgene expression in a subset of cells (see at least Statements of the Inventions and Some further key Advantages of the Invention). Naldini et al also stated explicitly “Expression vectors as described herein comprise regions of nucleic acid containing sequences capable of being transcribed. Thus, sequences encoding mRNA, tRNA and rRNA are included within this definition” (paragraph 20); and “As a proof-of-principle that miRNA can be used to de-target transgene expression from particular cell types, we developed an LV which can provide robust expression in hepatocytes and other non-hematopoietic cells, while preventing expression from hematopoietic cells. This design is particularly relevant for systemic gene therapy in which the host immune response against the transgene limits therapeutic efficacy (Brown and Lillicrap, 2002). Studies from our laboratory and others indicate that a major factor contributing to the induction of a transgene-specific immune response following gene transfer is related to the site of transgene expression (Brown et al, 2004b; Follenzi et al., 2004). Vectors that are expressed in APCs of the hematopoietic system, such as macrophages and dendritic cells, are known to effectively trigger anti-transgene immune responses (De Geest et al., 2003)” (paragraph 47). In an exemplification, Naldini et al constructed a miRNA-regulated lentiviral vector by inserting four tandem copies of a 23 bp sequence (mirT) with perfect complementarily to either mir-30a, mir-142-5p or mir-142-3p into the 3’-untranslated region (3’UTR) of a GFP expression cassette driven by the ubiquitously expressed PGK promoter (paragraph 276 and Fig. 1a).
Additionally, Adamis et al also taught the use of a variety of biocompatible and biodegradable polymers such as poly (lactic acid), poly (glycolic acid), poly (lactic-co-glycolic acid), and poly (caprolactone) for a sustained or controlled release of an aptamer (e.g., RNA aptamers, DNA aptamers, or aptamers having a mixed of both RNA and DNA aptamers) depending on the rate of drug release required in a particular treatment regimen (see at least Abstract; Summary of the Invention; particularly page 21, line 30 continues to line 11 at page 22).
It would have been obvious for an ordinary skilled artisan to modify the teachings of Kariko et al by also further incorporating into the modified polyribonucleotide a micro-RNA binding site in the 3’UTR which is followed by a poly(A) tail; as well as further introducing the modified polyribonucleotide into a delayed/sustained release matrix such as poly (lactic acid), poly (glycolic acid), poly (lactic-co-glycolic acid), and poly (caprolactone) prior to the administration step based on the rate of drug release required by a treatment regimen; in light of the teachings of Naldini et al and Adamis et al as presented above.
An ordinary skilled artisan would have been motivated to further carry out the above modifications because Naldini et al already taught that transgene expression can be engineered with miRNAs target sequence in order to be recognized by endogenous miRNAs cell type specific, and thus regulating transgene expression in a subset of cells. Naldini et al successfully demonstrated that miRNA can be used to de-target transgene expression from particular cell types, particularly APCs of the hematopoietic system, such as macrophages and dendritic cells, are known to effectively trigger anti-transgene immune responses. Please also note that the primary Kakiko reference already taught the use of a modified polyribonucleotide containing a poly(A) tail, and in an exemplification demonstrated that the addition of a polyA tail to pseudouridine-containing mRNA further increases translation efficiency (Example 10, particularly paragraph [00225]). Moreover, Adamis et al already taught the use of a variety of biocompatible and biodegradable polymers such as poly (lactic acid), poly (glycolic acid), poly (lactic-co-glycolic acid), and poly (caprolactone) for a sustained or controlled release of an aptamer depending on the rate of drug release required in a particular treatment regimen.
An ordinary skilled artisan would have a reasonable expectation of success to carry out the above modifications in light of the teachings of Kariko et al, Naldini et al and Adamis et al; coupled with the level of skill for an ordinary skilled artisan in the relevant art.
The modified method resulting from the combined teachings of Kariko et al, Naldini et al and Adamis et al as set forth above is indistinguishable from and encompassed by the presently claimed method.
Therefore, the claimed invention as a whole was prima facie obvious in the absence of evidence to the contrary.
Amended claims 49, 51-53 and 56-60 are rejected under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Kariko et al (WO 2007/024708; IDS) in view of Geall et al (US 2011/0300205). This is a modified rejection necessitated by Applicant’s amendment.
The instant claims are directed to a method of decreasing binding of a polyribonucleotide to retinoic acid-inducible gene I (RIG-1), comprising: (a) providing a polyribonucleotide comprising a sequence which encodes a protein or protein fragment, wherein the polyribonucleotide contains a combination of unmodified and modified nucleotides, wherein 5 to 50%, preferably 5 to 30%, of uridine nucleotides in the polyribonucleotide are 2-thiouridine, and 5 to 50%, preferably 5 to 30%, of cytidine nucleotides in the polyribonucleotide are 5-methylcytidine, wherein the remainder of the adenosine triphosphate, cytidine triphosphate, guanosine triphosphate and uridine triphosphate in the polyribonucleotide does not include a modified nucleoside, and wherein the modified nucleotides and their percentages are selected to minimize binding of the polyribonucleotide to RIG-1 relative to a control polyribonucleotide that does not comprise the modified nucleotides; (b) providing the polyribonucleotide so produced; and (c) administering the polyribonucleotide to a subject or cell, wherein the protein or protein fragment encoded by the polyribonucleotide is expressed in the subject or cell, and wherein the polyribonucleotide has decreased binding to RIG-1 relative to a control polyribonucleotide that does not comprise the modified nucleotides; the same method further comprising encapsulating said polyribonucleotide in a nanoparticle or a cationic lipid, or further comprising the step of introducing the polyribonucleotide into a delayed release matrix before the step of administration.
