DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Applicant’s submission filed on March 23, 2026 has been entered and considered. Rejections and/or objections not reiterated from the previous action mailed December 23, 2025 are hereby withdrawn. The following rejections and/or objections are either newly applied or are reiterated and are the only rejections and/or objections presently applied to the instant application. The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Election/Restrictions
Applicant’s election of Group 1, claims 1-10 in the reply filed on November 11, 2025 is acknowledged. It is noted that Applicant did not indicate traversal in the response. Accordingly, the election is being treated as without traverse.
Claims 11-19 were previously withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected inventions, there being no allowable generic or linking claim.
Applicant previously elected the following species: EIF2AK2 (claim 2), E3L of vaccinia virus (claim 3), Zα domain from E3L vaccinia virus operably linked to a dsRNA-binding domain from Influenza A virus NS1 protein (claim 4), a dsRNA-binding region from EIF2AK2 protein deleted of its carboxyl-terminal kinase domain and Skp-1 interacting domains from BTRCP, FBW7, SPK2 (claim 6a and b), and SEQ ID NO: 20 (claim 9).
Rejoinder
In view of the prior art, the non-elected species of orthologous dsRNA binding domains such as the dsRNA binding domain of E3L protein from vaccinia virus (claim 6a) was previously rejoined.
Claims 1-10 have been amended. Claims 11-19 have been canceled. Claims 25-33 are newly added. Claims 1-10 and 25-33 are examined on the merits.
Priority
The instant application is a 35 U.S.C 371 national stage filing of the International Application No. PCT/EP2021/071698 filed on August 3, 2021. The instant application claims foreign priority under 35 U.S.C 119(a)-(d) to European Patent Application EP20305899.5, filed on August 4, 2020. Receipt is acknowledged of a certified copy of the foreign patent application as required by 37 CFR 1.55.
Information Disclosure Statement
The information disclosure statements (IDS) submitted on January 27, 2023 are in compliance with the provisions of 37 CFR 1.97 and is being considered by the examiner.
Applicant is reminded that the listing of references in the specification is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
Claim Objections
Claim 26 is objected to because of the following informalities: Claim 26 appears to be missing the article before bacteriophage DNA-dependent RNA polymerase and should read “wherein the DNA-dependent RNA polymerase is a bacteriophage DNA-dependent RNA polymerase”. Appropriate correction is required.
Claim Rejections - 35 USC § 112
Newly added claim 26, which depends from claim 2, recites the limitation "the target host cell protein" in line 1. There is insufficient antecedent basis for this limitation in the claim as the prior recitation is to “a target host cell protein involved in the regulation of the phosphorylation level of eIF2α. Appropriate correction is required.
Claim Rejections - 35 USC § 103
This is a new rejection necessitated by Applicant’s amendment. However, this rejection shares substantial similarity to the rejection as previously set forth in the office action dated December 23, 2025. Any aspect of Applicant’s traversal that pertains to the rejection as newly set forth will be provided following the new statement of rejection.
Claims 1, 10, 25-26 and 33 are rejected under 35 U.S.C. 103 as being unpatentable over Jais et al. (2019, C3P3-G1: first generation of a eukaryotic artificial cytoplasmic expression system. Nucleic acids research, 47(5), 2681-2698, hereafter “Jais”).
With regard to claim 1, Jais teaches a chimeric cytoplasmic capping prone phage polymerase (C3P3-G1) expression system (Abstract) which can be used in mammalian cells for in cellulo transcription of DNA templates (Pg. 2692, right col., Discussion, 1st para.) wherein the C3P3-G1 expression system comprises an mRNA capping enzyme and a DNA-dependent RNA polymerase (Abstract).
Jais does not teach constitutively or transiently downregulating the phosphorylation level of eIF2α in the host cell. However, Jais does teach that the C3P3-G1 system has incomplete translational efficiency (Pg. 2693, right col., last para.) and details various processes which are known to play a role in translation, including teaching that phosphorylation of eIF2α inhibits translation (Pg. 2694, left col.).
Therefore, it would have been obvious to one having ordinary skill in the art, before the effective filing date of the claimed invention, that downregulating phosphorylation of eIF2α would increase translation with a reasonable expectation of success based Jais’ teachings that phosphorylation eIF2α inhibits translation. A skilled artisan would have been motivated to apply decreasing phosphorylation of eIF2α to the C3P3-G1 system as taught by Jais since Jais teaches that the C3P3-G1 system has incomplete translational efficiency and further teaches that phosphorylation of eIF2α inhibits translation, which would improve the translational efficiency of the C3P3-G1 system.
With regard to claim 10, the C3P3-G1 expression system taught by Jais generates 5’-capped and 3’ polyadenylated transcripts (Pg. 2693, left col.). Jais teaches additional modifications to the C3P3 system for improving polyadenylation including tethering of a poly(A) polymerase to the C3P3 transcripts (Pg. 2693, right col., 1st para., lines 16-19).
