DETAILED ACTION
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Applicant’s Request for Continued Examination, Amendment and Arguments/Remarks received on 10 November 2025 have been entered.
Claims 1-3, 5-10, 13-14, 16-24, 27, and 29-33 were previously pending in the application. Claims 31-33 have been cancelled, and new claims 34-39 have been added by Applicant. Claims 1-3, 5-10, 13-14, 16-24, 27, 29-30, and 34-39 are currently pending in the application. Claim 1 is an independent claim.
Claims 1-3, 5-10, 13-14, 16-24, 27, 29-30, and 34-39 are currently pending and under examination in the instant application. An action on the merits follows.
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA . The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Priority
The present application is a CON of 17/855,423, filed 30 June 2022, now abandoned, which is a CON of 17/499,232, filed 12 October 2021, now U.S. Patent No. 11,672,874, which is a CIP of International Application No. PCT/US2020049240, filed 03 September 2020, which claims priority to U.S. Provisional Application Nos. 62/895,441, filed 03 September 2019, 62/908,800, filed 01 October 2019, and 63/039,261, filed 15 June 2020.
Thus, the earliest possible priority for the instant application is 03 September 2019.
Information Disclosure Statement
The information disclosure statement filed 10 November 2025 has been considered by the Examiner. Examiner notes the filing of IDS Size Fee assertions for the IDS filed 10 November 2025, as required under 37 CFR 1.98, indicating that no IDS size fee is required under 37 CFR 1.17(v) at this time.
37 CFR 1.821-1.825
This application contains sequence disclosures that are encompassed by the definitions for nucleotide and/or amino acid sequences set forth in 37 CFR 1.821(a)(1) and (a)(2), see for example Figure 3B. However, this application fails to comply with the requirements of 37 CFR 1.821 through 1.825 for the reason(s) set forth below and on the Notice to Comply With Requirements For Patent Applications Containing Nucleotide Sequence And/Or Amino Acid Sequence Disclosures which is attached to this communication.
Specifically, Figure 3B discloses amino acid and/or nucleotide sequences but do not include any sequence identifiers. If the unidentified sequences of Figure 3B are included in the submitted sequence listing, Applicant must amend the claims and specification and/or drawings to comply with the sequence identification requirements. Alternatively, if the unidentified sequences of Figure 3B are not included in the presently submitted sequence listing, Applicant must submit an updated sequence listing in compliance with 37 CFR 1.821(c)-(d) and 37 CFR 1.825(b). See also the attached Notice to Comply.
APPLICANT IS GIVEN A THREE MONTH EXTENDABLE PERIOD WITHIN WHICH TO COMPLY WITH THE SEQUENCE RULES, 37 CFR 1.821-1.825. Failure to comply with these requirements will result in ABANDONMENT of this application under 37 CFR 1.821 (g). Extension of time may be obtained by filing a petition accompanied by the extension fee under the provisions of 37 CFR 1.136. In no case may an applicant extend the period for response beyond the six month statutory period. Applicant is requested to return a copy of the attached Notice to Comply with the response.
Required response – Applicant must provide: (For specifications with noncompliant sequences)
A substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3) and 1.125 inserting the required sequence identifiers, including into the Brief Description of the Drawings, consisting of:
A copy of the previously-submitted specification, with deletions shown with strikethrough or brackets and insertions shown with underlining (marked-up version);
A copy of the amended specification without markings (clean version); and
A statement that the substitute specification contains no new matter.
Drawings
The drawings are objected to for the following reasons: 37 CFR 1.84 (u)(1) states “Partial views intended to form one complete view, on one or several sheets, must be identified by the same number followed by a capital letter.”
In the current case, the view numbers for the partial views for Figure 5 that appear on several sheets are followed by "continued" instead of a capital letter such as FIG. 5A, FIG. 5B, etc.
Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
Claim Objections
Amended, previously presented and original claims 6, 8-10, 21-23 are newly objected to because of the following informalities: claims 6 and 9-10 each recite the abbreviation “ORF1p” without first writing out the term for which it is an abbreviation. Claims 8-10 each recite the abbreviation “ORF2p” without first writing out the term for which it is an abbreviation. Claims 21-23 each recite the abbreviation “UTR” without first writing out the term for which it is an abbreviation. Appropriate correction is required.
