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 .
Priority
Acknowledgement is made of Applicant’s claim for priority from US provisional application 62/776,322 filed 12/06/2018.
Application Status
This Application is the National Stage of Application PCT/US19/65046 filed 12/06/2019 under 35 U.S.C. § 371.
Amendments to claims filed 02/13/2026 are hereby acknowledged. Claims 2-4, 6, 8, 11-14,16-34, 37-39, 41-42, 44-55, 57-67, 69-71, 73, 74, 77-80, 82-84 and 86-87 are cancelled. Claims 1, 68, 72, 75-76, 81 and 85 are currently amended. Claims 88 and 89 are newly added.
Claims 36, 40, 43, 56, 68, 72, 75-76, 81 and 85 are withdrawn from further consideration based upon election after restriction requirement. Newly added claim 89 depending from withdrawn claim 68 is also withdrawn as also drawn to a non-elected invention. Claims 1, 5, 7, 9-10, 15, 35, 36, 40, 43, 56, 68, 72, 75-76, 81, 85, 88 and 89 are currently pending.
Therefore, claims 1, 5, 7, 9-10, 15, 35 and 88 are under examination in this office action.
Any objection or rejection not reiterated herein has been overcome by Applicant’s amendments and is therefore withdrawn.
Applicant’s amendments and arguments have been thoroughly reviewed, but are not persuasive to place the claims in condition for allowance for the reasons that follow.
Claim Objections
Claim 35 is objected to because of the following informalities: PAH, ABCA3 and ATP8B1 are cited twice in the claim: “OTC, ASL, PAH, ABCB4, ABCB11, PAH, AGL, CFTR, MUT, PCCA, PCCB, ASS1, FAH, HMBS, ATP7B, PFIC2, LDLR, G6PC, AGXT, FXN, PAL, BCKDHA, BCKDHB, DBT, UGT1A1, SLC25A13, CD46, CFH, CFI, FIX, FVII, FVIII, C2, C3, C5, GCHD, CBS, MPI, LNL, SERPING1, UROC1, SMPD1, GLA, GAA, GRHPR, ATP8B1, SERPINC1, PROS1, GBA, ACADVL, HFE, BCKDA, CDG1B, SERPINA1, BMPR2, ENG, ACVR1, SMAD4, BMPR9, HBB, FLCN, HSP1, AP3B1, HPS3, HPS4, HPS5, HPS6, DTNBP1, BLOC1S3, PLDN, AP3D1, BRAF, NF-1, SLC34A2, FBN1, COL1A1, COL1A2, COL1A3, COL5A1, COL5A2, ADAMTS2, PLOD1, TNXB, ABCA3, SP-B, SP-C, GBA, NPC1, NPC2, FOXF1, NKX2-1, SFTPB, SFTPC, ABCA3, CSF2RA, SFTPD, MUCSB, BMPR2, EIF2AK4, CSF2RB, DNAH5, DNAI1, DNAH11, AKR1D1, AMACR, ATP8B1, CYP7A1, FOXC2, GATA2, GHR, HSD3B7, IGFALS, IKBKG, JAG1, KIF11, NOTCH1, NOTCH2, NR1H4, SOX18, TJP2, P53, P73, P63, VIPAS39, VPS33B, EPO, ARG1, CPS1, NAGS, NOS, KRAS, OX40L, IL12, VEGF-A, MMA, TTR, PCSK9, AT, and ALAS1”.
Appropriate correction is required.
Specification
The disclosure is objected to because of the following informalities: The sequence disclosures located in Specification, § [0160] (i.e., SEQ ID NOs: 1799 and 1921) can appear as Specific deficiencies in Sequence Requirements.
Table on page 25 lists number 1799 as an mRNA Construct No with SEQ ID NO: 73 and Table on page 27 lists number 1921 as an mRNA Construct No with SEQ ID NO: 119.
Appropriate correction is required.
Claim Rejections - 35 USC § 112
Claim Rejections - 35 USC § 112(a)
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Written Description requirement
Claims 1, 5, 7, 9-10, 15 and 35 are rejected under 35 U.S.C. §112(a) or 35 U.S.C. §112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention. This rejection is modified as necessitated by Applicants’ amendments.
MPEP 2163.II.A.3.(a).i) states, “Whether the specification shows that applicant was in possession of the claimed invention is not a single, simple determination, but rather is a factual determination reached by considering a number of factors. Factors to be considered in determining whether there is sufficient evidence of possession include the level of skill and knowledge in the art, partial structure, physical and/or chemical properties, functional characteristics alone or coupled with a known or disclosed correlation between structure and function, and the method of making the claimed invention”.
For claims drawn to a genus, MPEP § 2163 states the written description requirement for a claimed genus may be satisfied through sufficient description of a representative number of species by actual reduction to practice, reduction to drawings, or by disclosure of relevant, identifying characteristics, i.e., structure or other physical and/or chemical properties, by functional characteristics coupled with a known or disclosed correlation between function and structure, or by a combination of such identifying characteristics, sufficient to show the applicant was in possession of the claimed genus. See Eli Lilly, 119 F.3d at 1568, 43 USPQ2d at 1406.