With regard to newly added claim 25, Jais teaches wherein the mRNA capping enzymes can be an NP868R African swine fever virus capping enzyme (Pg. 2682, right col., last para.), which is considered to reasonably read on a cap-0 canonical capping enzyme.
With regard to newly added claim 26, Jais teaches that the C3P3-G1 enzyme can comprise a bacteriophage K1E RNA polymerase (Pg. 2693, left col., 1st para.), which is considered to reasonably read on a bacteriophage DNA dependent RNA polymerase.
With regard to newly added claim 33, which depends from claim 10, Jais teaches additional modifications to the C3P3 system for improving polyadenylation including tethering of a poly(A) polymerase to the C3P3 transcripts (Pg. 2693, right col., 1st para.).
While Jais is silent as to how the poly(A) polymerase will be tethered to the C3P3 transcript, Jais teaches use of the lambda N peptide for tethering of the capping enzyme (Pg. 2682, right col., last para. and Fig. 4A).
Therefore, it would have been obvious to one having ordinary skill in the art, before the effective filing date of the instant invention, to choose a lambda N peptide to tether a poly(A) polymerase to the C3P3 transcripts. Since Jais teaches that future versions of the C3P3 system will have improved polyadenylation and suggests the addition of a poly(A) polymerase (Pg. 2693, right col, 1st para.) and Jais’ C3P3 system already uses a lambda N peptide for tethering of other fusion protein components (Pg. 2682, right col., last para. and Fig. 4A), a skilled artisan would have recognized that a lambda N peptide could be chosen in order to tether a poly(A) polymerase to the fusion protein with a reasonable expectation of success.
Response to Arguments
Applicant's arguments filed March 23, 2026 have been fully considered but they are not persuasive.
Applicant traverses the rejection of claim 1 under 35 U.S.C. 103 over Jais (Pg. 13-18) based on Applicant’s assertion that Jais fails to teach each and every limitation of claim 1 (Pg. 13). Applicant summarizes the teachings of Jais that the C3P3-G1 expression system exhibit incomplete maturation of transcripts in HEK-293 cells and that Jais acknowledges an imperfection in the C3P3-G1 system of incomplete translational efficiency of C3P3 transcripts (Pg. 14). Applicant asserts that Jais details four theories for additional investigation: 1) incomplete mRNA modifications (i.e., 5’ capping and/or 3’ polyadenylation), 2) post-transcriptional modifications such as methylation, 3) missing host cell proteins that participate in translation, and 4) inappropriate intracellular positioning of C3P3 transcripts (Pg. 15).
First, Applicant asserts that although Jais teaches translation issues with the C3P3 system, Jais does not teach that constitutively or transiently downregulating eIF2α phosphorylation in the host cell would have an effect on translational efficiency and therefore, a skilled artisan would not consider eIF2α phosphorylation as the cause of the translational inefficiency (Pg 1, last para. and Pg. 16, 1st sentence.). Second, Applicant asserts that based on the normal polysome profiling (used as an indicator of global inhibition of host-cell translation) in Fig. 10 of Jais, a skilled artisan would conclude that translational inefficiencies of the C3P3-G1 system are not due to global inhibition of host-cell translation and are not linked to an increase in phosphorylation of eIF2α (Pgs. 16 -17) and thus would not consider downregulation of eIF2α phosphorylation as a mechanism to address the translational inefficiencies exhibited by the C3P3-G1 system (Pg. 18). Applicant points to the instant specification showing drastically altered polysome profiling in the “2nd generation” C3P3-G2 system, which comprises the addition of a poly(A) polymerase, and the instant specification’s assertion that these results indicate eIF2α hyperphosphorylation and traverses that is it these instant results, which are not present in Jais, which would lead a skilled artisan to modify the phosphorylation of eIF2α.
Applicant also traverses the rejection of claim 10 under 35 U.S.C. 103 over Jais (Pg. 18-19), based on the assertion that Jais’ C3P3-G1 system, which did not include a poly(A) polymerase produced normal polysome profiling and led the Jais group to conclude translational inefficiencies of the C3P3-G1 system are not due to global inhibition of host-cell translation and therefore are not linked to eIF2α phosphorylation. Applicant’s traversal is based on the assertion that it is the altered polysome profile exhibited after addition of the poly(A) polymerase which would lead a skilled artisan to modify the phosphorylation of eIF2α.
Applicant's arguments have been fully considered but they are not persuasive.