Additionally, claim 22 newly recites “(I)” on lines 2 and 19. The second recitation of “(I)” appears to be a typographical error for “(II)”. Appropriate correction is required.
Claim Rejections - 35 USC § 112(b)
The rejection of cancelled claim 33 under 35 U.S.C. 112(b) as failing to particularly point out and distinctly claim the subject matter which the inventor(s) regards as the invention for reciting “without an enrichment of the target human cell population”, is withdrawn.
Amended, previously presented, original, and new claims 1-3, 5-10, 13-14, 16-24, 27, 29-30, and 34-39 are newly 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. Claims 2-3, 5-10, 13-14, 16-24, 27, 29-30, and 34-39 are included in this rejection due to their dependence on independent claim 1.
Amended independent claim 1 now recites, “wherein the one or more isolated exogenous RNA molecules do not comprise an antibiotic selection marker” in lines 6-7, which is indefinite because it is unclear whether the one or more isolated exogenous RNA molecules are meant to not comprise any one particular antibiotic selection marker, wherein they can comprise any number of antibiotic resistance markers as long as there is at least one marker which they do not comprise, or whether the one or more isolated exogenous RNA molecules are meant to not comprise any antibiotic selection markers. As such, the metes and bounds of the claim cannot be determined.
Similarly, new claim 39 recites, “wherein the one or more isolated exogenous RNA molecules do not comprise a selection marker” in lines 1-2, which is indefinite because it is unclear whether the one or more isolated exogenous RNA molecules are meant to not comprise any one particular selection marker, wherein they can comprise any number of selection markers as long as there is at least one marker which they do not comprise, or whether the one or more isolated exogenous RNA molecules are meant to not comprise any selection markers. As such, the metes and bounds of the claim cannot be determined.
Previously presented claim 8 recites, “wherein the polypeptide encoded by the RNA sequence of the mobile genetic element comprises human ORF1p or a functional fragment thereof” in lines 1-2, which is indefinite because it is unclear whether Applicant intends for the ORF1p and the polypeptide with TPRT activity to be a fusion protein or whether “the polypeptide encode by the RNA sequence of the mobile genetic element” is meant to refer to a different polypeptide than the polypeptide with TPRT activity. As such, the metes and bounds of the claim cannot be determined.
Previously presented claim 9, which depends on claim 8, which in term depends on independent claim 1, recites, “wherein the human ORF1p or the functional fragment thereof and the human ORF2p or the functional fragment thereof are translated from different open reading frames of the same isolated exogenous RNA molecule” in lines 1-3, which is indefinite because claim 8 recites, “wherein the polypeptide encoded by the RNA sequence of the mobile genetic element comprises human ORF1p or a functional fragment thereof” in lines 1-2, and independent claim 1 recites, “a mobile genetic element comprising an RNA sequence encoding a polypeptide with target-primed reverse transcriptase (TPRT) activity” in lines 11-12. Therefore, a limitation that that the two proteins are translated from different open reading frames of the same isolated exogenous RNA molecule conflicts with the limitation of 8 that the ORF1p is in the same polypeptide as the polypeptide with target-primed reverse transcriptase (TPRT) activity. As such, the metes and bounds of the claim cannot be determined.
Previously presented claim 10, which depends on claim 8, which in term depends on independent claim 1, recites, “wherein different RNA molecules encode the human ORF1p or functional fragment thereof and the human ORF2p or the functional fragment thereof” in lines 1-2, which is indefinite because claim 8 recites, “wherein the polypeptide encoded by the RNA sequence of the mobile genetic element comprises human ORF1p or a functional fragment thereof” in lines 1-2, and independent claim 1 recites, “a mobile genetic element comprising an RNA sequence encoding a polypeptide with target-primed reverse transcriptase (TPRT) activity” in lines 11-12. Therefore, a limitation that that the two proteins are translated from different isolated exogenous RNA molecules conflicts with the limitation of 8 that the ORF1p is in the same polypeptide as the polypeptide with target-primed reverse transcriptase (TPRT) activity. As such, the metes and bounds of the claim cannot be determined.
Claim Rejections - 35 USC § 112(a)- Written Description
The rejection of amended, previously presented, original, and cancelled claims 1-3, 5-10, 13-14, 16-24, 27, and 29-33 under 35 U.S.C. 112(a) for containing 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, at the time the application was filed, had possession of the claimed invention, for reciting “wherein the exogenous therapeutic polypeptide is expressed in at least 2% of cells in the target human cell population” in claim 1, which is new matter, is withdrawn in view of Applicant’s amendments to the claims such that amended claim 1 no longer recites, “wherein the exogenous therapeutic polypeptide is expressed in at least 2% of cells in the target human cell population”.