Nature of the invention:
Claim 1 recites: “A modified protein having an amino acid sequence derived from an amino acid sequence of a human wild-type protein, wherein:
The human wild-type protein has a localization signal peptide located at a terminal portion, wherein the amino acid sequence of the localization signal peptide has been modified in the modified protein by insertion of an amino acid at the +3 position of the localization signal peptide as compared to the human wild-type protein;
the amino acid sequence if the human wild-type protein has been modified to remove one or more ubiquitination sites that is (a) present in an N terminal portion of the amino acid sequence of the human wild-type protein, but (b) not present in a homologous mouse wild-type protein; and wherein the sequence of the modified protein differs from both the human wild-type and homologous mouse wild-type proteins; and
the sequence of the modified protein differs from both the human wild-type and homologous mouse wild-type proteins.”
It is therefore expected in the instant application a disclosure of species comprising each limitation claimed, a disclosure of a wide variety and different proteins in which a localization signal peptide exists in human and has been modified to become different from the wild-type human in a terminal portion, wherein the sequence of the modified protein differs from both the human wild-type and homologous mouse wild-type.
It is expected that Applicant discloses a consensus sequence for modification in a terminal region, a uniformed structure that can be specifically and concretely named as Applicant’s invention, and a list of modifiable proteins with the same structure of the claimed invention, belonging to a group species of the claimed invention. Here, in claim 35, the list of proteins provided, only have in common that they are proteins containing a N-terminal sequence that is a signal peptide; however these proteins are not from the same pathways, same class of proteins, e.g., endoplasmic reticulum proteins, or nuclear proteins, or enzymes active on same substrates or in same pathways. These proteins are diverse in structures and functions, with little in common.
Therefore, It is expected a disclosure of one or more modifications that can be performed to remove one or more ubiquitination sites as disclosed in a consensus. It is expected a reduction to practice with modification of representatives species for protein classes and/or signal pathways. It is expected that those modifications are not naturally occurring according to the broadest reasonable interpretation of the limitation, and not modifications that can occur from natural selection and in naturally occurring isoforms or variants. It is expected an enumeration of residues in sequence for modified proteins, and examples of sequences for more than one protein.
The state of the art:
1)- Regarding ubiquitination sites in N-terminal regions; difference in
human wild-type and mouse wild-type proteins:
One example of proteins that is listed in Specification as part of therapeutic protein suitable for modification as described (see page 45) is Desmocollin 3 (DSC3), which is a tumor suppressing gene, important in human embryogenesis for epidermis development and is frequently mutated in human skin diseases.
Kim (Kim, D.S. et al. “Gains of ubiquitylation sites in highly conserved proteins in the human lineage”. BMC Bioinformatics, Vol. 13 (2012), p: 306) teaches a large variety of proteins that are specifically modified in human and have a different ubiquitination site that is not present in mouse (see title, Table 1 and examples in Figures 3-4).
Kim teaches that of 281 ubiquitylation sites examined, 269 sites in 243 human proteins were acquired along the human lineage during primate evolution (see page 4, right column).
Kim teaches that Desmocollin 3 is an example of proteins in which ubiquitylation sites appear to be human-specific (see Table 1, No. 67).
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DSC3 is associated to skin disorders and cancer, involved in cell adhesion and cell proliferation.
Naturally occurring mutations in proteins to select for a variant useful for cell proliferation or metastasis are therefore numerous and expected.
However, the specific modification that is part of the invention is not clearly described in the claims, nor in the Specification. An enumeration of residues is not shown for the alteration of this protein, so that it would be concluded that the claimed composition is not a naturally occurring product, but an engineered/synthetic protein with a modified signal peptide and a modified ubiquitination site.
Regarding ubiquitination sites and Lysine residues in N-terminal region:
Akimov (Akimov, V. et al. “UbiSite approach for comprehensive mapping of lysine and N-terminal ubiquitination sites”. Nature Structural & Molecular Biology, Vol. 25 (July, 2018), pp: 631-640) teaches a method using an antibody (UbiSite) recognizing C-terminal 13 amino acids of ubiquitin, for identifying proteins that are ubiquitinated in N-terminal region, in human cell lines (see abstract). Akimov identifies 63,000 unique ubiquitination sites in 9,200 proteins in the two human cell lines (see abstract and Figure 1C). Further selection using Akimov’s antibody identifies 104 proteins modified in the N-terminal region (see page 636, right column, second paragraph; see Figure 6).
Gallo (Gallo, L.H. et al. “Novel Lys63-linked ubiquitination of IKKβ induces STAT3 signaling”. Cell Cycle, Vol. 13, No. 24 (2014), pp: 3964-3976) teaches not all Lysine residues can be ubiquitinated.