Kariko et al already disclosed at least an in vitro transcribed RNA molecule (e.g., mRNA) comprising pseudouridine and/OR a modified nucleoside, wherein the modified nucleoside is m5C (5-methylcytidine), m5U, m6A, s2U (2-thiouridine), pseudouridine (ψ) or 2’-O-methyl-U, including a modified RNA molecule encoding Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) or EPO for treating cystic fibrosis and anemia in a subject, respectively; wherein the modified RNA molecule has reduced immunogenicity and/or enhancing translation efficiency (Abstract; Summary of the Invention; particularly paragraphs 5-7, 10-11, 17, 21-22, 43, 47-48, 56, 62, 68-69, 71, 74, 76, 87-88, 100, 102, 114, 143, 207-208, 212, 214, 218, 275-276 and Examples 2, 7, 10-15 and 23). Kariko et al stated explicitly “In another embodiment, an RNA, oligoribonucleotide, or polyribonucleotide molecule of methods and compositions of the invention comprises a combination of 2 or more of the above modifications. In another embodiment, the RNA molecule or oligoribonucleotide molecule comprises a combination of 3 or more of the above modifications” (paragraph 71); and “In another embodiment, 0.1% of the residues of a given nucleotide (uridine, cytidine, guanosine, or adenine) are modified. In another embodiment, the fraction of the nucleotide is 0.2%....In another embodiment, the fraction is 5%. In another embodiment, the fraction is 6%. In another embodiment, the fraction is 8%. In another embodiment, the fraction is 10%....In another embodiment, the fraction is 20%. In another embodiment, the fraction is 25%. In another embodiment, the fraction is 30%. In another embodiment, the fraction is 35%. In another embodiment, the fraction is 40%. In another embodiment, the fraction is 45%. In another embodiment, the fraction is 50%” (paragraph 74). Kariko et al also taught that the modified RNA molecule is encapsulated in a nanoparticle, and methods for nanoparticle packaging are well known in the art (paragraph 118 and claim 7); and/or the modified RNA molecule is mixed with a cationic lipid transfection reagent (paragraphs 94-95). Kariko et al also disclosed a method for inducing a mammalian cell to produce a recombinant protein comprising contacting the mammalian cell with the above in vitro-synthesized modified RNA; and a treatment method for a subject comprising contacting a cell of the subject with the above in vitro-synthesized modified RNA encoding a therapeutic protein such as CFTR and EPO (paragraphs 10-11, 17, 104 and 114). In an exemplification, Kariko et al disclosed in vitro transcription reactions being performed in which 1 or 2 of the 4 nucleotide triphosphates (NTP) were substituted with a corresponding nucleoside-modified NTP (Example 2; paragraphs 187 and 194); and Fig. 2B below shows that nucleoside modifications such as m6A, m5C, m5U, s2U and pseudouridine, alone and in combination, reduce immunogenicity of RNA as mediated by TLR3, TLR7 and TLR8 signaling in comparison to unmodified, in vitro transcribed RNA..
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In another exemplification, Kariko et al determined the minimal frequency of particular modified nucleosides (1%, 10%, 50%, 90%, 99% and 100% of m6A, pseudouridine, or m5C) that is sufficient to reduce immunogenicity, and Figure 5 below demonstrated that pseudouridine and modified nucleosides reduce the immunogenicity of RNA molecules, even when present as a small fraction of the residues (Example 7; and paragraphs 212-214).
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Kariko et al further demonstrated that exemplary pseudouridine- and m5C-modified RNAs were translated almost 10-times and 4 times more efficiently, respectively, than unmodified mRNA in cultured cells (Example 11 and Figure 10); and in vivo (Example 12; paragraphs 231-233; Figure 11; and Examples 13-15, Figures 13-15).
Kariko et al did not teach explicitly a method using a polyribonucleotide encoding a protein or protein fragment, that contains a combination of 5 to 50% of uridine nucleotides as 2-thiouridine nucleotides and 5 to 50% of cytidine nucleotides as 5-methylcytidine nucleotides, wherein the modified nucleotides and their percentages are selected to minimize binding of the polyribonucleotide to RIG-1 relative to a control polyribonucleotide that does not comprise the modified nucleotides, and wherein the polyribonucleotide has decreased binding to RIG-1 relative to a control polyribonucleotide that does not comprise the modified nucleotides; or the same method further comprising the step of introducing the polyribonucleotide into a delayed release matrix before the step of administration.
At the effective filing date of the present application (07/31/2009), Geall et al already taught that when unmodified RNA is delivered to cells by viral or non-viral delivery, the RNA is recognized as foreign nucleic acid by endosomal and cytoplasmic immune receptors, such as the toll-like receptors 3, 7 and 8 of the endosomes, retinoic acid-induced gene (RIG-1), melanoma differentiation-associated gene-5 (MDA-5) and laboratory of genetics and physiology-2 (LGP2) receptors of the cytoplasm (paragraph 45). Geall et al also taught that stimulation of the above immune receptors by a self-replicating RNA that does not include modified nucleotides is expected to modulate the immune response which could impact expression of gene products encoded by the RNA, amplification of and adjuvant effect of the self-replicating RNA, the immune response to encoded proteins (i.e., decreased potency of vaccine), and could also lead to safety concerns, such as injection site irritation, inflammation and/or pain (paragraph 45). Accordingly, Geall et al disclosed self-replicating RNA molecules that contain modified nucleotides to avoid or reduce stimulation of endosomal and cytoplasmic immune receptors when the self-replicating RNA is delivered into a cell, wherein 0.01%-25% of a particular nucleotide are modified nucleotides, or wherein 0.01%-25% of two, three or four particular nucleotides are substituted nucleotides, and wherein the self-replicating RNA molecule comprising at least one of modified nucleosides selected from a group that includes 2-thiouracil, 5-methylcytosine, pseudouridine, N6-methyladenosine among others and any combination thereof (see at least Abstract; Summary of the Invention; particularly paragraphs 8-9, 16-20, 24, 45-52, 67-69; and Example 3). Moreover, Geall et al also disclosed that the use of various non-toxic, biodegradable polymers (matrix) to form microparticles to encapsulate or adsorb RNA, including poly(lactides) (“PLA”), copolymers of lactide and glycolide such as poly (D,L-lactide-coglycolide) (“PLG”), and co-polymers of D,L-lactide and caprolactone; all of which are known in the prior art as “delayed release matrix/polymer” (paragraphs [0097]-[0101]).
It would have been obvious for an ordinary skilled artisan to modify the teachings of Kariko et al by also producing a polyribonucleotide that encodes a therapeutically useful protein, that contains a combination of 5 to 50% of uridine nucleotides as 2-thiouridine nucleotides and 5 to 50% of cytidine nucleotides as 5-methylcytidine nucleotides, with the remainder of the nucleotides in the polyribonucleotide contains no modified nucleosides; and the polyribonucleotide has reduced immunogenicity and/or enhancing translation efficiency for treatment of a subject, wherein such modified polyribonucleotide also has decreased binding to RIG-1 relative to a control polyribonucleotide that does not comprise the modified nucleotides; as well as further introducing the modified polyribonucleotide into a delayed release matrix in the form of microparticles comprised of poly(lactides), poly(D,L-lactide-coglycolide) and/or co-polymers of D,L-lactide and caprolactone prior to the step of administration; in light of the teachings of Geall et al as presented above.