Previously presented and original claims 6 and 8-10 are newly 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.
Claim 6 depends on independent claim 1 and recites, “wherein the polypeptide with TPRT activity comprises a human ORF2p or a functional fragment thereof” in lines 1-2. Claim 8 depends on claim 6 and recites, “wherein the polypeptide encoded by the RNA sequence of the mobile genetic element comprises human ORF1p or a functional fragment thereof” in lines 1-3. Claim 9 depends on claim 8 and recites, “wherein the human ORF1p or the functional fragment thereof and the human ORF2p or the functional fragment thereof are translated from different open reading frames of the same isolated exogenous RNA molecule” in lines 1-3. Claim 10 depends on claim 8 and recites, “wherein different RNA molecules encode the human ORF1p or functional fragment thereof and the human ORF2p or functional fragment thereof” in lines 1-2.
The specification recites, “in some embodiments, the polypeptide encoded by the sequence of the mobile genetic element comprises one or more long interspersed nuclear element (LINE) polypeptides, wherein the one or more LINE polypeptides comprises: (i) human ORF1p or a functional fragment thereof, and (ii) human ORF2p or a functional fragment thereof” [0005]. The specification provides additional recitations of the sequences of the invention comprising sequences of or encoding ORF1p or a fragment/functional fragment thereof and/or ORF2p or a fragment/functional fragment thereof [0021-0022, 0026, 00118, 00383]. The specification further teaches “in some embodiments, the ORF2p or functional fragment thereof lacks endonuclease activity or comprises a mutation selected from the group consisting of S228P and Y1180A, and/or wherein the ORF1p or functional fragment comprises a K3R mutation” [0029].
The specification does not recite any limitation definition for “functional fragment”; nor does the specification recite any particular functions which are necessary to be retained within the functional fragment of ORF1p or ORF2p, other than the indication that the nuclease activity of ORF2p is not a required function. The specification does not teach the structural limitations for fragments which retain the recited function(s). The specification does not provide guidance as to the amino acids which could be modified such that the sequence would retain the necessary function(s), nor does the specification teach a minimum size or minimal region(s) necessary to retain the recited function(s).
The examples in the specification teach co-expressing the mRNA encoding a transgene of interest with a nucleic acid sequence encoding an ORF2p protein or both ORF1p and ORF2p proteins in cis or trans configurations (Example 1) [00593-00594, Figure 1-2]. Example 2 teaches a circularized mRNA configuration for the mRNA as taught in Example 1 [00595-00596, Figure 3].
Example 3 teaches designs for modifying an ORF2 to comprise an mRNA-binding domain (i.e., MS2 coat protein (MCP)) of a heterologous protein to generate a chimeric ORF2 to recognize alternative mRNAs [00598-00600, Figure 4A-B]. Example 3 also teaches designs for an ORF2 mutated to remove its ability to recognize and bind to RNA sequences such that a fused protein is used to direct ORF2 to alternative mRNA target sequences [00601, Figure 4C].
Example 4 teaches exemplary plasmid designs, wherein the plasmid encodes ORF1p and ORF2p wherein no alterations are taught for ORF1, and wherein ORF2 is enhanced alternatively through addition of fused domains (and not truncated), replacement of the ORF2 RT domain with the Group II intron’s reverse transcriptase domain, or chimerism of ORF2 domains compiled from multiple species [00607, Figure 5].
Examples 7-9 present integration data using unmodified ORF1 and ORF2 [00611-00617, Figure 8-11].
Examples 10 and 14 teach modification to the ORF2 protein sequence to enhance retrotransposition by mRNA, including an NLS fusion, replacing human ORF2 with Minke whale ORF2, and an MCP fusion, none of which removed sequence from the ORF1 or ORF2 sequences [00618, 00628, 00631, 00634, Figure 15, 22, 24, 27]. Example 14 additionally teaches designs for fusing the full-length ORF2 with a DNA binding domain of a heterologous zinc finger protein (ZFP) or with a CRISPR nuclease [00632, Table 4, Figure 25]. Example 14 further teaches designs for modifying the reverse transcriptase domain of ORF2 for increasing its efficiency by fusing the ORF2 reverse transcriptase domain with a DNA binding domain of a heterologous protein, such as those listed in Tables 5A and 5B, a MegaTAL nuclease, a CRISPR-CAS nuclease, a TALEN, R2 retroelement binding zinc finger binding domain, or a DNA binding domain that can bind to repetitive elements such as Rep78 AAV [00633, 00636, Table 7, Figure 26, 28].