Gallo teaches that Lys147, Lys171, Lys418, Lys555 and Lys703 of IKKβ are ubiquitinated (see abstract). However, an alignment between the IKKB isoform 1 of human and mouse sequences shows multiple differences in sequences, including differences in Lysine residues in the N-terminal region, see below (Qy, Query : Human IKKβ isoform 1; Db, Database, Mouse IKKβ isoform 1; underline and bolded are the Lysine and arginine in Human or mouse sequences that are in N-term region and are NOT ubiquitinated):
Query Match 93.3%; Score 3676; DB 1; Length 757;
Best Local Similarity 92.7%;
Matches 700; Conservative 26; Mismatches 29; Indels 0; Gaps 0;
Qy 1 MSWSPSLTTQTCGAWEMKERLGTGGFGNVIRWHNQETGEQIAIKQCRQELSPRNRERWCL 60
||||||| ||||||||||||||||||||||||||| ||||||||||||||||:||:||||
Db 1 MSWSPSLPTQTCGAWEMKERLGTGGFGNVIRWHNQATGEQIAIKQCRQELSPKNRDRWCL 60
Qy 61 EIQIMRRLTHPNVVAARDVPEGMQNLAPNDLPLLAMEYCQGGDLRKYLNQFENCCGLREG 120
|||||||| ||||||||||||||||||||||||||||||||||||:||||||||||||||
Db 61 EIQIMRRLNHPNVVAARDVPEGMQNLAPNDLPLLAMEYCQGGDLRRYLNQFENCCGLREG 120
Qy 121 AILTLLSDIASALRYLHENRIIHRDLKPENIVLQQGEQRLIHKIIDLGYAKELDQGSLCT 180
|:|||||||||||||||||||||||||||||||||||:||||||||||||||||||||||
Db 121 AVLTLLSDIASALRYLHENRIIHRDLKPENIVLQQGEKRLIHKIIDLGYAKELDQGSLCT 180
Qy 181 SFVGTLQYLAPELLEQQKYTVTVDYWSFGTLAFECITGFRPFLPNWQPVQWHSKVRQKSE 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 SFVGTLQYLAPELLEQQKYTVTVDYWSFGTLAFECITGFRPFLPNWQPVQWHSKVRQKSE 240
Qy 241 VDIVVSEDLNGTVKFSSSLPYPNNLNSVLAERLEKWLQLMLMWHPRQRGTDPTYGPNGCF 300
||||||||||| ||||||||:||||||||||||||||||||||||||||||| |||||||
Db 241 VDIVVSEDLNGAVKFSSSLPFPNNLNSVLAERLEKWLQLMLMWHPRQRGTDPQYGPNGCF 300
Qy 301 KALDDILNLKLVHILNMVTGTIHTYPVTEDESLQSLKARIQQDTGIPEEDQELLQEAGLA 360
:||||||||||||:|||||||:||||||||||||||| |||:|||| | ||||||||||
Db 301 RALDDILNLKLVHVLNMVTGTVHTYPVTEDESLQSLKTRIQEDTGILETDQELLQEAGLV 360
Qy 361 LIPDKPATQCISDGKLNEGHTLDMDLVFLFDNSKITYETQISPRPQPESVSCILQEPKRN 420
|:||||||||||| | ||| ||||||||||||||| |||||:||||||||||||||||||
Db 361 LLPDKPATQCISDSKTNEGLTLDMDLVFLFDNSKINYETQITPRPQPESVSCILQEPKRN 420
Qy 421 LAFFQLRKVWGQVWHSIQTLKEDCNRLQQGQRAAMMNLLRNNSCLSKMKNSMASMSQQLK 480
|:||||||||||||||||||||||||||||||||||:|||||||||||||:||| :||||
Db 421 LSFFQLRKVWGQVWHSIQTLKEDCNRLQQGQRAAMMSLLRNNSCLSKMKNAMASTAQQLK 480
Qy 481 AKLDFFKTSIQIDLEKYSEQTEFGITSDKLLLAWREMEQAVELCGRENEVKLLVERMMAL 540
||||||||||||||||| |||||||||||||||||||||||| |||||:|| ||||||||
Db 481 AKLDFFKTSIQIDLEKYKEQTEFGITSDKLLLAWREMEQAVEQCGRENDVKHLVERMMAL 540
Qy 541 QTDIVDLQRSPMGRKQGGTLDDLEEQARELYRRLREKPRDQRTEGDSQEMVRLLLQAIQS 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 541 QTDIVDLQRSPMGRKQGGTLDDLEEQARELYRRLREKPRDQRTEGDSQEMVRLLLQAIQS 600
Qy 601 FEKKVRVIYTQLSKTVVCKQKALELLPKVEEVVSLMNEDEKTVVRLQEKRQKELWNLLKI 660
||||||||||||||||||||||||||||||||||||||||:|||||||||||||||||||
Db 601 FEKKVRVIYTQLSKTVVCKQKALELLPKVEEVVSLMNEDERTVVRLQEKRQKELWNLLKI 660
Qy 661 ACSKVRGPVSGSPDSMNASRLSQPGQLMSQPSTASNSLPEPAKKSEELVAEAHNLCTLLE 720
||||||||||||||||| |||| |||||||||:| :|||| ||||||||||| ||: ||
Db 661 ACSKVRGPVSGSPDSMNVSRLSHPGQLMSQPSSACDSLPESDKKSEELVAEAHALCSRLE 720
Qy 721 NAIQDTVREQDQSFTALDWSWLQTEEEEHSCLEQA 755
:|:||||:|||:||| ||||||| |:|| ||||
Db 721 SALQDTVKEQDRSFTTLDWSWLQMEDEERCSLEQA 755
Regarding ubiquitination sites differences in human and mouse:
Gallo teaches that difference in sequences in Human and mouse exist naturally, transforming Lysine into arginine. However, these changes are not correlated to the ability of the protein to be ubiquitinated (see above).