An ordinary skilled artisan would have been motivated to carry out the above modifications because Kariko et al already taught an in vitro transcribed RNA molecule (e.g., mRNA) comprising pseudouridine and/OR a modified nucleoside, wherein the modified nucleoside is m5C (5-methylcytidine), m5U, m6A, s2U (2-thiouridine), pseudouridine (ψ) or 2’-O-methyl-U, including a modified RNA molecule encoding Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) or EPO for treating cystic fibrosis and anemia in a subject, respectively; the RNA molecule comprises a combination of 2 or more of the above modifications, wherein the fraction of a given nucleotide (uridine, cytidine, guanosine or adenine) to be modified can be 5%, 10%, 25%, 30% or 50%; coupled with exemplary modified RNA molecules with 1%, 10%, 50%, 90%, 99% or 100% of m6A, pseudouridine or 5-methyl cytidine (m5C) have reduced immunogenicity even when modified nucleosides are present as a small fraction of the RNA molecules, and exemplary RNA molecules with m6A, m5C, m5U, s2U and pseudouridine nucleosides, alone or in combination, specifically the combination of m5C and pseudouridine modified nucleosides, reduce immunogenicity of RNA mediated by TLR3, TLR7 and TLR8 signaling relative to control unmodified RNA molecules. Additionally, since Geall et al already taught that when unmodified RNA is delivered to cells by viral or non-viral delivery, the RNA is recognized as foreign nucleic acid by endosomal and cytoplasmic immune receptors, such as the toll-like receptors 3, 7 and 8 of the endosomes, retinoic acid-induced gene (RIG-1), melanoma differentiation-associated gene-5 (MDA-5) and laboratory of genetics and physiology-2 (LGP2) receptors of the cytoplasm; and such modified RNA molecule containing a combination of 5 to 50% of uridine nucleotides as 2-thiouridine nucleotides and 5 to 50% of cytidine nucleotides as 5-methylcytidine nucleotides of Kariko et al is structurally indistinguishable from the polyribonucleotide used in the claimed method of the present application, and such modified RNA polyribonucleotide would also possess a decreased binding to RIG-1 relative to a control unmodified polyribonucleotide and/or whether Kariko et al recognized or not that such modified RNA molecule is selected for minimizing binding of the polyribonucleotide to RIG-1 relative to an unmodified RNA molecule; particularly Kariko et al already demonstrated that exemplary RNA molecules with m6A, m5C, m5U, s2U and pseudouridine nucleosides, alone or in combination such as the combination of m5C and pseudouridine modified nucleosides, reduce immunogenicity of RNA mediated by TLR3, TLR7 and TLR8 signaling relative to control unmodified RNA molecules. Moreover, Geall et al already disclosed the use of various non-toxic, biodegradable polymers (matrix) to form microparticles to encapsulate or adsorb RNA, including poly(lactides) (“PLA”), copolymers of lactide and glycolide such as poly (D,L-lactide-coglycolide) (“PLG”), and co-polymers of D,L-lactide and caprolactone; all of which are known in the prior art as “delayed release matrix/polymer”.
An ordinary skilled artisan would have a reasonable expectation of success to carry out the above modifications in light of the teachings of Kariko et al and Geall et al; coupled with the level of skill for an ordinary skilled artisan in the relevant art.
The modified method resulting from the combined teachings of Kariko et al and Geall et al as set forth above is indistinguishable from and encompassed by the method as claimed of the present application.
Therefore, the claimed invention as a whole was prima facie obvious in the absence of evidence to the contrary.
Claims 54-55 are rejected under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Kariko et al (WO 2007/024708; IDS) in view of Geall et al (US 2011/0300205) as applied to claims 49, 51-53 and 56-60 above, and further in view of Naldini et al (US 2010/0041737; IDS).
The combined teachings of Kariko et al and Geall et al were presented above. However, none of the cited references teach or suggest that the modified polyribonucleotide further includes a micro-RNA binding site (claim 54), preferably the micro-RNA binding site is 3’ to the sequence encoding a protein or protein fragment and 5’ from a 3’ polyA tail (claim 55).
At the effective filing date of the present application (07/31/2009), Naldini et al already taught vectors (e.g., DNA or RNA vectors, viral vectors such as lentiviral vectors as well as non-viral vectors) for transgene expression for gene transfer and therapy can be engineered with miRNAs target sequence in order to be recognized by endogenous miRNAs cell type specific, thus regulating transgene expression in a subset of cells (see at least Statements of the Inventions and Some further key Advantages of the Invention). Naldini et al also stated explicitly “Expression vectors as described herein comprise regions of nucleic acid containing sequences capable of being transcribed. Thus, sequences encoding mRNA, tRNA and rRNA are included within this definition” (paragraph 20); and “As a proof-of-principle that miRNA can be used to de-target transgene expression from particular cell types, we developed an LV which can provide robust expression in hepatocytes and other non-hematopoietic cells, while preventing expression from hematopoietic cells. This design is particularly relevant for systemic gene therapy in which the host immune response against the transgene limits therapeutic efficacy (Brown and Lillicrap, 2002). Studies from our laboratory and others indicate that a major factor contributing to the induction of a transgene-specific immune response following gene transfer is related to the site of transgene expression (Brown et al, 2004b; Follenzi et al., 2004). Vectors that are expressed in APCs of the hematopoietic system, such as macrophages and dendritic cells, are known to effectively trigger anti-transgene immune responses (De Geest et al., 2003)” (paragraph 47). In an exemplification, Naldini et al constructed a miRNA-regulated lentiviral vector by inserting four tandem copies of a 23 bp sequence (mirT) with perfect complementarily to either mir-30a, mir-142-5p or mir-142-3p into the 3’-untranslated region (3’UTR) of a GFP expression cassette driven by the ubiquitously expressed PGK promoter (paragraph 276 and Fig. 1a).
It would have been obvious for an ordinary skilled artisan to further modify the combined teachings of Kariko et al and Geall et al by also further incorporating into the modified polyribonucleotide a micro-RNA binding site in the 3’UTR which is followed by a poly(A) tail; in light of the teachings of Naldini et al as presented above.
An ordinary skilled artisan would have been motivated to further carry out the above modification because Naldini et al already taught that transgene expression can be engineered with miRNAs target sequence in order to be recognized by endogenous miRNAs cell type specific, and thus regulating transgene expression in a subset of cells. Naldini et al successfully demonstrated that miRNA can be used to de-target transgene expression from particular cell types, particularly APCs of the hematopoietic system, such as macrophages and dendritic cells, are known to effectively trigger anti-transgene immune responses. Please note that the primary Kakiko reference already taught the use of a modified polyribonucleotide containing a poly(A) tail, and in an exemplification demonstrated that the addition of a polyA tail to pseudouridine-containing mRNA further increases translation efficiency (Example 10, particularly paragraph [00225]).