Regarding Applicant’s assertion that Jais teaches four theories for additional investigation including theory 2) post-transcriptional modifications such as methylation, it is noted that Jais teaches that these types of modifications could be critical for translational efficiency and details that the absence of these modifications could shut down host-cell translation including via a pathway involving lack of inhibition of PKR which then phosphorylates eIF2α and results in translational inhibition (Pg. 2694, left col., last para.). Further, Jais teaches that the proposed hypotheses Therefore, Jais provides specific guidance regarding opportunities to modify the C3P3-G1 system in order to overcome the translational inefficiencies, including pointing to the phosphorylation of eIF2α as inhibiting translation and thus, a skilled artisan could have easily envisioned downregulation of eIF2α phosphorylation as a potential solution to the translational inefficiency of Jais’ C3P3-G1 system. Although, Jais does state that “no frank modifications of ribosome distribution patterns were brought into focus in the polysome profile analysis” and Jais indicates that this finding does not support the hypothesis of global inhibition of host-cell translation which would be expected based on a strong interferon-α response, Jais also suggests that closer investigation of the polysomal fractions is warranted (Pg. 2694, left col., last para.) before suggesting the above-mentioned four theories for additional investigation which includes modifications to eIF2α phosphorylation. Additionally, although Jais’ assertion is that “no frank modifications are present” in the polysome profile, the meaning of “frank modifications” is not clear and Fig. 10 of Jais appears to show at least some differences in polysome profiles which are not quantified.
The polysome profiling was performed between 24 and 48 hours after transfection yet Jais teaches that peak luciferase mRNA buildup occurs at day 4 with the C3P3-G1 system (See Fig. 9B). There is no reported polysome profiling corresponding to day 4 when untranslated mRNA is highest and it is likely that polysome profiling performed at that time would have indicated larger alterations. Further, a skilled artisan, in attempting to reduce the pool of untranslated mRNA based on Jais’ results, would have inhibited phosphorylation of eIF2α on day 4. Applicant is reminded that the claimed method is broadly worded and does not require expression or introduction of the chimeric protein and inhibition of inhibited phosphorylation of eIF2α on the same day.
Furthermore, although drastic alterations of the polysome profile were not seen in the C3P3-G1 system, they appear to be due to the addition of a poly(A) polymerase in the C3P3-G2 system (See instant Example 1), and a skilled artisan would have expected alterations in the polysome profile after the addition of a poly(A) polymerase as poly(A) tails are known to increase mRNA stability, thereby increasing the amount of untranslated mRNA which would be reflected in polysome profile alterations. Further, since the C3P3-G2 system has more stable mRNA due to the poly(A) tail, it is expected that peak buildup of untranslated mRNA would occur earlier and therefore the polysome profiling in the instant specification for the C3P3-G2 system may more accurately coincide with and reflect the buildup of untranslated mRNA when compared to the polysome profiling of the C3P3-G1 system which is not performed during the time of peak mRNA buildup. Additionally, Applicant’s instant specification indicates that increased eIF2α phosphorylation is present in the C3P3-G1 system (Fig. 6 and Example 2(c)) despite the fact that no “frank changes” were see in the polysome profile in the C3P3-G1 system, indicating that the polysome profile is not necessarily a reliable indicator in the present system.
Thus, a skilled artisan would not necessarily have concluded that Jais teaches away from eIF2α phosphorylation nor would a skilled artisan conclude that eIF2α phosphorylation was not to responsible for translational inefficiencies of the C3P3-G1 system. Additionally, since Jais provides the suggestion that eIF2α phosphorylation could be responsible for translational efficiency, the instantly claimed modifications regarding downregulation of eIF2α phosphorylation are considered to be easily envisioned by one having ordinary skill in the art.
Claims 2-6 and 27-31 are rejected under 35 U.S.C. 103 as being unpatentable over Jais as applied to claim 1 above, in view of Langland and Jacobs (2004, Inhibition of PKR by vaccinia virus: role of the N-and C-terminal domains of E3L. Virology, 324(2), 419-429); and in further view of Guerra et al. (2011, Host-range restriction of vaccinia virus E3L deletion mutant can be overcome in vitro, but not in vivo, by expression of the influenza virus NS1 protein. PLoS One, 6(12), hereafter “Guerra”); Ahn et al. (1990, Identification of rpo30, a Vaccinia Virus RNA Polymerase Gene with Structural Similarity to a Eucaryotic Transcription Elongation Factor. Mol Cell Bio, 10(10),5433-5441); Li and Koromilas (2001, Dominant negative function by an alternatively spliced form of the interferon-inducible protein kinase PKR. J. of Biol.Chem., 276(17), 13881-13890) and Ly and Ikegami (2016, Rift Valley fever virus NSs protein functions and the similarity to other bunyavirus NSs proteins. Virology journal, 13(1), 118).
With regard to claims 2, 3, and newly added claim 27 as detailed above, Jais teaches a chimeric cytoplasmic capping prone phage polymerase (C3P3-G1) expression system comprising an mRNA capping enzyme and a DNA-dependent RNA polymerase for use in mammalian systems. Jais further teaches that the C3P3-G1 system has incomplete translational efficiency and provides support for reducing the level of phosphorylation of eIF2α in order to increase translation as increased phosphorylation of eIF2α is known to reduce translation.
Jais does not teach introduction of a polypeptide or nucleic acid molecule encoding the polypeptide which modulates the activity or expression of EIF2AK2 (i.e., PKR, see Pg 10 of the specification), which is involved in the regulation of phosphorylation of eIF2α, nor does Jais teach wherein the polypeptide is E3L from vaccinia virus.