Samelson (Samelson, L.E. et al. US 2012/0114700 A 1; published May 10, 2012; listed on IDS filed 08/17/2021; previously cited) teaches an isolated, non-naturally occurring linker for activation of T cells (LAT) polypeptide comprising at least one amino acid substitution at amino acid 52 or 204 (see Claim 1, page 42 of 43). Samelson teaches the modification of LAT (Linker for Activation of T-cells), mutating ubiquitylation sites for increase stability of the protein (see “[A]bstract” section). Samelson also teaches that the sequence in the N-terminus of human LAT is different compared to mouse LAT (see SEQ ID NO: 1 and SEQ ID NO: 2, page 38 of 43). Samelson teaches a mouse wild type LAT protein, homologous to the wild-type human protein (see SEQ ID NO: 1 and SEQ ID NO: 2, page 38 of 43).
Samelson teaches the position to mutate the ubiquitylation site is different in Human and in mouse (human, positions amino acid 52 and 204; mouse, positions amino acid 53 and 121).
Samelson teaches in its claim 1 “at least one amino acid substitution”; Samelson teaches in its claim 2 (page 42 of 43), that the amino acid substitution comprises substitution of lysine with arginine.
However, according to the amended claim 1, Samuelson’s modified LAT protein does not qualify as a suitable modified protein since the ubiquitination site modified is not located in N-terminal region. However, the modified LAT protein was stabilized by Samuelson’s modification.
Baldi ( Baldi, L. et al. The Journal of Biological Chemistry, Vol. 271, No. 1 (Jan. 5, 1996), pp: 376-379; previously cited) teaches a polypeptide , namely IƘB-α, modified by amino acid substitutions compared to the human wild-type protein, wherein two adjacent ubiquitination sites (Lys 21 and Lys 22) were mutated (see abstract section).
Baldi teaches that, in the chicken, IƘB-α homolog does not contain the lysine residue Lys 21 (see page 378, right column, last ¶).
Baldi teaches that site-directed mutagenesis was performed on the NH2-terminal lysines including those at positions 21 ,22, 38, 47, 67 and 87 (see page 377, “[R]esults” section, lines 1-12). Baldi teaches that the lysine residues in IƘB-α are changed into arginine (see page 377, “[R]esults” section, lines 12-13 and figure 1). Baldi teaches stabilization of the protein, however, the difference in sequences are between Human and chicken in this case, not Human and mouse.
2- Regarding signal peptide and mutations in signal peptides:
Gutierrez Guarnizo (Gutierrez Guarnizo, S.A. et al. “Pathogenic signal peptide variants in the human genome”. NAR Genomics and Bioinformatics, Vol. 5, No.4 (2023), pp: 1-17) teaches that in eukaryotic cells, the most numerous targeting signals include signal peptides, N-terminal amino acid sequences that direct the targeting and translocation of many secreted and membrane proteins to the endoplasmic reticulum (see page 1, “Introduction” section, first paragraph). Gutierrez Guarnizo also teaches that in database searched, there are 3,607 genes encoding protein with annotated signal peptides. However, using predictive algorithm SignalP6.0m 4,142 different proteins were found with signal peptides and that proteins with cleavable signal peptides contribute to 20 % of the total human proteome (page 5, “Results” section, left column).
Gutierrez Guarnizo also teaches that there are missense variants modifying signal peptides (SNPs) and that only 83% of signal peptides are found with the same length and about 17% have alternative lengths (see page 5, right column, last paragraph);
[Examiner interprets that there are also insertion mutations or deletion mutations variants.]
Gutierrez Guarnizo teaches that 65 thousands signal peptide mutations were uncovered, and over 11 thousand were classified as pathogenic (see abstract). [Examiner interprets “pathogenic” as “occurring naturally in altered environmental conditions (cellular microenvironment) and diseases”.]
Gutierrez Guarnizo teaches in Figure 2 that the N-terminal region of a signal peptide is a median of 3 amino acids in length and that 18% of detected missense variants are located in this N-region (Figure 2F). This analysis was performed on a total of 65,655 protein sequences in the whole human genome.
Therefore, it is likely that there are pathogenic variants occurring at +1, +2 or +3 positions in the signal peptide, and occurring naturally in the human proteome.
3- Summary:
Kim teaches that there are at least 269 ubiquitylation/ubiquitination sites in 243 human proteins that are different in mouse homologs. Kim did this work in 2012. Improved algorithms with different results are expected in the literature.
Gutierrez Guarnizo teaches that there are 11 thousand signal peptides that are mutated. Gutierrez Guarnizo teaches evidence that there are pathogenic signal peptide that would have mutations in the first 3 amino acids.
Collectively, these references show that the genus of proteins as claimed can be a large one comprising thousands or at least hundreds of proteins in the human proteome that can occur naturally.
When considering the proteins comprising a signal peptide in N-terminal region and a ubiquitination site at the N-terminal that is different from mouse homologous sequence, and that can be modified synthetically, the claim is drawn to an even broader genus and a large amount of species.
What the Specification does and does not teach:
The Specification teaches only OTC as a modified protein in the Drawings and in
Examples.