An ordinary skilled artisan would have a reasonable expectation of success to carry out the above modification in light of the teachings of Kariko et al, Geall et al and Naldini et al; coupled with the level of skill for an ordinary skilled artisan in the relevant art.
The modified method resulting from the combined teachings of Kariko et al, Geall et al and Naldini et al as set forth above is indistinguishable from and encompassed by the presently claimed method.
Therefore, the claimed invention as a whole was prima facie obvious in the absence of evidence to the contrary.
Response to Argument
Applicant’s arguments related to the above modified 103 rejections in the Amendment filed on 08/04/2025 (pages 4-11) and in the previous Amendment filed on 12/05/2024 (pages 4-13) have been fully considered, but they are respectfully not found persuasive for the reason discussed below.
A. New limitation “and wherein the modified nucleotides and their percentages are selected to minimize binding of the polyribonucleotide to RIG-1”.
Applicant argued that the Office has failed to set forth a prima facie case of obviousness over the amended claims for failing to provide convincing articulated reasoning with some rational underpinning related to producing the claimed polyribonucleotides, including the selection of modified nucleotides and their percentages to minimize binding of the polyribonucleotide to RIG-1. Particularly, Kariko fails to even mention RIG-1 in the document. Thus, currently amended claims are novel and non-obvious over Kariko. With respect to the rejection based on Kariko and Geall, Applicant argued that Geall does not disclose the binary combination of 5-methylcytidine and 2-thiouridine in the claimed percentages, nor link either of the 5-methylcytidine or 2-thiouridine and any substitution percentage in a polyribonucleotide with minimizing the binding of the polyribonucleotide to RIG-1. Consequently, Geall fails to remedy the deficit of Kariko. With respect to supplementary teachings of Naldini and/or Adamis, Applicant argued that none of these supplementary references remedy the shortcomings of Kariko or the combination of Kariko and Geall.
First, please refer to the above modified 103 rejections for details. With respect to the new limitation, since the modified RNA molecule containing a combination of 5 to 50% of uridine nucleotides as 2-thiouridine nucleotides and 5 to 50% of cytidine nucleotides as 5-methylcytidine nucleotides of Kariko et al is structurally indistinguishable from the polyribonucleotide used in the claimed method of the present application, such modified RNA polyribonucleotide would also possess a decreased binding to RIG-1 relative to a control unmodified polyribonucleotide and/or whether Kariko et al recognized or not that such modified RNA molecule is selected for minimizing binding of the polyribonucleotide to RIG-1 relative to an unmodified RNA molecule, particularly Kariko et al already demonstrated that exemplary RNA molecules with m6A, m5C, m5U, s2U and pseudouridine nucleosides, alone or in combination such as the combination of m5C and pseudouridine modified nucleosides, reduce immunogenicity of RNA mediated by TLR3, TLR7 and TLR8 signaling relative to control unmodified RNA molecules. Please, also note that where, as here, the claimed and prior art products are identical or substantially identical, or are produced by identical or substantially identical processes, the PTO can require an applicant to prove that the prior art products do not necessarily or inherently possess the characteristics of his claimed product. See In re Ludtke. Whether the rejection is based on "inherency" under 35 USC 102, or "prima facie obviousness" under 35 USC 103, jointly or alternatively, the burden of proof is the same, and its fairness is evidenced by the PTO's inability to manufacture products or to obtain and compare prior art products. In re Best, Bolton, and Shaw, 195 USPQ 430, 433 (CCPA 1977) citing In re Brown, 59 CCPA 1036, 459 F.2d 531, 173 USPQ 685 (1972). Accordingly, the method of administering a modified RNA polyribonucleotide containing a combination of 5 to 50% of uridine nucleotides as 2-thiouridine nucleotides and 5 to 50% of cytidine nucleotides as 5-methylcytidine nucleotides of Kariko et al to a mammalian cell in a subject is also indistinguishable from a method of decreasing binding of polyribonucleotide to RIG-1 as claimed.
Second, with respect to the rejection based on Kariko and Geall it should be noted that none of the cited references individually has to teach every limitation of the instant claims. It is also apparent that Applicant considered each of the cited references in total isolation one from the other, and did not consider the specific combination of Kariko and Geall. Particularly, Geall et al already taught that when unmodified RNA is delivered to cells by viral or non-viral delivery, the RNA is recognized as foreign nucleic acid by endosomal and cytoplasmic immune receptors, such as the toll-like receptors 3, 7 and 8 of the endosomes, retinoic acid-induced gene (RIG-1), melanoma differentiation-associated gene-5 (MDA-5) and laboratory of genetics and physiology-2 (LGP2) receptors of the cytoplasm. This means that modified RNA would be less recognized as foreign nucleic acids by at least RIG-1 (or less binding to RIG-1).
Third, Naldini and/or Adamis were cited primarily to supplement the teachings of Kariko or the combination of Kariko and Geall for the limitations recited in dependent claims.