Examples 15-16 teaches the use of full-length, unmodified ORF1 and ORF2 proteins [00639-00644, Figure 29-38].
Although the examples teach designs for domain swapping of the endonuclease domain or the reverse transcriptase domain of ORF2, the examples do not teach any fragments of ORF1. Additionally, the example designs provided for the domain swapping configurations do not provide details related to which amino acids constitute the ORF2 domains to remove or which are necessary to remain. Further, the specification does not provide any examples of an ORF2 protein which comprises a functional TPRT domain and a nonfunctional EN domain as required by independent claim 1, and particularly does not teach any ORF2 fragments which comprise a functional TPRT domain and a nonfunctional EN domain.
Therefore, a written description for a functional fragment of an ORF1p or an ORF2p is lacking within the application as filed. As such, claims 6 and 8-10 fail to comply with the written description requirement and do not reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, at the time the application was filed, had possession of the claimed invention.
Amended claim 22 is newly 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 is a new matter rejection.
The applicant is reminded that an amendment to the claims or the addition of a new claim must be supported by the description of the invention in the application as filed. In re Wright, 866 F.2d 422, 9 USPQ2d 1649 (Fed. Cir. 1989). New or amended claims which introduce elements or limitations which are not supported by the as-filed disclosure violate the written description requirement. See, e.g., In re Lukach, 442 F.2d 967, 169 USPQ 795 (CCPA 1971); In re Smith, 458 F.2d 1389, 1395, 173 USPQ 679, 683 (CCPA 1972).
Claim 22 was amended to recite, “wherein
the RNA sequence that is a promoter sequence is downstream of the RNA sequence that is a 5’ UTR sequence,
the RNA sequence that is a 5’ UTR sequence is downstream of the RNA sequence encoding the exogenous human therapeutic polypeptide,
the RNA sequence that is a 3’ UTR sequence is upstream of the RNA sequence encoding the exogenous human therapeutic polypeptide, and
the RNA sequence that is a poly A sequence is upstream of the RNA sequence that is a 3’ UTR sequence and downstream of the mobile genetic element”
in lines 22-30.
However, the instant disclosure does not provide any teachings, explicitly or implicitly, to support this new limitation. The specification does not recite any teachings for an expression cassette comprising a promoter sequence, a 5’UTR sequence, an exogenous human therapeutic polypeptide coding sequence, a 3’UTR sequence, and a poly A sequence, wherein the expression cassette sequences correspond to the non-template coding strand gene sequence which are in the order recited in amended claim 22.
The specification teaches expression cassettes which comprise reverse complement sequences which are ordered 5’ (upstream) to 3’ (downstream), relative to the orientation of the isolated exogenous RNA molecule, as poly A, 3’ UTR, exogenous therapeutic coding sequence, 5’ UTR, promoter; such that the expression cassette is in the reverse/antisense orientation relative to the isolated exogenous RNA molecule and cannot express from the isolated exogenous RNA molecule [0019]. This orientation, as taught by the specification, allows for expression of the exogenous polypeptide only after retrotransposition into the genomic DNA [00640]. The specification further teaches inclusion of an antisense GFP or ATAK gene [00167, 00189, 00640, 00643, Figure 1C, 1D, 2B, 5-6, 8-11, 13, 15-18].
The specification also teaches that the transgene can be co-expressed with a nucleic acid sequence encoding an ORF2p protein in the sense orientation [00593-00594, Figure 1C, 2A]. Such a sense orientation expression necessarily requires that the gene be arranged in a promoter-5’UTR-transgene coding sequence-3’ UTR-poly A orientation to achieve expression of the transgene.
Neither the specification nor the drawings teach expression in a sense orientation wherein the arrangement 5’ to 3’ (upstream to downstream) is poly A-3’ UTR-transgene coding sequence-5’ UTR-promoter. Additionally, such an orientation would not result in expression of the transgene either from the isolated exogenous RNA molecule or from the retrotransposed genomic DNA sequence.