Briefly, the examples show an OTC modified protein in hepatocyte cell lines (Figures 1A-B), the stability of the protein modified with different compounds at different times (Figures 2-4). Applicant teaches the use of different mRNA constructs modified and expressed in hepatocytes and mice, testing for stability in Figures 5-8. Applicant teaches the expressed modified OTC protein in mitochondrial versus cytosolic fractions of cells isolated from mice treated with the mRNAs (Figures 9-12).
The Specification list hundreds of proteins under the heading “Therapeutic Proteins” (see [0127]). The Specification list exemplary therapeutic proteins alphabetically from AIBG (page 34) to ZZZ3 (page 80).
The Specification narrows the list on page 80, [0128]. However, these proteins are clearly different from one another, involved in different functions and pathways, and may or may not have a homologous mouse sequence comprises a ubiquitylation site in N-terminal region that is different in the wild-type human protein.
The Specification further teaches that the removal of predicted ubiquitination sites preferably comprises replacing N-terminus residues that have been found to support ubiquitination such as asparagine, arginine, leucine, lysine or phenylalanine with residues that have been found to be stabilizing against ubiquitination such as alanine, glycine, methionine, serine, threonine, valine and proline (see page 1, lines 28-19 and page 2, lines 1-3).
Therefore, the residues considered by Applicant are not limited to Lysine residues. It is also known that not all Lysine residues are systematically ubiquitinated. Gallo teaches example of ubiquitination sites and not all Lysines in the sequence can be ubiquitinated.
The Specification teaches LAT as part of suitable protein for the invention (see page 55, line 1). However, according to Samuelson, the ubiquitination sites in N-terminal region of LAT are not really different between Human and mouse.
Also, it is not clear what qualifies the proteins listed in the Specification for belonging to the claimed invention, according to claim 1’s limitations. The proteins are clearly different in structure, however they are included as part of the invention. It is not clear what modification renders the listed proteins eligible as part of the claimed invention. Applicant does not provide with a consensus sequence for proteins “suitable’ for modification, wherein the residues to be modified are clearly pointed out, so that one of ordinary skills in the art could identify the residues in a protein of interest and replicate the invention on any protein of the list.
Conclusion:
Taking into consideration the factors outlined above, including the nature of the
invention, the state of the art, the guidance provided by the applicant and the specific example, it is the conclusion that Applicant does not possess the invention as claimed in claim 1. There is no specific written consensus sequence for ubiquitination and conserved residue modifications and steps within the Specification that would lead one with ordinary skills in the art to a different conclusion.
Response to Arguments
Applicant's arguments filed 02/13/2026 have been fully considered but they are not persuasive.
Applicant states that “MPEP 2163 instead provides that the "written description requirement for a claimed genus may be satisfied through sufficient description of a representative number of species by actual reduction to practice . .. or by disclosure of relevant, identifying characteristics" such as "by functional characteristics coupled with a known . . .correlation between function and structure" (emphasis added)”.
In response, MPEP 2163 (B) also states “While there is no in haec verba requirement, newly added claims or claim limitations must be supported in the specification through express, implicit, or inherent disclosure”.
The Genus claimed by Applicant is a large one in which species are gathered under a Markush claim (claim 35), having in common the fact that these are proteins that are ubiquitinated and important for their potential use as therapeutic proteins. However, the proteins are very different in their functions in signal pathways and in their importance at different levels of a cell’s life. Their folding patterns and interacting partners are different, all depending upon specific structures; therefore, any specific modification leading to a patentable and novel composition, should have a minimum description of structure, showing how different the claimed composition is structurally different from those mutants/variants in the prior art. A consensus target sequence for modification would effectively lead to a better understanding of what Applicant’s invention is.
Applicant just stated in the Specification: “Suitable codon optimization tools, algorithms and services are known in the art.” ([0101]). Therefore, Applicant teaching is : go and use them. This Specification does not provide a satisfactory description for demonstrating that Applicant possess the species claimed.
Scope of enablement
Claims 1, 5, 7, 9, 10 and 15 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being partially enabling for the functional limitation of “wherein the human wild-type protein is ornithine transcarbamylase (OTC) does not reasonably provide enablement for any of the other proteins listed in claim 35. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make the invention commensurate in scope with these claims. This is a new rejection necessitated by Applicants’ amendments
The test of enablement is whether one skilled in the art could make and use the claimed invention from the disclosures in the specification coupled with information known in the art without undue experimentation (United States v. Telectronics., 8 USPQ2d 1217 (Fed. Cir. 1988)). Whether undue experimentation is needed is not based upon a single factor but rather is a conclusion reached by weighing many factors. These factors were outlined in Ex parte Forman, 230 USPQ 546 (Bd. Pat. App. & Inter. 1986) and again in In re Wands, 8 USPQ2d 1400 (Fed. Cir. 1988), and the most relevant factors are indicated below:
Nature of the Invention:
Regarding claim 1, it recites “A modified protein having an amino acid sequence derived from an amino acid sequence of a human wild-type protein, wherein:
The human wild-type protein has a localization signal peptide located at a terminal portion, wherein the amino acid sequence of the localization signal peptide has been modified in the modified protein by insertion of an amino acid at the +3 position of the localization signal peptide as compared to the human wild-type protein;
the amino acid sequence of the human wild-type protein has been modified to remove one or more ubiquitination sites that is (a) present in an N terminal portion of the amino acid sequence of the human wild-type protein, but (b) not present in a homologous mouse wild-type protein; and wherein the sequence of the modified protein differs from both the human wild-type and homologous mouse wild-type proteins; and
the sequence of the modified protein differs from both the human wild-type and homologous mouse wild-type proteins.”