B. Unexpected Superior Results?
Applicant argued that the Examiner has ignored the data presented in the post-filing Kormann reference (Nature Biotechnology 29:154-157, 2011; IDS) stating “We found that replacement of only 25% of uridine and cytidine with 2-thiouridine and 5-methyl-cytidine synergistically decreased mRNA binding to pattern recognition receptors, such as TLR3, TLR7, TLR8 and RIG-1, in human peripheral blood mononuclear cells (PBMCs) (Fig 1a).” (page 1, right column). The Fig 1a in the Kormann reference corresponds to Figure 2C of the as-filed specification. Additionally, Applicant argued that Figure 1d in the Kormann reference (corresponding to Figure 1C of the present application) also demonstrates synergistically enhanced stability of red fluorescent protein (RFP) mRNA comprising modified nucleotides as claimed. Accordingly, the examiner has improperly ignored important evidence depicting the synergistic enhancement of mRNA modified according to the claimed invention by making and maintaining the 103 rejections over Kariko et al and Kariko et al in view of Geall et al. Applicant also argued that the Kariko reference fails to disclose the claimed method, and completely fails to even mention RIG-1. Thus, the Kariko reference is incapable of singling out the binary combination of nucleotide modifications and their amounts, and can only be potentially arrived at by picking and choosing which arrive at the unexpected superior results demonstrated in Applicant’s specification and accompanying Figures 1A/1D, as further evidenced by the data presented in the post-filing Kormann reference. In the latest response dated 08/04/2025, Applicant argued that Applicant is not required to compare the claimed invention with subject matter that does not exist in the prior art, including providing evidence for the superior results asserted by Applicant. Unlike Kariko disclosing a polyribonucleotide in which 100% of the uridines are 2-thiouridine, a polyribonucleotide in which 100% of the cytidines are 5-methylcytidine, and a polyribonucleotide in which 100% of uridines are pseudo-uridine (Example 2 in Kariko), Applicant has tested the claimed invention against art that is closer than that cited by the examiner with a polyribonucleotide comprising 50% uridine nucleotides as 2-thiouridine and a polyribonucleotide comprising 50% cytidine nucleotides as 5-methylcytidne as depicted in Figures 1B and 1C of the present application; and the examiner has improperly dismissed the markedly reduced binding to RIG-1 (Figure 2C of the present application) and markedly increased total expression (Figures 1B and 1C of the present application). Applicant also argued when taking the data of Figures 1B, 1C and 11C together, it is clear that Applicant has demonstrated unexpected results over more than half of each claimed percentage range of m5C and s2U, particularly a clear trend of increasing expression can be seen when the content of s2U/m5C diminishes from 100% to 50% to 25% in Figures 1B and 1C; coupled with the already demonstrated unexpected superior results over more than half of each percentage range, an ordinary skilled artisan would have expected these superior results to extend to polyribonucleotides having less than 25% m5C/s2U and greater than 5%, 7.5% and 10% m5C/s2U, as currently claimed.
First, Figure 2C of the present application (corresponding to Fig 1a in the Kormann reference) was reproduced below. It is noted that the Figure does not contain any data for m5C(0.25) polyribonucleotide nor does it contain data for s2U(0.25) polyribonucleotide; and therefore an ordinary skill in the art would not be able to conclude whether the effect observed for the s2U(0.25)m5C(0.25) polyribonucleotide in the Figure is “synergistic”, “additive”, or even “non-additive”, let alone “unexpected superior” results as asserted by Applicant.
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The examiner also notes that there might be a typo error in Figure 2C, namely should the label “s2(0.5)m5C(0.25)” be s2(0.25)m5C(0.25)? By stating that replacement of only 25% of uridine and cytidine with 2-thiouridine and 5-methyl-cytidine synergistically decreased mRNA binding to pattern recognition receptors such as TLR3, TLR7, TLR8 and RIG-1, without supporting data is not sufficient! Even assuming that there is an unexpected superior result, such result can only be assigned to the s2U(0.25)m5C(0.25) polyribonucleotide, and not to any other modified polyribonucleotide as encompassed broadly by the rejected claims.
Second, Figure 1C of the present application (does not correspond to Figure 1d of the Kormann reference in the levels of modified nucleosides) and Figure 1B of the present application were reproduced below.
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The Examiner notes that both Figures 1B-C do not contain any data for s2U(0.25) and/or m5C(0.25) polyribonucleotides. Thus, an ordinary skill in the art can only assess the effect of the s2U(0.5)m5C(0.5) polyribonucleotide and s2U(1.0)m5C(1.0) polyribonucleotide relative to their respective polyribonucleotide containing individual modification (e.g., s2U or m5C) at respective amount (e.g., 0.5 or 1.0) in the Figures. In both Figures 1B-C, there is certainly no synergistic effect on % RFP positive cells observed for s2U(0.5)m5C(0.5) polyribonucleotide and s2U(1.0)m5C(1.0) polyribonucleotide. Although the total RFP expression value for the s2U(0.5)m5C(0.5) polyribonucleotide is higher than the sum of the total RFP expression values for the s2U(0.5) and m5C(0.5) polyribonucleotides in Figure 1C, this “synergistic effect” appears to be dependent on mouse epithelial lung MLE12 cells used for transfection. This is because no “synergistic effect” could be observed in Figure 1B when the same polyribonucleotides were transfected in human lung A549 cells; instead the total RFP expression value for the s2U(0.5)m5C(0.5) polyribonucleotide is less than the sum of the total RFP expression values for the s2U(0.5) and m5C(0.5) polyribonucleotides. Once again, should there be any “synergistic/unexpected” result, it must be commensurate with the scope of the claims. Furthermore, it is noted that the instant claims recite specifically the limitation “A method of decreasing binding of a polyribonucleotide to retinoic acid-inducible gene I (RIG-1)….wherein the polyribonucleotide has decreased binding to RIG-1 relative to a control polyribonucleotide that does not comprise the modified nucleotide”. Data in both Figures 1B-C of the present application are not related to RIG-1 binding.
Third, please refer the 103 rejection of record that is based on Kariko et al alone or the 103 rejection that is based on Kariko et al in view of Geall et al for details. With respect to the issue that the Kariko reference fails to mention even RIG-1, since the method of administering a modified RNA polyribonucleotide containing a combination of 5 to 50% of uridine nucleotides as 2-thiouridine nucleotides and 5 to 50% of cytidine nucleotides as 5-methylcytidine nucleotides of Kariko et al to a mammalian cell in a subject is indistinguishable from the claimed method of the present application, such method necessarily results in decreasing binding of the modified polyribonucleotide to RIG-1 as claimed. Please, also note that where, as here, the claimed and prior art products are identical or substantially identical, or are produced by identical or substantially identical processes, the PTO can require an applicant to prove that the prior art products do not necessarily or inherently possess the characteristics of his claimed product. See In re Ludtke. Whether the rejection is based on "inherency" under 35 USC 102, or "prima facie obviousness" under 35 USC 103, jointly or alternatively, the burden of proof is the same, and its fairness is evidenced by the PTO's inability to manufacture products or to obtain and compare prior art products. In re Best, Bolton, and Shaw, 195 USPQ 430, 433 (CCPA 1977) citing In re Brown, 59 CCPA 1036, 459 F.2d 531, 173 USPQ 685 (1972).
Fourth, Figure 11C of the present application was also reproduced below. Once again, it is noted that the Figure does not contain any data for m5C(0.25) polyribonucleotide nor does it contain data for s2U(0.25) polyribonucleotide; and therefore an ordinary skill in the art would not be able to conclude whether the RIG-1 binding effect observed for the s2U(0.25)m5C(0.25) polyribonucleotide in the Figure is “synergistic”, “additive”, or even “non-additive”, let alone “unexpected superior” results as asserted by Applicant.
