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 .
Election/Restrictions
Applicant’s election without traverse of Group I in the reply filed on 7/27/2026 is acknowledged.
Claims 90-99 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to nonelected inventions, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 7/27/2026.
Applicant also elected (b) residues 33, 39, and 47 of MAAP consensus sequence for alteration without traverse.
Claims 83-89 are under consideration in this office action.
Claim Objections
Claim 85 is objected to because of the following informalities: In the last line, the claim recites, “residues numbers”. This is grammatically incorrect. Amending it to recites, “residue numbers” would be remedial. Appropriate correction is required.
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.
Claim 85 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.
Claim 85 recites, “preferably from residues numbers 39 to 103”. “Preferably” renders limitations following it indefinite because it is not apparent if the limitations that follow preferably are intended to further limit “the neurogenic transcription factor” or if they are exemplary, thus non-limiting.
Claim Rejections - 35 USC § 112
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.
Claims 83-89 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.
When determining if a recited genus has adequate written description for a genus:
(1) the broadest reasonable interpretation of the genus is determined;
(2) the disclosure is examined to determine if the specification has provided a representative number of species to describe the complete structure of the genus;
(3) the disclosure is examined to determine whether a representative number of species have been sufficiently described by other relevant characteristics, specified features and functional attributes that would distinguish different members of the claimed genus; and
(4) the state of the art is examined to the determine if it supports/supplement the genus description in the specification in a manner that would demonstrate the application was in possession of the claimed genus at the time of effectively filing.
Breadth of the claimed Genus:
Claim 1 is a genus of AAV genome. The breadth includes any AAV genome of any serotype. The AAV genome is recited to have “a mutation that reduces expression of the full-length wild-type MAAP yet maintains expression of VP1, wherein said mutation introduces at least one spot codon to stop any translation, in any part of translation, to stop translation of full-length wild-type MAAP. As such, the mutation as claimed introduces any stop coding, canonical or non-canonical, to anywhere in the genome to impart complete stop of MAAP translation completely or partially so as a full-length MAAP is not expressed. The introduction of the stop codon can be in anywhere in the nucleic acid coding sequence of any MAAP gene variant known. The introduction of the stop codon can be outside of the MAAP in a gene known to be upstream in the induction of MAAP gene expression that disrupts MAAP gene expression fully. As such, the breadth of adding any stop codon to any part of the MAAP gene coding sequence or an upstream regulator that would result in stopping expression of MAAP is a broad genus with a structurally diverse and large number of different mutant AAV genome species.
Specification Description: (citations from pre-grant publication)
[0089] We serendipitously found a way to both increase production while also making a better-quality vector. Using our new approach, one can increase viral production 72 hours after infection by 300-400%. The resulting viruses are more stable, showing less capsid degradation ≥72 hours after infection. The resulting virus, if designed to be a gene therapy vector (i.e., if it is recombinant and includes a “transgene” or therapeutic foreign gene), shows also improved genome packaging at 72 h. Furthermore, the resulting gene therapy vector is expected to achieve improved transduction efficiency (expression of the therapeutic transgene in the target cells).
[0090] While our discovery is important industrially, we came across it while doing research more academic or theoretical in nature. Doing a virion characterization and whole-genome analysis of AAV, we identified a novel protein encoded by a non-canonical start codon and then discovered that the protein in fact is expressed in wild-type AAV. We named the novel protein “DS”. While carrying out our studies the same protein was described by Ogden et. al. (2019) who named it as a membrane-associated accessory protein (MAAP). For consistency, we also use the name MAAP herein instead of DS.
[0091] We found that mutation of the non-canonical start codon leads to inactivation of this new protein. We similarly found that the introduction of multiple stop codons in the N-terminus of the accompanying ORF also leads to protein inactivation.
[0092] We then compared AAVs that were modified to include stop codons to inactivate this new protein to wild-type AAV. We found that 24 h after infection, wt-AAV produces higher viral titers than do viruses modified to include stop codons to inactivate the new protein. This is perhaps not surprising, because it implies that the wild-type gene gives some kind of selective advantage viz mutants lacking a working copy of that gene.
[0093] Surprisingly, however, we found that when we extended the period of culturing infected cells to 72 h, our mutant null viruses produced higher viral genome (vg) titers than did wt-AAV2. This is surprising because it implies that a wild-type gene has a deleterious selective disadvantage viz mutants lacking a working copy of that gene.