Langland and Jacobs teach wherein the process of translation in eukaryotic cells is regulated by the phosphorylation of eIF2α (Abstract) and wherein protein synthesis is regulated by PKR which, when activated, phosphorylates eIF2α which blocks translation (Pg. 419, right col., last para.). Langland and Jacobs teach wherein the vaccina virus E3L protein binds and masks dsRNA synthesized during viral replication which blocks activation of PKR, preventing phosphorylation eIF2α by PKR and the resultant translation inhibition, thus allowing for protein translation (Pg. 420, left col., 2nd para.). Additionally, Langland and Jacobs teach that use of vaccina virus constructs comprising a deletion of the E3L gene results in activation of PKR and phosphorylation of eIF2α (Pg. 420, right col., Results, 1st para.) Thus, Langland and Jacobs teach wherein expression of E3L of vaccinia virus reduces the activity of PKR which results in decreased phosphorylation of eIF2α thereby allowing for translation. Further, Langland and Jacobs teach that the E3L vaccinia virus protein comprises a highly-conserved dsRNA binding domain at the C-terminus (Pg. 424, right col., 1st para.) and that the N-terminus (i.e., the Zα domain) plays a role in inhibition of PKR activation (Pg. 425, right col., lines 4-6).
Therefore, it would have been obvious to one having ordinary skill in the art, before the effective filing date of the claimed invention, to apply the E3L protein of vaccinia virus which is able to reduce phosphorylation of eIF2α via reduced activation of PKR as taught by Langland and Jacobs to the C3P3-G1 expression system comprising an mRNA capping enzyme and a DNA-dependent RNA polymerase as taught by Jais with a reasonable expectation of success. Since Jais teaches that the C3P3-G1 system has incomplete translational efficiency and further teaches that phosphorylation of eIF2α inhibits translation and Langland and Jacobs teach that E3L protein of vaccinia virus which is able to reduce phosphorylation of eIF2α via reduced activation of PKR, a skilled artisan would have been motivated to combine these teachings in order to increase the translational efficiency of the C3P3-G1 system via blocking phosphorylation of eIF2α leading to a C3P3-G1 system having improved translational ability.
With regard to claim 4, 28, and newly added claim 29, as detailed above, the combined teachings of Jais and Langland and Jacobs teach a C3P3-G1 expression system comprising an mRNA capping enzyme and a DNA-dependent RNA polymerase for use in mammalian systems wherein expression of the E3L protein of vaccinia virus is able to inhibit activation of PKR (i.e. EIF2AK2) thereby preventing the phosphorylation of eIF2α which is well known in the art as a negative regulator of translation. Langland and Jacobs further teach that E3L of vaccinia virus comprises a C-terminus which functions as a dsRNA binding domain and an N-terminus, or Zα domain, which plays an important role in inhibition of PKR activation.
The combined teachings of Jais and Langland and Jacobs do not teach wherein the Zα domain of E3L of vaccinia virus is operably linked to a dsRNA binding domain from Influenza A virus NS1.
Guerra teaches wherein the NS1 protein from influenza A virus (Pg. 3, Engineering of the VACV recombinant viruses) comprises an N-terminal domain which binds to dsRNA and to PKR in order to inhibit phosphorylation activity (Pg. 2, left col., last para and Pg. 5, left col., 2nd full para.) and wherein the NS1 protein has functional properties which are similar to the E3 protein of vaccinia virus (Pg. 2, left col., 3rd full para.). Guerra further teaches wherein NS1 was able to functionally replace E3 in vitro (Pg. 2, right col. 1st full para.) and that mice infected with wildtype vaccina virus comprising the wildtype E3L gene showed severe signs of infection and died a few days after inoculation whereas mice infected with vaccina virus comprising a deleted E3L gene replaced by NS1 did not show signs of infection and survived (Pg. 2, right col., 2nd full para.).
Therefore, it would have been obvious to one having ordinary skill in the art, before the effective filing date of the claimed invention, to substitute the ds-RNA binding domain of Influenza A NS1 as taught by Guerra for the ds-RNA binding domain of E3 of vaccinia virus as taught by Langland and Jacobs with a reasonable expectation of success. As Guerra teaches that ds-RNA binding domain of Influenza A NS1 is functionally equivalent to E3L and is similarly able to inhibit PKR activity (and therefore subsequent phosphorylation of eIF2α) and also provides the benefit of reduced toxicity to mammalian systems compared to wildtype vaccinia virus, a skilled artisan would have been motivated to combine the Zα domain of E3L which is required for reduced phosphorylation of eIF2α by PKR as taught by Langland and Jacobs with the NS1 ds-RNA binding domain comprising has similar properties to E3 as taught by Guerra in order to generate a system which is less toxic to mammals.
With regard to claim 5, as detailed above, the combined teachings of Jais and Langland and Jacobs teach a C3P3-G1 expression system comprising an mRNA capping enzyme and a DNA-dependent RNA polymerase for use in mammalian systems wherein expression of the E3L protein of vaccinia virus is able to inhibit activation of PKR (i.e. EIF2AK2) thereby preventing the phosphorylation of eIF2α which is well known in the art as a negative regulator of translation. Langland and Jacobs further teach that E3L of vaccinia virus comprises a C-terminus which functions as a dsRNA binding domain and an N-terminus, or Zα domain, which plays an important role in inhibition of PKR activation.