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Accordingly, none of the data presented in Figures 1B, 1C, 2C and 11C of the present application alone or in combination show any “superior/synergistic” decreased binding to RIG-1 exhibited by any of the claimed modified polyribonucleotide as asserted by Applicant. Once again, the instant claims recite specifically the limitation “A method of decreasing binding of a polyribonucleotide to retinoic acid-inducible gene I (RIG-1)….wherein the polyribonucleotide has decreased binding to RIG-1 relative to a control polyribonucleotide that does not comprise the modified nucleotide”. Please also note Kariko et al already taught that their modified RNA molecules have reduced immunogenicity and/or enhancing translation efficiency.
B. Genus/Species
I. Once again, Applicant argued that the Office fails to identify any express disclosure within Kariko et al or Geall et al of a binary combination of 2-thiouridine and 5-methycytidine, much less in the required percentage. Rather, the Office cites Kariko et al for polyribonucleotides that comprise a single type of modified nucleoside or combinations of modified nucleosides that necessarily include pseudouridine. Additionally, the Kariko reference fails to mention RIG-1 at all in the disclosure. In regard to paragraph [0074] of Kariko reference, Applicant argued that it fails to specifically identify s2U as a modification but instead refers to fractions of any given nucleotide which can be modified, and such a recitation captures any modification to any of uridine, cytidine, guanosine, or adenine, as well as percentages ranging from 0.1 to 100%. In regard to Figure 2B of Kario reference, Applicant argued that it demonstrates the results of a polyribonucleotide comprising 100% substitution of s2U for uridines therein, which percentage and lack of combination with m5C falls outside the scope of the claimed invention and consequently would not have motivated an ordinary skilled artisan to arrive at the claimed polyribonucleotide. In regard to Figure 5 of Kariko reference, Applicant argued that it fails to identify or test s2U, and thus would have failed to motivate the ordinary skilled artisan to arrive at the claimed species of ribonucleotide which requires substitution of s2U for 5-50%, 5-30%, 7.5-25%, or 25% uridine residues in the polyribonucleotide. Accordingly, when taken together, Applicant asserts that one of ordinary skill in the art would not have been motivated to practice the claimed method of reducing binding of RIG-1 by providing and administering to a subject or a cell the claimed polyribonucleotide containing a combination of unmodified and modified nucleotides wherein 5 to 50% of uridine nucleotides in the polyribonucleotide are 2-thiouridine, and 5 to 50% of cytidine nucleotides in the polyribonucleotide are 5-methylcytidine, wherein the remainder of the adenosine triphosphate, cytidine triphosphate, guanosine triphosphate and uridine triphosphate in the polyribonucleotide does not include a modified nucleoside, given the enormous genus presented in the prior art and the unpredictability of the results.
First, the examiner has identified numerous trail markers in the teachings of Kariko et al (see at least the 103 rejections of record and Section B. II below) such that it would have been obvious to an ordinary skilled in the art to recognize that the teachings of Kariko et al would encompass or modify the teachings of Kariko et al to prepare at least an in vitro transcribed RNA molecule coding for a therapeutically useful protein, which comprises a combination of 5 to 50% of uridine nucleotides as 2-thiouridine nucleotides and 5 to 50% of cytidine nucleotides as 5-methylcytidine nucleotides, that have reduced immunogenicity and/or enhanced translation efficiency. Additionally, since the modified RNA molecule containing a combination of 5 to 50% of uridine nucleotides as 2-thiouridine nucleotides and 5 to 50% of cytidine nucleotides as 5-methylcytidine nucleotides of Kariko et al is structurally indistinguishable from the polyribonucleotide used in the claimed method of the present application, such modified RNA polyribonucleotide would also possess a decreased binding to RIG-1 relative to a control unmodified polyribonucleotide, particularly Kariko et al already demonstrated that exemplary RNA molecules with m6A, m5C, m5U, s2U and pseudouridine nucleosides, alone or in combination such as the combination of m5C and pseudouridine modified nucleosides, reduce immunogenicity of RNA mediated by TLR3, TLR7 and TLR8 signaling relative to control unmodified RNA molecules. Please, also note that where, as here, the claimed and prior art products are identical or substantially identical, or are produced by identical or substantially identical processes, the PTO can require an applicant to prove that the prior art products do not necessarily or inherently possess the characteristics of his claimed product. See In re Ludtke. Whether the rejection is based on "inherency" under 35 USC 102, or "prima facie obviousness" under 35 USC 103, jointly or alternatively, the burden of proof is the same, and its fairness is evidenced by the PTO's inability to manufacture products or to obtain and compare prior art products. In re Best, Bolton, and Shaw, 195 USPQ 430, 433 (CCPA 1977) citing In re Brown, 59 CCPA 1036, 459 F.2d 531, 173 USPQ 685 (1972).
Second, the teachings of Kariko et al are not necessarily limited only to the paragraph [0074], Figures 2B and 5. It also apparent that Applicant did not consider the totality of Kariko’s teachings. Please refer to the numerous trail markers disclosed in the Kariko reference that are discussed in detail in Section B. II below.
II. Size of Genus/Predictability
Applicant argued that the paragraphs preceding paragraph [0071] in the Kariko reference recite 98 different modified nucleosides, thus when paragraph [0071] references “the above modifications” it references an enormous list of modified nucleosides from which one of ordinary skill in the art could select binary, tertiary, quaternary and higher combinations. Applicant also argued that even assuming one of skill in the art to focus on binary combinations, the possible list of different binary combinations from the 98 different modified nucleosides can be calculated to be 4,753, whereas the total number of possible binary, tertiary and quaternary combinations when considered is an enormous 3,769,129. Similarly, the genus of Geall et al is no less expansive with a total of 741 to greater than 4,950 possible binary combinations of modified nucleosides from the list of Geal et al. Applicant argued that the Office fails to address the almost infinite size of the genus each of the Kariko et al and Geall et al. Applicant also argued a disclosure of polyribonucleotides containing 2-thiouridine or 5-methylcytidine individually provides no data on combinations thereof, nor works to limit the expansive genus disclosed in the Kariko reference. Applicant further argued that based on Figure 2B and Figure 3B of the Kariko reference, an ordinary skill in the art would have concluded that this technology is unpredictable and that providing a polyribonucleotide comprising m5C and another modified nucleotide would not have reliably provided enhanced effect over a polyribonucleotide comprising only a single modified nucleotide, such as m5C. Accordingly, one of ordinary skill in the art would not have been motivated to arrive at the presently claimed invention, and that the disclosure of Kariko et al and/or Geall et al amount only to an invitation to experiment and that cannot form the basis of a prima facie case of obviousness of a species.