Applicant also claims in claims 5 and 7 that the modified protein of claim 1 comprises one or more ubiquitination sites removed by changing an amino acid residues to an arginine residue.
Applicant also claims (claim 10) that the localization signal peptide directs translocation of the wild-type protein to an intracellular organelle, e.g., a mitochondrion.
Claim 15 is drawn to modifying the protein further by changing the localization signal peptide in +1 or +2 position of the signal peptide to a stabilizing amino acid selected from alanine, glycine, methionine, serine, threonine, valine and proline.
Applicant claims in claim 35 that the modified protein can be any protein from the group consisting of: OTC, ASL, PAH, ABCB4, ABCB11, PAH, AGL, CFTR, MUT, PCCA, PCCB, ASS1, FAH, HMBS, ATP7B, PFIC2, LDLR, G6PC, AGXT, FXN, PAL, BCKDHA, BCKDHB, DBT, UGT1A1, SLC25A13, CD46, CFH, CFI, FIX, FVII, FVIII, C2, C3, C5, GCHD, CBS, MPI, LNL, SERPING1, UROC1,
SMPD1, GLA, GAA, GRHPR, ATP8B1, SERPINC1, PROS1, GBA, ACADVL, HFE, BCKDA, CDG1B, SERPINA1, BMPR2, ENG, ACVR1, SMAD4, BMPR9, HBB, FLCN, HSP1, AP3B1, HPS3, HPS4, HPS5, HPS6, DTNBP1, BLOC1S3, PLDN, AP3D1, BRAF, NF-1, SLC34A2, FBN1, COL1A1, COL1A2, COL1A3, COL5A1, COL5A2, ADAMTS2, PLOD1, TNXB, ABCA3, SP-B, SP-C, GBA, NPC1, NPC2, FOXF1, NKX2-1, SFTPB, SFTPC, ABCA3, CSF2RA, SFTPD, MUCSB, BMPR2, EIF2AK4, CSF2RB, DNAH5, DNAI1, DNAH11, AKR1D1, AMACR, ATP8B1, CYP7A1, FOXC2, GATA2, GHR, HSD3B7, IGFALS, IKBKG, JAG1, KIF11, NOTCH1, NOTCH2, NR1H4, SOX18, TJP2, P53, P73, P63, VIPAS39, VPS33B, EPO, ARG1, CPS1, NAGS, NOS, KRAS, OX40L, IL12, VEGF-A, MMA, TTR, PCSK9, AT, and ALAS1.
Guidance of the Specification:
The Specification teaches about possible changes for modifying a protein of the
List claimed in claim 35.
The Specification teaches about changing and codon-optimizing the open reading frame to have a theoretical minimum of uridines possible, and having a 5’ cap, a 5’UTR, a 3’UTR, a modified ORF and a 3’ poly (A) tail (see [0011]).
When referring to possible 5’UTR to swap in a recombinant construct to obtain encoding an optimized modified protein, Applicant teaches the use of genes expressed in Arabidopsis thaliana in Table 2 ([0011]).
However, when aligning the sequence taught for one of the gene in Table 2, e.g., AT1G58420 (SEQ ID NO: 6), one of ordinary skills can notice that only 21 residues are perfectly identical, while the sequence is 28 residues in length. See results for BLAST (NCBI) below (Query= instant application’s SEQ ID NO: 6; Sbjct = AT1G58420):
Arabidopsis thaliana Uncharacterized conserved protein UCP031279 (AT1G58420), mRNA
Sequence ID: NM_104622.3Length: 951Number of Matches: 1
Range 1: 212 to 232GenBankGraphicsNext MatchPrevious Match
Alignment statistics for match #1
Score
Expect
Identities
Gaps
Strand
42.1 bits(21)
1.3
21/21(100%)
0/21(0%)
Plus/Plus
Query 1 ATTATTACATCAAAACAAAAA 21
|||||||||||||||||||||
Sbjct 212 ATTATTACATCAAAACAAAAA 232
Applicant does not explain why this random portion of the 5’UTR gene is selected for modifying the 5’UTR of a sequence, and how the extra “GCCGCCA” sequence was selected and presented as A. thaliana sequence. In contrast, AT1G67090 sequence provided in Table 2 (SEQ ID NO: 125) is 100% identical over its whole length, to the gene it comes from (GenBank sequence ID: AY06271.1), see below:
Arabidopsis thaliana ribulose bisphosphate carboxylase, small subunit (At1g67090; F5A8.1) mRNA, complete cds
Sequence ID: AY062711.1Length: 772Number of Matches: 1
Range 1: 6 to 26GenBankGraphicsNext MatchPrevious Match
Alignment statistics for match #1
Score
Expect
Identities
Gaps
Strand
42.1 bits(21)
0.43
21/21(100%)
0/21(0%)
Plus/Plus
Query 1 CACAAAGAGTAAAGAAGAACA 21
|||||||||||||||||||||
Sbjct 6 CACAAAGAGTAAAGAAGAACA 26
However, the sequence was selected to start at nucleotide 6 of the 5’UTR of the gene.