While Langland and Jacobs teach wherein the E3L protein of vaccinia virus is able to inhibit of PKR activation, Langland and Jacobs are silent as to the specific amino acid sequence of the E3L protein of vaccinia virus. Ahn teaches an E3L RNA binding protein from vaccinia virus comprising a Z binding domain (Fig. 2), which has at least 90% amino acid sequence identity with instantly claimed SEQ ID NO: 16.
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Therefore, it would have been obvious to one having ordinary skill in the art, before the effective filing date of the claimed invention, to choose the amino acid sequence of the vaccinia virus E3L RNA binding protein as taught by Ahn for the E3L protein of vaccinia virus which plays an important role in inhibition of PKR activation as taught by Langland and Jacobs with a reasonable expectation of success. A skilled artisan would have recognized that the amino acid sequence of E3L RNA binding protein from vaccinia virus as taught by Ahn could have been chosen for the E3L protein of vaccinia virus as taught by Langland and Jacobs with the predictable result using an E3L protein from vaccinia virus which is able to inhibit activation of PRK and therefore also reduce phosphorylation of eIF2α.
With regard to claims 6, 30, and newly added claim 31, as detailed above, the combined teachings of Jais and Langland and Jacobs teach a C3P3-G1 expression system comprising an mRNA capping enzyme and a DNA-dependent RNA polymerase for use in mammalian systems wherein introduced expression of the E3L protein of vaccinia virus is able to inhibit activation of PKR (i.e. EIF2AK2) thereby preventing the phosphorylation of eIF2α which is well known in the art as a negative regulator of translation. Additionally, Langland and Jacobs teach that the vaccinia virus E3L gene encodes a protein comprising a highly-conserved dsRNA binding domain which functions to bind and “mask” dsRNA thereby preventing activation of PKR and phosphorylation of eIF2α (Pg. 420, left col., 2nd para.).
While the combination of Jais and Langland and Jacobs provide support for introduction of polypeptides, specifically E3L, which are able to modulate the activity of PRK which in turn regulates the phosphorylation of eIF2α, the combination of Jais and Langland and Jacobs does not teach wherein the polypeptide is a chimeric protein comprising the E3L dsRNA-binding region and a Skp1-interacting domain from BTRCP, FBW7, and SPK2.
Ly and Ikegami teach wherein NSs proteins from Rift Valley fever virus promote degradation of PKR via the cullin 1-Skp1-Fbox E3 ligase complex which promotes ubiquitination (Abstract). Ly and Ikegami teach that NSs proteins assist in increased translation by blocking eIF2α phosphorylation by PKR (Pg. 6, left col., 1st para., lines 6-9). Additionally, Ly and Ikegami teach that the specific E3 ligase complex responsible for PKR degradation by NSs involves interaction between NSs protein and cullin-1 via Skp1 and F-box proteins FBXW11 (also called βTrCP2) and BTRC (also called βTrCP1), which are functional paralogs of the BTRC/ βTrCP gene (Pg. 6, left col., 3rd para., lines 1-2 and Pg. 6, right col., 1st para., also Fig. 3). Thus, Ly and Ikegami teach that Skp1 interacting domains from βTrCP are involved in binding, ubiquitination, and resultant degradation of PKR which, in turn, reduces eIF2α phosphorylation and promotes translation.
Therefore, it would have been obvious to one having ordinary skill in the art, before the effective filing date of the claimed invention, to use an E3 ligase protein comprising a Skp1 interacting domain from BTRCP for degradation of PKR as taught by Ly and Ikegami in a chimeric protein comprising the dsRNA binding domain of a vaccinia virus E3L protein which is capable of regulating eIF2α phosphorylation via PKR as taught by the combined teachings of Jais and Langland and Jacobs with a reasonable expectation of success. A skilled artisan would have recognized that the dsRNA binding domain of a vaccinia virus E3L protein which reduces PKR activation as taught by Langland and Jacobs and the E3 ligase protein comprising a Skp1 interacting domain from BTRCP which signals degradation of PKR as taught by Ly and Ikegami could be used to reduce eIF2α phosphorylation thereby increasing translation. A skilled artisan would have been motivated to make this combination in order to generate a chimeric enzyme system with increased translational ability as Jais teaches that the C3P3-G1 expression system has incomplete translational efficiency and that increased phosphorylation of eIF2α is known to reduce translation. Since Jais teaches use of chimeric enzymes which can be introduced to a host cell for increased translation, one having ordinary skill in the art could easily envision generation of other chimeric proteins.
With regard to claim 6 and newly added claim 30, as detailed above, the combined teachings of Jais and Langland and Jacobs teach a C3P3-G1 expression system comprising an mRNA capping enzyme and a DNA-dependent RNA polymerase for use in mammalian systems wherein introduced expression of the E3L protein of vaccinia virus is able to inhibit activation of PKR (i.e. EIF2AK2) thereby preventing the phosphorylation of eIF2α which is well known in the art as a negative regulator of translation.