Therefore, the disclosure does not provide support for the limitation newly added to amended claim 22 reciting a reverse order of elements for an expression cassette of the RNA sequence encoding the exogenous human therapeutic polypeptide, and the limitations represent new matter.
Claim Rejections - 35 USC § 112(a)- Enablement
The rejection of cancelled claim 33 under 35 U.S.C. 112(a) for failing to comply with the enablement requirement is withdrawn.
Amended, previously presented, original, and new claims 1-3, 5-10, 13-14, 16-24, 27, 29-30, and 34-39 are newly rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the enablement requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to enable one skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention.
The claims are drawn to a method of expressing an exogenous human therapeutic polypeptide from a genomically integrated DNA sequence of a cell of a target human cell population. The method comprises (a) contacting one or more isolated exogenous RNA molecules to the target human cell population, wherein a cell of the target human cell population uptakes the one or more isolated exogenous RNA molecules and wherein the one or more isolated exogenous RNA molecules do not comprise an antibiotic selection marker and do comprise: (i) an RNA sequence that is a reverse complement of a sequence encoding the exogenous human therapeutic polypeptide or an RNA sequence encoding the therapeutic polypeptide; (ii) a mobile genetic element comprising an RNA sequence encoding a polypeptide with target-primed reverse transcription (TFPR) activity, wherein the polypeptide with TPRT activity comprises an RT domain and an endonuclease (EN) domain, wherein (I) the EN domain of the polypeptide with TPRT activity does not have EN activity, (II) the mobile genetic element is not operably linked to a promoter, and (III) the mobile genetic element is derived from a mammal; (iii) at least one guide RNA sequence or a polynucleotide encoding the at least one guide RNA sequence, wherein the at least one guide RNA sequence targets a target site of the genomic DNA in the cell of the target human cell population; and (iv) a sequence encoding a Cas9 nickase. The method also comprises (b) translating the mobile genetic element to produce the polypeptide with TPRT activity, (c) reverse transcribing the RNA sequence that is the reverse complement of the sequence encoding the therapeutic polypeptide or the RNA sequence encoding the therapeutic polypeptide via the TPRT activity of the polypeptide translated in (b), thereby producing a DNA sequence complementary to the RNA sequence which was reverse transcribed. Additionally, the method comprises (d) integrating the DNA sequence encoding the therapeutic polypeptide or the DNA sequence that is the reverse complement of the sequence encoding the therapeutic polypeptide into the target site, and (d) expressing the exogenous human therapeutic polypeptide from the genomically integrated DNA sequence encoding the therapeutic polypeptide or the DNA sequence which is the reverse complement of the sequence encoding the therapeutic polypeptide in the cell of the target human cell population, wherein the therapeutic polypeptide is stably expressed in the cell.
The specification fails to enable one skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention with respect to the following three (3) issues:
1) the specification fails to enable the combination of a sequence encoding a polypeptide with TPRT activity which does not have EN activity with a sequence encoding a Cas9 nickase for integrating a DNA sequence into the genome of a target cell.
2) the specification fails to enable integrating a DNA sequence into the genome of a target cell to express an exogenous human therapeutic without the inclusion of both a sequence encoding an ORF1p polypeptide and an inter-ORF sequence.
3) the specification fails to enable the expression of a therapeutic polypeptide from an expression cassette having the order and orientation of expression cassette components as recited in amended claim 22 (I) [second (I)].
Regarding issue 1), the specification discloses designs for the use of a LINE1 ORF2p protein having TPRT activity and an endonuclease domain which is mutated to lack endonuclease activity [0027-0029, 0122, 00146, 00364], wherein the endonuclease function of the ORF2p is substituted or complemented with an alternative/ additional/ heterologous nuclease domain, including a Cas9 domain [0027-0029, 00364, 00632, 00636, Table 7, Figure 25, 28]. Example 14 specifically teach designs for replacing the endonuclease activity of the ORF2p with an alternative endonuclease for the retrotransposon-mediated integration of a transgene into genomic DNA by fusion of the alternative nuclease domain to the ORF2p protein, as illustrated in Figures 25 and 28, including Cas nucleases as the alternative nuclease [00632, 00636]. The specification also teaches wherein the additional nuclease domain has reduced nuclease activity [00122, 00364], or wherein the additional nuclease is a Cas9 nickase [00381].