[0094] Further, we were surprised to find that our mutant (null) viruses show more robust capsid integrity or durability, and reduced capsid degradation over time. In contrast, wt-AAV displayed specific proteolytic fragments that are visible in immunoblotting.
[0095] Further, we found that our mutant (null) viruses show increased relative concentrations of VP1 and VP2 in the resulting capsids.
[0096] Further, we expect that our mutant (null) viruses are more infective than are wild-type AAV. Without intending to be bound by theory, we posit that mutant virus will be more infective due to higher capsid integrity, notably due to the VP1 protein. The VP1 unique domain encodes a phospholipase A2 domain, “PLA2.” This phospholipase is critical for AAV endosomal escape during the infection.
[0097] Similarly, we expect that our mutant (null) viruses, when engineered to contain a transgene, produce improved transgene expression. Without intending to be bound by theory, we posit that this improved transgene expression is because VP1 delivers the transgene to the infected cell nucleus for expression and our mutant (null) viruses have an increased relative concentration of VP1.
[0098] Although we do not wish to be bound theory, the MAAP C-terminus contains three basic-amino-acid-rich (BR) clusters, KKIR (MAAP2BR1), RRKR (MAAP2BR2) and RNLLRRLREKRGR (MAAP2BR3) that could be involved in the cellular localisation of MAAP. Indeed, similar BR clusters were shown to act as nuclear localisation signal (NLS) for AAP. The nearly complete deletion of the MAAPBR3 did not provide evidence of impaired nuclear localisation, so it remains that the MAAP2BR1 and MAAP2BR2 domains may still have allowed for membrane association. The lack of only the last ten amino acids at the MAAP C-terminus boosted VP and capsid levels and reduced capsid degradation while the total deletion of MAAP2BR3 fully prevented the degradation of AAV2 capsid. Proteasome inhibition plays a role during AAV infection and the addition of protease inhibitor can prevent capsid antigen presentation, as well enhance viral transduction. Furthermore, AAV capsid has been shown to be able to auto-cleave in acidic conditions, and AAV capsids are subjected to proteasome-involved post-translational modifications (PTMs) during wt-AAV production, including ubiquitination. These PTMs could potentially be used as signals to initiate host cell defence or to down-regulate new capsids via ubiquitination and subsequent proteasomal degradation. In addition to a possible impact of MAAP on the cellular degradation processes, the AAV stabilising effect of MAAP could be achieved by protecting the capsids from entering into subcellular localisations where the degradation process takes place. However, we did not observe an effect of MAAP on capsid egress from the nucleus within 24 hpt. Accordingly, in certain embodiments, MAAP peptides of the invention exclude, partially or fully, one or more of the BR clusters, particularly MAAP2BR3.
[0099] We thus found a surprising way to increase the industrial production of AAV vectors by inactivating expression of this novel wild-type protein. We also found a surprising way to increase the shelf-life stability of AAV viruses by inactivating expression of the wild-type protein. Our findings are industrially valuable because AAV can be therapeutically used as a gene therapy vector. Thus, our findings provide a way to make AAV gene therapy vectors (e.g., non-complimentary or self-complementary AAVs, AAVs with engineered ITRs, etc.) in larger quantity and with greater stability than previously possible.
[0100] By inactivating expression of the wild-type protein, one can increase viral production 72 hours after infection by 300-400%. The resulting viruses are more stable, showing less capsid degradation ≥72 hours after infection. The resulting virus has as high a percentage of full capsids as wild-type, and perhaps an even higher percentage of full capsids than wild type. Furthermore, we expect that the resulting gene therapy vector will achieve improved transduction efficiency (expression of the therapeutic transgene in the target cells).
[0223] We found that the first CTG encountered on the MAAP reading frame is the principal translation initiation codon for MAAP. The CTG translates to a leucine in MAAP (L1 on the full-length protein). On VP1 frame (−1 to MAAP), at this site is CCT that translates to P27.
[0224] To analyze non-canonical start codon usage in the context of the wild-type genome, we mutated the first potential non-canonical start codon of MAAP (CTG, L1 on the full-length protein) to CGG (translating to R1). This abolished its potential start codon function. In response, we found that MAAP production fell to levels undetectable using Western blot.