While the combination of Jais and Langland and Jacobs provide support for introduction of polypeptides which are able to modulate the activity of PRK which in turn regulates the phosphorylation of eIF2α, the combination of Jais and Langland and Jacobs does not teach wherein the polypeptide is a chimeric protein comprising a dsRNA-binding region for EIF2AK2 (i.e., PKR) protein deleted of its carboxyl-terminal kinase domain and Skp1-interacting domain from BTRCP, FBW7, and SPK2.
Li and Koromilas teach an alternatively spliced form of PKR which results in a truncated protein that retains just the dsRNA binding motifs and exhibits a dominant negative function by inhibiting PKR autophosphorylation and eIF2α phosphorylation (Abstract). Li and Koromilas teach that the truncated PKR protein comprising the dsRNA binding motifs (Pg. 13883, left col., 1st para., lines 17-20) which are located at the N-terminus (Pg. 13883, left col., 1st para., lines 6-7) is able to “self-associate” and bind to wildtype PKR (Pg. 13883, right col., 3rd para., lines 3-5 and 11-13). This is considered to reasonably read on a dsRNA binding region of EIK2AK2 protein deleted of its carboxyl-terminal kinase domain which is capable of selectively binding to EIK2AK2. Li and Koromilas further teach that the truncated PKR protein exhibits a dominant negative function both via inhibition of autophosphorylation (Pg. 13884, left col., lines 1-2 and right col., lines 4-6) and inhibition of eIF2α phosphorylation (Pg. 13885, left col., 1st full para., lines 13-14 and Pg. 13885, right col., 1st para., lines 1-3).
Therefore, it would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention, to substitute the alternatively spliced PKR protein comprising only dsRNA binding domains which is capable of binding to PKR and regulating phosphorylation of eIF2α as taught by Li and Koromilas for the E3L protein of vaccinia virus which is able to inhibit activation of PKR and regulate phosphorylation of eIF2α as taught by Langland and Jacobs with a reasonable expectation of success. A skilled artisan would have recognized that substitution with the alternatively spliced PKR protein comprising just the dsRNA binding domains as taught by Li and Koromilas would create the predictable result of use of polypeptide which is capable of binding to PKR and regulating phosphorylation of eIF2α.
While Jais teaches generation of a chimeric enzyme, the combined teachings of Jais, Langland and Jacobs, and Li and Koromilas do not teach a chimeric protein comprising a Skp1-interacting domain from BTRCP, FBW7, and SPK2.
Ly and Ikegami teach wherein NSs proteins from Rift Valley fever virus promote degradation of PKR via the cullin 1-Skp1-Fbox E3 ligase complex which promotes ubiquitination (Abstract). Ly and Ikegami teach that NSs proteins assist in increased translation by blocking eIF2α phosphorylation by PKR (Pg. 6, left col., 1st para., lines 6-9). Additionally, Ly and Ikegami teach that the specific E3 ligase complex responsible for PKR degradation by NSs involves interaction between NSs protein and cullin-1 via Skp1 and F-box proteins FBXW11 (also called βTrCP2) and BTRC (also called βTrCP1), which are functional paralogs of the BTRC/ βTrCP gene (Pg. 6, left col., 3rd para., lines 1-2 and Pg. 6, right col., 1st para., also Fig. 3). Thus, Ly and Ikegami teach that Skp1 interacting domains from βTrCP are involved in binding, ubiquitination, and resultant degradation of PKR which, in turn, reduces eIF2α phosphorylation and promotes translation.
Therefore, it would have been obvious to one having ordinary skill in the art, before the effective filing date of the claimed invention, to use an E3 ligase protein comprising a Skp1 interacting domain from BTRCP for degradation of PKR as taught by Ly and Ikegami in a chimeric protein comprising an alternatively spliced PKR protein comprising only dsRNA binding domains which is capable of binding to PKR and regulating eIF2α phosphorylation as taught by the combined teachings of Jais and Li and Koromilas with a reasonable expectation of success. A skilled artisan would have recognized that both the alternatively spliced PKR protein comprising only dsRNA binding domains which is capable of binding to PKR and regulating phosphorylation of eIF2α as taught by Li and Koromilas and the E3 ligase protein comprising a Skp1 interacting domain from BTRCP which signals degradation of PKR as taught by Ly and Ikegami could be used to reduce eIF2α phosphorylation thereby increasing translation. A skilled artisan would have been motivated to make this combination in order to generate a chimeric enzyme system with increased translational ability as Jais teaches that the C3P3-G1 expression system has incomplete translational efficiency and that increased phosphorylation of eIF2α is known to reduce translation. Since Jais teaches use of chimeric enzymes which can be introduced to a host cell for increased translation, one having ordinary skill in the art could easily envision generation of other chimeric proteins.
Response to Arguments
Applicant's arguments filed March 23, 2026 have been fully considered but they are not persuasive.