However, neither the specification nor the drawings provide any examples wherein Applicant actually combined the EN(-) ORF2p (nor any other polypeptide with TPRT activity) protein with any other nuclease domain. In particular, neither the specification nor the drawings provide any data for any combination of a polypeptide having TPRT activity with any Cas nuclease, including a lack of data for any Cas9 nickase.
In support of an argument of unexpected results, Applicant has referenced a post-filing publication, Wang, which teaches the combination of an EN(-)-ORF2p protein with a Cas9 nickase for the targeted integration of a transgene into a genomic DNA sequence [Wang et al. 2025, EMBO Reports, 26, 1062-1683, IDS]. However, the Wang publication does not support an enabling disclosure for the instantly claimed invention. Firstly, Wang teaches testing two alternative Cas9 nickases, Cas9D10A and Cas9H840A, wherein only the nCas9H840A was found to function in combination with an EN(-)-ORF2p to achieve genomic integration of a transgene [column 8 ¶ 1, Figure 2B, 3A]. In fact, Wang explicitly states, “When we transfected CREATE mRNA and the two AAVS1-targeting sgRNAs into HEK293T cells stably expressing either Cas9D10A and Cas9H840A nickase, we observed GFP expression and integration exclusively in the presence of Cas9H840A nickase (Figure 3A), consistent with the model that hybridization of the PBS sequences with 3’-flap released from non-target strand nickase is required for priming cDNA synthesis (Fig. 1B)” [column 8 ¶ 1]. The instant specification merely recites “a Cas nickase” in [00381] or even more generically that an endonuclease may have reduced nuclease activity [00364]. The specification does not disclose either of the specific Cas9 nickases taught by Wang (Cas9D10A and Cas9H840A), and moreover does not disclose the specific Cas9 nickase which Wang teaches to be the only one able to combine with an endonuclease-deficient ORF2p for targeted genomic retrotransposon-mediated integration.
Also note that Wang teaches the use of 293T cells which have been engineered to stably express the Cas9 nickase variants, which were generated by transfection of PiggyBacTM vectors expressing the Cas9 nickase H840A or D10A variants [column 4 ¶ 3, column 22 ¶ 4]. As such, the method of Wang does not comprise contacting a human cell with an RNA molecule comprising a sequence encoding the Cas9 nickase as required by the instant claims.
Additionally, independent claim 1 recites “at least one guide RNA sequence or a polynucleotide encoding the at least one guide RNA sequence” in lines 19-20. However, Wang teaches that two guide RNAs are necessary to achieve the targeted genomic integration by targeting two sites (pBS1 and PBS2, respectively) flanking the insertion site [column 3 ¶ 3, Figure 1A]. However, the specification merely recites “In some embodiments, the second protein is a Cas nickase, wherein the retrotransposable system further comprises introducing a guide RNA” [00381]. Nowhere in the specification nor in the drawings does the instant disclosure teach the requirement for targeting two positions in the genome using two guide RNAs to achieve genomic integration.
Accordingly, the specification does not provide an enabling disclosure for using an endonuclease deficient ORF2p in combination with a Cas9 nickase for promoting the retrotransposon-mediated genomic integration, and subsequent expression, of a transgene in a target human cell population. Additionally, Applicant’s post-filing publication (i.e., Wang) does not cure the deficiency of the disclosure in that Wang teaches required elements which are not disclosed in the instantly filed disclosure.
Further, at the time of filing, the art of combining an endonuclease deficient ORF2p with a Cas9 nickase to facilitate targeted genomic integration was still considered unpredictable. Ade teaches that Cas9D10A is unable to replace an ORF2p endonuclease domain in an ORF2p fusion protein comprising a mutated EN domain to achieve targeted retrotransposon-mediated genomic integration [Ade et al. 2018, Gene, Vol. 642, 188-198, cited in a prior action, column 14 ¶ 2-3, Table 2, Figure 5]. Specifically, Ade teaches that no target-specific insertion was observed from blasticidin-resistant colonies generated by the nickase-ORF2endo- fusion [column 14 ¶ 2]. Further, Ade states, “Overall, these data demonstrate that using components of the CRISPR/Cas9 system is not an efficient approach for redirecting ORF2p-driven retrotransposition events to specific locations“ [column 15 ¶ 2] and that “our strategy to use the Cas9 to provide the cleavage instead of the ORF2 endonuclease also failed to support retrotransposition” [column 17 ¶ 4].