Applicant does not explain, nor gives a specific rule for selecting the 5’UTR sequence from a different gene in A. thaliana to modify the coding sequence of the selected protein of interest.
The Specification teaches generally the process of design (starting at [0097]), stating: “Further, nucleotide sequence of any region of the mRNA or DNA template may be codon optimized. Codon optimization methods are known in the art and may be useful in efforts to achieve one or more of several goals. These goals include to match codon frequencies in target and host organisms to ensure proper folding, to bias GC content to increase mRNA stability or reduce secondary structures, to minimize tandem repeat codons or base runs that may impair gene construction or expression, to customize transcriptional and translational control regions, to insert or remove protein trafficking sequences, to remove/add post translation modification sites in encoded protein (e.g. glycosylation sites), to add, remove or shuffle protein domains, to insert or delete restriction sites, to modify ribosome binding sites and mRNA degradation sites, to adjust translational rates to allow the various domains of the protein to fold properly, or to reduce or eliminate problematic secondary structures within the mRNA. Suitable codon optimization tools, algorithms and services are known in the art.” ([0101]).
Applicant teaches decreasing/changing uracil numbers, distribution and clustering to the sequence encoding a protein of interest to modify the Toll-Like Receptor response (innate immunity) ( [0106]-[0114]).
Applicant specifies in the Specification “The removal of predicted ubiquitination sites preferably comprises replacing N-terminus residues that have been found to support ubiquitination such as asparagine, arginine, leucine, lysine or phenylalanine with residues that have been found to be stabilizing against ubiquitination such as alanine, glycine, methionine, serine, threonine, valine and proline.” (see Specification, page 1, lines 28-29 and page 2, lines 1-3).
The Drawings and Figures only present data obtained with one specific protein: Ornithine Transcarbamylase (OTC), i.e., a codon optimized cDNA constructs with SEQ ID NO: 4 (see Table 1, [0058]; Table 6 and [0117]).
The Specification teaches from [0005] that “For example, stabilization of a modified ornithine transcarbamylase (OTC) protein of SEQ ID NO: 4 in this manner is particularly advantageous for preserving the stability of the modified OTC protein during its transport from the cytosol to the mitochondria wherein it exerts its enzymatic activity.”
So, clearly, the Specification is drawn to ONE specific protein, which is OTC.
State of the Art:
Regarding ubiquitination modification, Samelson (Samelson, LE. et al. US
2012/0114700 A 1; published May 10, 2012; listed on IDS filed 08/17/2021) teaches an isolated, non-naturally occurring linker for activation of T cells (LAT) polypeptide comprising at least one amino acid substitution at amino acid 52 or 204 (see Claim 1, page 42 of 43). Samelson teaches the modification of LAT (Linker for Activation of T-cells), mutating ubiquitylation sites for increase stability of the protein (see "Abstract" section). Samelson also teaches that the sequence in the N-terminus of human LAT is different compared to mouse LAT (see SEQ ID NO: 1 and SEQ ID NO: 2, page 38 of 43). Samelson teaches the position to mutate the ubiquitylation site is different in Human and in mouse (human, positions amino acid 52 and 204; mouse, positions amino acid 53 and 121 ). Samelson teaches in claim 1 "at least one amino acid
substitution"; Samelson teaches in claim 2 (page 42 of 43), that the amino acid
substitution comprises substitution of lysine with arginine.
Regarding modifying residues at positions +1 or +2 positions in sequence flanking a signal peptide, Güler-Gane (Güler-Gane,G. et al. “Overcoming the refractory expression of secreted recombinant proteins in mammalian cells through modification of the signal peptide and adjacent amino acids”. PloS ONE, Vol.11, No.5 (2016), p:e0155340; previously cited ) teaches modifying residues at positions + 1 or +2 in flanking heterologous signal peptide in SEAP (secreted alkaline phosphatase) model
protein (title and abstract). Güler-Gane also teaches that changing the amino acid
specifically at+ 1 position with an alanine has a positive effect on the function of the
signal peptide, i.e. secretion level (see abstract; see page 5, first ,r of "[R]esults"
section). Güler-Gane teaches that adding an alanine in the Secrecon signal (Table 1) or two alanine residues lead to increased secretion of SEAP compared to the native signal
(see figure 2B). Güler-Gane also teaches unfavorable residues (see page 8).
Regarding modification of the signal peptide, MacIntyre (MacIntyre, S. et al. “Export incompatibility of N-terminal basic residues in a mature polypeptide of Escherichia coli can be alleviated by optimising the signal peptide.” Mol.Gen. Genet. Vol. 221 (1990), pp: 466-474) teaches optimizing the signal peptide at the N-terminal region, for proper protein trafficking (see title). MacIntyre teaches that one of ordinary skills in the art could modify the signal peptide composition in amino acids to obtain a protein that can be, or prevented from being exported to the outer membrane, using OmpA of Escherichia coli as an example. MacIntyre teaches that the export depends on number of basic residues versus acid residues in the terminal region, which can be manipulated using insertional mutagenesis (see Abstract).