Applicant traverses the rejection of claim 1 under 35 U.S.C. 103 over Jais in view of the additional references of Langland and Jacobs, Guerra, Ahn, Li and Koromilas, and Ly and Ikegami (PG. 19) do not cure the deficiencies of Jais based on Applicant’s traversal as detailed above and thus would not teach, suggest, or render obvious any of claims 2-6.
Applicant’s arguments have been fully considered by are not persuasive based on the reasons stated in the response to arguments for claims 1 and 10 above.
Claims 7, 8 and 32 are rejected under 35 U.S.C. 103 as being unpatentable over Jais as applied to claims 1 and 10 above in view of Sahin et al. (WO 2014071963 A1, hereafter “Sahin”) and in further view of Li and Koromilas (2001, Dominant negative function by an alternatively spliced form of the interferon-inducible protein kinase PKR. J. of Biol.Chem., 276(17), 13881-13890).
With regard to claims 7-8, as detailed above, Jais teaches a chimeric cytoplasmic capping prone phage polymerase (C3P3-G1) expression system comprising an mRNA capping enzyme and a DNA-dependent RNA polymerase for use in mammalian systems. Jais further teaches that the C3P3-G1 system has incomplete translational efficiency and provides support for reducing the level of phosphorylation of eIF2α in order to increase translation as increased phosphorylation of eIF2α is known to reduce translation.
Jais does not teach wherein downregulation of phosphorylation of eIF2α comprises introducing at least two polypeptides or one or more nucleic acids encoding said polypeptides which modulate the activity or expression of at least two different target host cell proteins involved in regulation of phosphorylation of eIF2α. Jais also does not teach wherein one of the polypeptides inhibits the phosphorylation of eIF2α and wherein another of the polypeptides activates dephosphorylation of eIF2α.
Sahin teaches a method of expressing RNA generated by transcription of a DNA template (Pg. 20, lines 22-23) in a cell, the method comprising inhibiting intracellular IFN signaling in the cells (Abstract; Pg. 2, lines 30-32) wherein inhibiting intracellular IFN signaling comprises inhibiting the PKR pathway (Pg. 3, lines 24-25) via inhibition of eIF2α phosphorylation (Pg. 3, lines 27-28 and Pg. 4, lines 5-6) using a viral inhibitor of PKR (e.g. vaccinia virus E3). Sahin teaches wherein the viral inhibitor of PKR is provided to the cell as a nucleic acid encoding the inhibitor (Pg. 3, lines 9-12). This is considered to reasonably read on introducing a nucleic acid encoding a polypeptide which inhibits the phosphorylation of eIF2α. Additionally, Sahin teaches that the PKR pathway can also be inhibited by an agent which dephosphorylates eIF2α (e.g., herpes simplex virus ICP34.5) (Pg. 39, lines 1-3 and 11-12), which is considered to reasonably read on a polypeptide which activates dephosphorylation of eIF2α. Sahin further teaches that inhibiting intracellular IFN signaling via inhibition of the PKR pathway results in increased expression of the RNA in the host cell (Pg. 4, lines 32-33) and defines “increased expression” as translation of RNA to peptide (Pg. 24, lines 24-27). While Sahin does not directly teach introducing at least two polypeptides or one or more nucleic acid molecules encoding the polypeptides which modulate the activity or expression of at least two different target host cell proteins involved in the regulation of eIF2α wherein one polypeptide inhibits eIF2α phosphorylation and one polypeptide activates dephosphorylation of eIF2α, Sahin does teach introducing at least two polypeptides or nucleic acid molecules encoding at least two polypeptides which modulate the expression or activity of different pathways involved in the IFN signaling (i.e., extracellular and intracellular) (Pg. 5, lines 10-15; Example 2).
Therefore, it would have been obvious to one having ordinary skill in the art, before the effective filing date of the claimed invention, to modify Sahin’s embodiment of introducing two or more nucleic acids which encode polypeptides modulating two different pathways involved in IFN signaling to introduce two or more nucleic acids which encode polypeptides which modulate the activity or expression of two different host cell proteins involved in the regulation of eIF2α phosphorylation with a reasonable expectation of success. Since Sahin teaches that the PKR pathway can be inhibited via inhibition of eIF2α phosphorylation and by agent which dephosphorylates eIF2α, a skilled artisan could have easily combined introduction of a polypeptide or nucleic acid encoding which inhibits eIF2α phosphorylation with introduction of a polypeptide or nucleic acid encoding which dephosphorylates eIF2α, especially in light of Sahin’s teaching that inhibiting intracellular IFN signaling via inhibition of the PKR pathway results in increased expression of the RNA in the host cell. One having ordinary skill in the art would have been motivated to make this combination in order to determine whether inhibition of eIF2α phosphorylation in addition to dephosphorylation of eIF2α would have an increased effect on transcript expression in a host cell when compared to just inhibition of eIF2α phosphorylation or dephosphorylation of eIF2α.