Note that Applicant acknowledges in their Remarks filed 10 November 2025 that “the results presented by Ade show that a construct comprising a Cas nickase and a[n] endonuclease deficient ORF2p do not result in stable expression” [page 11 ¶ 1]. Applicant goes on to argue that Ade clearly teaches that a Cas9 endonuclease cannot be used as a substitute for an ORF2 endonuclease [page 11 ¶ 3]. As such, Applicant argues that “a skilled artisan would not have a reasonable expectation that using a construct with a sequence encoding ‘a polypeptide with TPRT activity… and an endonuclease (EN) domain [that] does not have endonuclease activity’ and ‘a sequence encoding a Cas9 nickase’ could be used to successfully integrate a sequence into a genomic target site of a cell according to the claimed method as amended” [page 12 ¶ 2]. Therefore, Applicant acknowledges the unpredictability taught in the prior art regarding the combination of an endonuclease-deficient ORF2p with a Cas9 nickase for promoting targeted integration into the genome of a human cell.
It is noted that the claims encompass a) wherein the Cas9 nickase is encoded on the same RNA molecule as the sequence encoding the polypeptide with TPRT activity, including encompassing a fusion protein of the TPRT protein and the Cas9 nickase as taught by Ade, as well as b) wherein the Cas9 nickase is expressed independently of the polypeptide with TPRT activity, including being present on a distinct RNA molecule and contacted at a distinct time. Although Wang does not teach contacting the human cell population with an mRNA comprising a sequence encoding a Cas9 nickase, the method of Wang does comprise contacting a human cell with a DNA molecule comprising a sequence encoding a Cas9 nickase, wherein the DNA molecule comprising a sequence encoding a Cas9 nickase is distinct from the CREATE mRNA comprising the sequence encoding the mutant ORF2p and which is contacted with the cell independently of and prior to contacting the human cell population with the CREATE mRNA. Therefore, Ade and Wang collectively teach away from combining an EN(-) mutant ORF2p with a Cas9D10A nickase either as a fusion protein or as an independent protein expressed within the cells.
Thus, at the time of filing, the skilled artisan did not consider the combination of an endonuclease deficient ORF2p with a Cas9 nickase for promoting the retrotransposon-mediated genomic integration, and subsequent expression, of a transgene in a target human cell population to be predictable. Applicant’s disclosure does not overcome this art recognized unpredictability as the disclosure does not provide sufficient guidance for combining an endonuclease deficient ORF2p with a Cas9 nickase for promoting the retrotransposon-mediated genomic integration, and subsequent expression, of a transgene in a target human cell population.
Regarding issue 2), the claims read broadly on any mobile genetic element comprising any RNA sequence encoding any polypeptide with TPRT activity. Additionally, the claims do not require any mobile genetic element components other than the sequence encoding the polypeptide with TPRT activity.
The specification teaches LINE1 mobile genetic elements which comprise coding sequences for ORF1p and ORF2p, and that other mobile elements, such as SINEs, including Alu elements, also require trans-factors for integration such that they can associate with the L1 ribonucleoproteins in trans to be retrotransposed by ORF1p and ORF2p [0005, 0089, 00269]. The only polypeptides with TPRT activity taught in the specification are ORF2p proteins [00267]. The drawings present schematics for the mobile genetic element, wherein the mobile genetic element comprises coding sequences for both ORF1p and ORF2p in cis or in trans [Figure 1, 5, 6, 8, 9, 10, 11, 13, 15, 16, 17, 18, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 53]. Additionally, the data presented in the Figures were generated using LINE1 mobile genetic elements comprising coding sequences for both ORF1p and ORF2p [00193-00199, 00201-00219, Figure 33-35, 37-53]. The specification further teaches that the ORF1p protein is essential for LINE1 integration [00268].
Applicant’s post-filing publication (i.e., Wang) does not overcome this deficiency in that Wang also teaches that the CREATE mRNA encodes both the ORF1p and the ORF2p proteins [column 3 ¶ 3-4], and that ORF1p is an RNA-binding protein that interacts with L1 mRNA transcripts [column 2 ¶ 3]. Further, Wang teaches that “In an attempt to reduce the overall size of the CREATE system and improve delivery efficiency, we deleted ORF1p from the CREATE construct” and that “Electroporation of dORF1 variant of CREATE mRNA did not result in successful integration in primary T cells, suggesting that exogenously expressed ORF1p is important for efficient integration” [column 14 ¶ 1]. Therefore, Wang teaches that the sequence encoding ORF1p is a critical element for a LINE1- based retrotransposon-mediated tool for integrating a transgene into the genome of a human cell.