[0239] MAAP Inactivation and Impact on AAV Production
[0240] The modification of MAAP, either by the mutation of the start codon or introduction of stop codons at various positions in the MAAP coding sequence led to a reduction of AAV productivity at 24 h post transfection. See FIG. 3. This reduction in AAV productivity was statistically significant when MAAP-L1 (CTG) was modified to MAAP-R1 (CGG), with a productivity decreasing to only 21% of that observed with wt-AAV2. When a stop codon was introduced in place of MAAP-Q9, productivity decreased to only 39% of that observed with wt-AAV2. We also observed a reduction to 15% and 20% of wild-type productivity (respectively) for AAV mutants for which MAAP-W103 or MAAP-W105 were mutated to stop codon. Introduction of stop codons in place of MAAP-S33-39-S47, MAAP-S65, led to a trend in the reduction of AAV titers to 76% and 61% of levels seen with compared to wt-AAV2. Introduction of a stop codon in place of amino acid residue number 90, 100, 106 or 110 produced no clear trend in difference viz results observed with the wild-type gene. See FIG. 3. Thus, 24 h post transfection the expression of MAAP protein provides a replicative advantage compared to AAV mutants in which the MAAP gene is inactivated. Our results differ from Ogden (2019), who observed that the MAAP mutants did not have reduced titers. However, they observed that MAAP mutants were out-competed unless complemented in trans with functional MAAP polypeptide. This conclusion fits with our observed results of MAAP providing a replicative advantage to wt-AAV2 at 24 h post transfection.
[0241] At 72 h time point, only MAAP mutants in which MAAP-W103 and MAAP-W105 were replaced by stop codons showed reduced titers, respectively 0.75 and 0.76-fold, compared to wt-AAV2. See FIG. 4. Surprisingly, however, we found that for all other studied MAAP mutants, AAV titers were improved, rather than decreased. This surprising increase in AAV titer was significant for mutants in which MAAP-S33-S39-S47, MAAP-E90, MAAP-L100, MAAP-L106, are replaced by stop codons, with titers respectively increased by 3.50, 4.62, 3.67, 4.08-fold compared to wt-AAV2. Thus, when production time (i.e., the time transfected cells are cultured to produce virus) is extended beyond 24 hours, inactivation of MAAP protein results in higher AAV titers than does wild-type AAV.
[0242] Next, we studied the effect of MAAP over-production to wt-AAV production, or to AAV mutant where MAAP-S33-S39-S47 were mutated to stop codons. At 24 h after transfection, when additional MAAP was expressed with wt-AAV2, we observed a 0.67-fold reduction in AAV titer compared to wt-AAV2. See FIG. 5, left. At 72 h time point, a 0.36-fold reduction compared to wt-AAV2 was observed. See FIG. 6, right. When we added to the wt-AAV reference cell a plasmid of similar size to the MAAP expression plasmid (to mimic the additional DNA mass provided by the addition of the MAAP expression plasmid), a 0.91-fold reduction compared to wt-AAV2 reference was observed at 24 h time point and 0.96-fold reduction at 72 h time point.
[0243] When we produced AAV for which MAAP-S33-S39-S47 are mutated to stop codons we observed a 1.66 fold increase in vg titers compared to wt-AAV2 at 24 h time point and 5.89-fold increase at 72 h time point.
[0244] MAAP added to AAV for which MAAP-S33-S39-S47 are mutated to stop codons resulted in vg titers similar to wt-AAV2 at 24 h time point and with 1.22-fold increase at 72 h time point. When the MAAP-S33-S39-S47 are mutated to stop codons and is complemented with plasmid of similar size to the MAAP expression plasmid, the titers were equal to the wt-AAV2 reference at 24 h time point and 3.80-fold higher at 72 h time point.
[0245] In summary, for culture periods longer than 24 hours, we surprisingly found that the over-expression of the MAAP protein resulted in reduced viral genome (vg) titers. This suggests that MAAP is required in a stoichiometric amount relative to another AAV protein(s), potentially the AAP or the VPs.
[0249] AAV for which MAAP-S33-S39-S47 are mutated to stop codons, MAAP-S65, MAAP-E90, MAAP-L100 and MAAP-L110 showed a trend of higher Kanamycin packaging compared to wt-AAV2 both at 24 h and at 72 h time points.