First, the examiner already identified numerous trail markers in the teachings of Kariko et al such that an ordinary skill in the art would not arrive at the presently claimed invention from an enormous list of different binary combinations derived from the disclosed 98 different modified nucleosides. For example, Kariko et al stated explicitly “In another embodiment, the present invention provides an in vitro-synthesized polyribonucleotide, comprising a pseudouridine or a modified nucleoside, wherein the modified nucleoside is m5C, m5U, m6A, s2U, ψ, or 2’-O-methyl-U” (paragraph [0048]; trail marker #1); “In another embodiment, the modified nucleoside of methods and compositions of the present invention is m5C (5-methylcytidine). In another embodiment, the modified nucleoside is m5U (5-methyluridine). In another embodiment, the modified nucleoside is m6A (N6-methyladenosine). In another embodiment, the modified nucleoside is s2U (2-thiouridine). In another embodiment, the modified nucleoside is ψ (pseudouridine). In another embodiment, the modified nucleoside is Um (2’-O-methyluridine)” (paragraph [0069]; trail marker #2); “In another embodiment, an RNA, oligoribonucleotide, or polyribonucleotide molecule of methods and compositions of the invention comprises a combination of 2 or more of the above modifications. In another embodiment, the RNA molecule or oligoribonucleotide molecule comprises a combination of 3 or more of the above modifications” (paragraph [0071]; trail marker #3); “In another embodiment, 0.1% of the residues of a given nucleotide (uridine, cytidine, guanosine, or adenine) are modified. In another embodiment, the fraction of the nucleotide is 0.2%....In another embodiment, the fraction is 5%. In another embodiment, the fraction is 6%. In another embodiment, the fraction is 8%. In another embodiment, the fraction is 10%....In another embodiment, the fraction is 20%. In another embodiment, the fraction is 25%. In another embodiment, the fraction is 30%. In another embodiment, the fraction is 35%. In another embodiment, the fraction is 40%. In another embodiment, the fraction is 45%. In another embodiment, the fraction is 50%” (paragraph [0074]; trail marker #4); along with the exemplifications showing nucleoside modifications that include m6A, m5C, m5U, S2U and pseudouridine alone or in combination being used to reduce immunogenicity of RNA in comparison to unmodified, in vitro transcribed RNA in Fig. 2B (trail marker #5) and determination of the minimal frequency of a particular modified nucleoside (e.g., 1%, 10%, 50%, 90%, 99% and 100% of m6A, pseudouridine or m5C) that is sufficient to reduce immunogenicity in Fig. 5 (trail marker #6). Based at least on these trail markers, it would have been obvious to an ordinary skilled in the art to recognize that the teachings of Kariko et al would encompass or modify the teachings of Kariko et al to prepare at least an in vitro transcribed RNA molecule coding for a therapeutically useful protein, which comprises a combination of 5 to 50% of uridine nucleotides as 2-thiouridine nucleotides and 5 to 50% of cytidine nucleotides as 5-methylcytidine nucleotides, while the remainder of the nucleotides in the RNA molecule contains no modified nucleosides; and the RNA molecule has reduced immunogenicity and/or enhanced translation efficiency. It appears that Applicant did not consider the totality of the Kariko reference, and apparently got stuck on the single paragraph [0070] listing 98 possible modified nucleosides.
Second, on the basis of paragraph [0074] an ordinary skill in the art would readily recognize that apart from the fraction of the residues of a given nucleotide being modified, the rest of the fraction for that given nucleotide is unmodified. For example, 25% of the cytidine residues in a polyribonucleotide are modified to 5-methylcytidine nucleotides, the rest of the cytidine residues in that polyribonucleotide remain unmodified or in the form of cytidine nucleotides.
Third, with respect to the alternative 103 rejection that is based on Kariko et al in view of Geall et al, the Geall reference was cited primarily to supplement the teachings of the primary Kariko reference on the limitation of “wherein the polyribonucleotide has decreased binding to RIG-1 relative to control polyribonucleotide that does not comprise the analogs”. Particularly, Geall et al taught that when unmodified RNA is delivered to cells by viral or non-viral delivery, the RNA is recognized as foreign nucleic acid by endosomal and cytoplasmic immune receptors, such as the toll-like receptors 3, 7 and 8 of the endosomes, retinoic acid-induced gene (RIG-1), melanoma differentiation-associated gene-5 (MDA-5) and laboratory of genetics and physiology-2 (LGP2) receptors of the cytoplasm (paragraph 45). Geall et al also taught that stimulation of the above immune receptors by a self-replicating RNA that does not include modified nucleotides is expected to modulate the immune response which could impact expression of gene products encoded by the RNA, amplification of and adjuvant effect of the self-replicating RNA, the immune response to encoded proteins (i.e., decreased potency of vaccine), and could also lead to safety concerns, such as injection site irritation, inflammation and/or pain (paragraph 45). Accordingly, Geall et al disclosed self-replicating RNA molecules that contain modified nucleotides to avoid or reduce stimulation of endosomal and cytoplasmic immune receptors when the self-replicating RNA is delivered into a cell, wherein 0.01%-25% of a particular nucleotide are modified nucleotides, or wherein 0.01%-25% of two, three or four particular nucleotides are substituted nucleotides, and wherein the self-replicating RNA molecule comprising at least one of modified nucleosides selected from a group that includes 2-thiouracil, 5-methylcytosine, pseudouridine, N6-methyladenosine among others and any combination thereof (see at least Abstract; Summary of the Invention; particularly paragraphs 8-9, 16-20, 24, 45-52, 67-69; and Example 3). Based on the specific trail markers and extensive teachings of Kariko et al already discussed above, an ordinary skilled in the art would not have to consider the possible enormous list of binary combinations determined by Applicant that was solely based on the single paragraph [0070] of Kariko et al listing numerous nucleosides and/or lists of possible modified nucleosides in the Geall reference that can be also used for a modified polyribonucleotide in other embodiments.
Fourth, Fig. 2B in an exemplification of Kariko et al shows clearly that nucleoside modifications such as m6A, m5C, m5U, s2U and pseudouridine, alone and in combination, reduce immunogenicity of RNA as mediated by TLR3, TLR7 and TLR8 signaling in comparison to unmodified, in vitro transcribed RNA. This Figure clearly shows that the exemplary combination of m5C/pseudouridine polyribonucleotide induces less IL-8 in comparison with m5C-containing polyribonucleotide and pseudouridine-containing polyribonucleotide, and of course much less IL-8 in comparison with unmodified polyribonucleotide. There is no unpredictability whatsoever regarding to the overall advantages/usefulness of the exemplary m5C/pseudouridine.