Regarding OTC, Yamaguchi (Yamaguchi, S. et al. “Mutations and
polymorphisms in the human Ornithine Transcarbamylase (OTC) gene”. Human Mutation, vol. 27 , no. 7 (2006), pp: 626-632; previously cited) teaches mutations and polymorphisms in human OTC leading to proteins with a different sequence from human wild-type protein, and specific to human diseases (see title). Therefore Yamaguchi teaches modified human OTC proteins.
Yamaguchi teaches naturally occurring mutations in the human sequence, substituting arginine residue at position 26, to a proline residue, or a leucine residue at position at position 43 to a proline, or a phenylalanine at position 48 to a serine (see Figure 1).
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554
412
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Greyscale
Ye (Ye. X. et al. Human Gene Therapy, Vol. 12 (2001), pp: 1035-1046;
previously cited ) teaches a leader sequence in OTC, i.e. a localization signal peptide (see Figure 1B). Ye teaches that the human OTC leader sequence, i.e. N-terminal region, is different in amino acid sequence of mouse.
Ye shows the sequence alignments of the N-terminus of human OTC with the mouse and rat OTC proteins ( see page 1037, Figure 1B and below). Ye teaches that there is a Phenylalanine at position 3 in the human sequence that is different in the mouse, and lysine (k) in position amino acid 34 that is present in the human sequence but not conserved in the rat and the mouse.
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228
556
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Greyscale
Ye teaches modified Ornithine transcarbamylase (OTC) protein, obtained using site-directed mutagenesis using wildtype OTC. Ye introduces single amino acid substitutions into wild-type OTC and obtains an R92Q and a R141Q arginine to glutamine conversion in the N-terminus (see page 1040, left column, “Coexpression of mutant and wild-type OTC in human or mouse
hepatocytes did not reduce expression of wild-type OTC activity" section, lines 7-15).
Therefore, Ye teaches that site-directed mutagenesis in N-terminus of OTC protein has been performed and are commonly used in prior art.
Oppliger Leibundgut (Oppliger Leibundgut, E. et al. “Ornithine Transcarbamylase Deficiency: Characterization of Gene Mutations and Polymorphisms”. Human Mutation, Vol. 8 (1996), pp: 333-339; previously cited) teaches that modifications at Lysine residues occur in diseases (see Tables 1 and 2). Oppliger Leibundgut therefore teaches that there is motivation to perform amino acid substitutions on lysine residues at the N-terminus of OTC to recreate and study the mutant found in patients, i.e. lysine at positions 46 and 88 taught in Tables 1 and 2.
Therefore, there are many examples of mutagenesis in the prior art in synthetic Biology to obtain/optimize therapeutic protein stability and production.
Breath of the Claims:
Claim 1 lists a set of elements that is part of the invention. Therefore, any human protein that differs in the N-terminal region when comparing with a mouse homolog, would qualify for this invention. This invention rely upon the ability of one of ordinary skills in the art to mutate such as protein, inserting a random amino acid at position +3 of the localization signal, and compensating the charge with another modification within the protein, and abrogating a ubiquitination site by changing lysine to arginine. There are to date, multiple algorithms and services that could be used to achieve such mutations and obtain a large number of variants for a single protein.
The level of skills in the art is high, therefore, the number of species encompassed by the invention could be large, in term of individual proteins and in term of variants obtained.
Experimentation required:
In order to practice the claimed invention, an immense amount of experimentation would be required to apply to the list of protein in claim 35.
Applicant does not give steps for specific manipulation to claim them as their invention. There is no consensus sequence for targeting. All the manipulations cannot be part of a novel invention since the alterations, i.e., insertional mutations, substitution mutagenesis, chemical modifications, are known in the art. However, Applicant claims a specific combination of manipulations without explaining what consensus sequences are to be used.
Applicant basically states: “Suitable codon optimization tools, algorithms and services are known in the art.” ([0101]).
These tools can give many different results in one researcher’s hand using one single protein. Yet, Applicant claims generally that all these results are part of their invention, when basically telling to one of ordinary skills in the art, to just “go and use them, and you’ll arrive at our invention”.
Applicant does not give information on how to use specific algorithms and other optimization tools to arrive at their specific invention. Applicant does not give explanation on how to obtain a modified 5’UTR using genes such as those in Arabidopsis thaliana, either.
Conclusion:
Taking into consideration the factors outlined above, including the nature of the invention, the breadth of the claims, the state of the art, the guidance provided by the applicant and the specific examples, it is the conclusion that an undue experimentation would be required to make and use the invention as claimed.
Response to Arguments
Applicant’s arguments, see Remarks , filed 02/13/2026, with respect to the rejections of claims 1, 5 and 7, and claims 1, 5, 9-10 and 35 under 35 U.S.C. 102(a)(1), and with respect to the rejections of claims 1, 5, 9-10, and 35 under 35 U.S.C. §103 have been fully considered and are persuasive. The rejections have been withdrawn.
Allowable Subject Matter
The following is a statement of reasons for the indication of allowable subject matter:
An modified OTC protein with the sequence identification number 4 (SEQ ID NO:4), corresponding to a modified protein according to claim 1, is free from prior art.
However, Claim 88 is objected to as being dependent upon a rejected base claim, but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims.
Conclusion
No claim is allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/A.D./Examiner, Art Unit 1636
/NANCY J LEITH/Primary Examiner, Art Unit 1636
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