[0250] When MAAP was added in complement to wt-AAV production or to AAV in which MAAP-S33-S39-S47 are mutated to stop codons, we observed an increase in Kanamycin resistance gene packaging compared to the wt-AAV without addition MAAP protein expression. See FIGS. 9 and 10.
[0255] Interestingly, for all constructs in which MAAP expression was inactivated, except the MAAP-L110 to stop mutant, we observed the disappearance of the three 32 kDa, 18 kDa and 14 kDa polypeptides (FIG. 13; FIG. 14). We also observed an increase in the quantity of VP1 and VP2 proteins. This suggests that the degradation products originated from the degradation of VP1 and VP2 specifically (FIG. 13; FIG. 14). However, the variant in which MAAP-L110 was mutated to stop codon still displayed the specific 18 kDa AAV capsid degradation product, but with lower intensity compared to wt-AAV2. This suggests that the specific proteolytic activity observed, when MAAP is expressed, is associated with its C-terminal part, more specifically with the basic amino acid-rich “BR3” domain (amino acid residues 107-119 on SEQ ID NO. 2), as only the MAAP-L110 to stop mutant, still encoding the BR3 domain, displayed the VP proteolytic fragment. When the MAAP was expressed in complement to the AAV mutant in which MAAP-S33-S39-S47 are replaced by stop codons, the proteolytic activity on the AAV capsid was restored. These data show that the virus preparation made from a genome that is MAAP-deleted or MAAP-truncated to eliminate the proteolytic domain, is physically different from virus made from a wild-type genome. Our finding here thus provides a way to increase both the yield of AAV, and the stability of the resulting AAV.
Thus the specification solely provides a handful of species examples, particularly the one specific AAV2 comprising MAAP-S33-S39-S47, that results in an AAV that the functional characteristics of reducing full length expression of wild-type yet maintaining expression of VP1. However, the genus of an AAV comprising at least one stop codon mutation anywhere in the genome to arrive at the function reduced expression of full-length wildtype MAAP yet maintains VP1 expression is vastly larger and more diverse. In particular it includes mutations to any MAAP proteins expression upstream regulatory proteins amongst others not described. As such, the specification fails to provide a representative number of species examples by relevant characteristics and traits to adequately describe the complete genus.
State of the Art: A search of the art does not impart additional species examples or descriptions to supplement the shortcomings of the specification. Further consistent with the art, the specification demonstrates that not all introduction of stop codon to the genome will predictably arrive at the function of reducing expression of full-length wild-type MAAP yet maintaining expression of VP1 as claimed. For example, the specification describes that a stop mutation MAAP-110 ablated the expression of VP1 This suggests that the specific proteolytic activity observed, when MAAP is expressed, is associated with its C-terminal part, more specifically with the basic amino acid-rich “BR3” domain (amino acid residues 107-119 on SEQ ID NO. 2), as only the MAAP-L110 to stop mutant, still encoding the BR3 domain, displayed the VP proteolytic fragment [0255]. Further Ogden et al 2019 (of record in IDS) describes MAAP protein expression when the first CTG star codon is mutated and when a stop codon is introduced into MAAP-Q6. Ogden et al 2019 also that MAAP mutants in the same manner described in the specification did not have the same reduced viral titers as seen in the instant application. As such, the art teaches that not all addition of stop codons, even into the MAAP gene sequence itself, predictably result in the claimed phenotypic function and other functions described in the instant application. Further, it is well established in the art that mutations introduces to the genome, especially outside of the target gene itself, do not predictably lead a desired phenotype and often have not impact at all.
As such, the genus of an AAV that has a mutation that reduced expression of full-length wild-type MAAP yet maintains expression of VP1, wherein said mutation introduces at least one stop codon to stop translation of full-length MAAP lacks written description because the small number of species examples described by the specification would not be representative of the complete structure of the genus. Further, the art does not provide sufficient examples to supplement the shortcomings of the application description and further describes an unpredictable correlation between genome mutations and desired phenotype. As such, an artisan of ordinary skill could not envision the complete genus in a manner that would convey that the application was in possession of the complete genus as claimed at the time of effective filing. As such, the claims lack adequate written description.
No claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to MARCIA STEPHENS NOBLE whose telephone number is (571)272-5545. The examiner can normally be reached M-F 9-5:30.
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MARCIA S. NOBLE
Primary Examiner
Art Unit 1632
/MARCIA S NOBLE/ Primary Examiner, Art Unit 1632
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