It would also have been obvious to one having ordinary skill in the art, before the effective filing date of the claimed invention, to combine use of introduction of at least two polypeptides or nucleic acids encoding which modulate the activity or expression of at least two different target proteins involved in regulation of eIF2α phosphorylation wherein one polypeptide inhibits eIF2α phosphorylation and the other polypeptide activates dephosphorylation of eIF2α as taught by Sahin with the C3P3-G1 expression system comprising an mRNA capping enzyme and a DNA-dependent RNA polymerase for use in mammalian systems as taught by Jais with a reasonable expectation of success. Since Jais teaches that the C3P3-G1 system has incomplete translational efficiency and further teaches that phosphorylation of eIF2α inhibits translation, a skilled artisan would have been motivated to make this combination in order to reduce eIF2α phosphorylation thereby producing a C3P3-G1 system with increased transcriptional ability.
With regard to newly added claim 32, as detailed above, the combination of Jais and Sahin teaches a C3P3-G1 expression system comprising an mRNA capping enzyme and a DNA-dependent RNA polymerase for use in mammalian systems via introduction of two or more nucleic acids which encode polypeptides which modulate the activity or expression of two different host cell proteins involved in the regulation of eIF2α phosphorylation, where in one polypeptide inhibits eIF2α phosphorylation and the other polypeptide activates dephosphorylation of eIF2α.
While Sahin teaches inhibition of the PKR pathway (Pg. 3, lines 24-25) via inhibition of eIF2α phosphorylation (Pg. 3, lines 27-28 and Pg. 4, lines 5-6), specifically, inhibition of PKR autophosphorylation and provides an embodiment using vaccinia virus E3L as the inhibitor of PKR (Pg. 4, 1st full para. and Pg 40, last para.), but is silent as to use of a PKR (i.e., EIF2AK2) inhibitor comprising the dsRNA binding domain from EIF2AK2 deleted of its carboxyl-terminal kinase domain or a biologically active fragment thereof.
Li and Koromilas teach an alternatively spliced form of PKR which results in a truncated protein that retains just the dsRNA binding motifs and exhibits a dominant negative function by inhibiting PKR autophosphorylation and eIF2α phosphorylation (Abstract). Li and Koromilas teach that the truncated PKR protein comprising the dsRNA binding motifs (Pg. 13883, left col., 1st para., lines 17-20) which are located at the N-terminus (Pg. 13883, left col., 1st para., lines 6-7) is able to “self-associate” and bind to wildtype PKR (Pg. 13883, right col., 3rd para., lines 3-5 and 11-13). This is considered to reasonably read on a dsRNA binding region of EIK2AK2 protein deleted of its carboxyl-terminal kinase domain which is capable of selectively binding to EIK2AK2. Li and Koromilas further teach that the truncated PKR protein exhibits a dominant negative function both via inhibition of autophosphorylation (Pg. 13884, left col., lines 1-2 and right col., lines 4-6) and inhibition of eIF2α phosphorylation (Pg. 13885, left col., 1st full para., lines 13-14 and Pg. 13885, right col., 1st para., lines 1-3).
Therefore, it would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention, to substitute the alternatively spliced PKR protein comprising only dsRNA binding domains at the N-terminus which is capable of binding to PKR and inhibit autophosphorylation as well as phosphorylation of eIF2α as taught by Li and Koromilas for the E3L protein of vaccinia virus which is able to inhibit activation of PKR and regulate autophosphorylation of PKR as taught by Sahin with a reasonable expectation of success. A skilled artisan would have recognized that substitution with the alternatively spliced PKR protein comprising just the N-terminal dsRNA binding domains as taught by Li and Koromilas would also inhibit autophosphorylation of PKR as well as provide the added benefit of inhibition eIF2α phosphorylation, thus increasing the inhibitory effect on eIF2α phosphorylation. A skilled artisan would have been motivated to make this substitution as Jais teaches that eIF2α phosphorylation inhibits translation (Pg. 2694, left col., last para.) and that the C3P3-G1 system suffers from translational inefficiency (Pg. 2693, right col., last para.).
Response to Arguments
Applicant's arguments filed March 23, 2026 have been fully considered but they are not persuasive.
Applicant traverses the rejection of claim 1 under 35 U.S.C. 103 over Jais in view of Sahin does not cure the deficiencies of Jais based on Applicant’s traversal as detailed above and thus would not teach, suggest, or render obvious any of claims 7 and 8.
Applicant’s arguments have been fully considered by are not persuasive based on the reasons stated in the response to arguments for claims 1 and 10 above.
Claim 9 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.
Response to Arguments
Applicant’s traversal regarding the rejection of claim 1 under 35 U.S.C. 103 over Jais in view of WO ‘811 and Langland and Jacobs (Pg. 19) is considered moot in light of Applicant’s amendment to claim 9 reciting an “amino acid sequence with at least 90% identity to SEQ ID NOs: 20 or 36”.
Conclusion
THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
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/ERIN V PAULUS/Examiner, Art Unit 1631
/ARTHUR S LEONARD/Examiner, Art Unit 1631
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