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Fig. 3B of the Kariko reference reproduced below also shows that the exemplary combination of m5C/pseudouridine polyribonucleotide is better than m5C-containing polyribonucleotide or s2U-containing polyribonucleotide in suppressing the induction of IL-12.
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The data on the TNF-alpha induction for m5C/pseudouridine polyribonucleotide, m5C-containing polyribonucleotide and pseudouridine-containing polyribonucleotide are all too low to make any meaningful comparison among them. Nevertheless, there is no unpredictability whatsoever regarding to the overall advantages/usefulness of the exemplary m5C/pseudouridine polyribonucleotide.
III. Examiner’s rebuttal arguments
(i) With respect to Figure 2B of Kariko et al, Applicant argued that this Figure demonstrates the unpredictability in the art regarding reduction of binding of modified mRNA to toll-like receptors, especially mRNA containing binary combinations of modified nucleotides. For example, Applicant argued that while an mRNA containing combination of m5C/pseudouridine modification provided some benefit in blocking TLR3 signaling compared to an mRNA containing only the pseudouridine modification, the same mRNA containing the combination of m5C/pseudouridine provided no statistically significant benefit in blocking TLR7 and TLR8 signaling over an mRNA containing only the pseudouridine modification. Similarly, Applicant argued that Figures 3A and 3B of Kariko et al demonstrate that mRNA which comprises m5C and pseudouridine provides no enhanced results when compared with mRNA containing only a pseudouridine. Thus, the Examiner cited figures evidence the unpredictability of the art, rather a reasonable expectation of success.
Please see the data in Figures 2B and 3B along with the Examiner’s responses in Section II above. Similar to Fig. 3B, Fig. 3A of the Kariko reference also shows the exemplary combination of m5C/pseudouridine polyribonucleotide is better than m5C-containing polyribonucleotide or s2U-containing polyribonucleotide in suppressing the induction of IL-12 in MDDC cells; while the data on the TNF-alpha induction for m5C/pseudouridine polyribonucleotide, m5C-containing polyribonucleotide and pseudouridine-containing polyribonucleotide are all too low to make any meaningful comparison among them. Based on these Figures, an ordinary skill in the art would reasonably conclude that there is no unpredictability whatsoever regarding to the overall advantages/usefulness of the exemplary m5C/pseudouridine polyribonucleotide relative to m5C-alone polyribonucleotide or pseudouridine-alone polyribonucleotide.
(ii) Applicant also argued that the Examiner has provided no evidence whatsoever to demonstrate that any of the results regarding TLR3, TLR7, TLR8, IL-12, or TNF-alpha will predict which single nucleotide modification or binary combination will predictably synergistically reduce mRNA binding to RIG-1; particularly Kariko et al fails to mention RIG-1 at all and thus no data to support the Examiner’s allegation of a reasonable prediction of successfully reducing RIG-1 binding using the claimed modified mRNA. Similarly, Geall et al fails to test a modified mRNA comprising the combination of m5C and 2sU, much less testing binding of such an mRNA to RIG-1. Accordingly, the Examiner has relied on improper hindsight reasoning.
First, with respect to the “synergistic effect” on reducing mRNA binding to RIG-1 please refer to the Examiner’s responses in Section A above. None of the data presented in Figures 1B, 1C, 2C and 11C of the present application alone or in combination show any “superior/synergistic” decreased binding to RIG-1 exhibited by any of the claimed modified polyribonucleotide as asserted by Applicant. It is noted that the instant claims recite specifically the limitation “A method of decreasing binding of a polyribonucleotide to retinoic acid-inducible gene I (RIG-1)….wherein the polyribonucleotide has decreased binding to RIG-1 relative to a control polyribonucleotide that does not comprise the modified nucleotide”.
Second, since the modified RNA molecule containing a combination of 5 to 50% of uridine nucleotides as 2-thiouridine nucleotides and 5 to 50% of cytidine nucleotides as 5-methylcytidine nucleotides of Kariko et al is structurally indistinguishable from the polyribonucleotide used in the claimed method of the present application, such modified RNA polyribonucleotide would also possess a decreased binding to RIG-1 relative to a control unmodified polyribonucleotide, particularly Kariko et al already demonstrated that exemplary RNA molecules with m6A, m5C, m5U, s2U and pseudouridine nucleosides, alone or in combination such as the combination of m5C and pseudouridine modified nucleosides, reduce immunogenicity of RNA mediated by TLR3, TLR7 and TLR8 signaling relative to control unmodified RNA molecules. Please, also note that where, as here, the claimed and prior art products are identical or substantially identical, or are produced by identical or substantially identical processes, the PTO can require an applicant to prove that the prior art products do not necessarily or inherently possess the characteristics of his claimed product. See In re Ludtke. Whether the rejection is based on "inherency" under 35 USC 102, or "prima facie obviousness" under 35 USC 103, jointly or alternatively, the burden of proof is the same, and its fairness is evidenced by the PTO's inability to manufacture products or to obtain and compare prior art products. In re Best, Bolton, and Shaw, 195 USPQ 430, 433 (CCPA 1977) citing In re Brown, 59 CCPA 1036, 459 F.2d 531, 173 USPQ 685 (1972).
Third, 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 refer to the above 103 rejections of record for more details.
(iii) Applicant further argued that in contrast to the data provided by Kariko et al, Applicant actually produced data which evidences the claimed modified mRNA synergistically increases mRNA stability and duration of expression as well as synergistically reduces RIG-1 binding. Thus, the presently claimed method and modified mRNA provide “unexpected” superior results, which would overcome even a prima facie case of obviousness.
Once again, with respect to the presented data on “synergistic effects” please refer to the Examiner’s responses in Section A above. Please also note that any “unexpected/surprising” result must be commensurate with the scope of the claims. However, none of the data presented in Figures 1B, 1C, 2C and 11C of the present application alone or in combination show any “superior/synergistic” decreased binding to RIG-1 exhibited by any of the claimed modified polyribonucleotide as asserted by Applicant. Once again, the instant claims recite specifically the limitation “A method of decreasing binding of a polyribonucleotide to retinoic acid-inducible gene I (RIG-1)….wherein the polyribonucleotide has decreased binding to RIG-1 relative to a control polyribonucleotide that does not comprise the modified nucleotide”.
Conclusion
No claim is allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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, 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 1633; 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