Accordingly, the specification does not provide an enabling disclosure for using an LINE1-based retrotransposon-mediated tool for integrating a transgene into the genome of a human cell for expression of the transgene in a target human cell population, wherein the mobile genetic element comprises an ORF2p encoding sequence in the absence of an ORF1p encoding sequence. Additionally, Applicant’s post-filing publication, Wang, does not cure the deficiency of the disclosure in that Wang teaches that ORF1p is a required element for a method according to the instant claims.
Further, at the time of filing, the art of using mobile genetic elements to facilitate targeted genomic integration in the absence of ORF1p was still considered unpredictable. Schumann teaches that both ORF1- and ORF2-encoded functional proteins are required for retrotransposition [Schumann, US20110045591A1, cited in a prior action, 0004].
Thus, at the time of filing, the skilled artisan did not consider the integration of a LINE1 element by ORF2p in the absence of ORF1p to be predictable. Applicant’s disclosure does not overcome this art recognized unpredictability as the disclosure does not provide sufficient guidance for using any element other than a LINE1 element or an Alu element, which each require both ORF1p and ORF2p, for promoting the retrotransposon-mediated genomic integration, and subsequent expression, of a transgene in a target human cell population.
Regarding issue 3), claim 22 was amended to recite, “(I) the RNA sequence encoding the exogenous human therapeutic polypeptide comprises an expression cassette comprising an RNA sequence that is a promoter sequence, an RNA sequence that is a 5' UTR sequence, an RNA sequence that is a 3' UTR sequence and an RNA sequence that is a poly A sequence; wherein (i) the RNA sequence that is a promoter sequence is downstream of the RNA sequence that is a 5' UTR sequence,(ii) the RNA sequence that is a 5' UTR sequence is downstream of the RNA sequence encoding the exogenous human therapeutic polypeptide, (iii) the RNA sequence that is a 3' UTR sequence is upstream of the RNA sequence encoding the exogenous human therapeutic polypeptide, and(iv) the RNA sequence that is a poly A sequence is upstream of the RNA sequence that is a 3' UTR sequence and downstream of the mobile genetic element.” However, as discussed above in the written description rejection under 35 U.S.C. 112(a), this combination of features is not disclosed in the specification and represents new matter. Further, this combination of features would result in an inoperable expression cassette.
The specification teaches an expression cassette encoding a transgene, such as GFP, wherein the expression cassette is in the reverse orientation and so comprising elements in a reverse order with respect to the isolated exogenous RNA molecule, wherein the elements are ordered (upstream to downstream) as follows: reverse complements of each of poly A, 3’ UTR, transgene encoding sequence, 5’ UTR, promoter [0019]. The specification also teaches wherein the expression cassette is in the same sense orientation with respect to the isolated exogenous RNA molecule [00593], but does not teach that such a construct is ordered (upstream to downstream) as follows: sense/coding strands of each of poly A, 3’ UTR, transgene encoding sequence, 5’ UTR, promoter. As is well known in the art, the coding sequence must be read in the 5’ to 3’ direction to generate a functional mRNA and subsequently a functional protein. Likewise, a promoter sequence must be upstream (5’) of a coding sequence on the coding (sense) strand to facilitate transcription of the coding sequence. By definition, a 5’ UTR must be upstream (5’) of a coding sequence and a 3’ UTR must be downstream (3’) of a coding sequence. Finally, a poly A sequence is also well known to be at the extreme 3’ end of a transcript [as seen for example in Figures 1, 2, 6, 7, and 8 of the instant drawings].
Applicant’s post-filing publication (i.e., Wang) does not overcome this deficiency in that Wang also teaches a GFP transgene which is in a reverse orientation with respect to the CREATE mRNA, wherein the EF1alpha promoter is downstream of the reverse complement of the GFP coding sequence and the SV40 poly(A) is upstream of the reverse complement of the GFP coding sequence [Figure 2].
Further, at the time of filing, the art teaches an inverted orientation for an expression cassette for expression of a transgene following retrotransposition, wherein the promoter for the transgene is downstream of the reverse complement of the transgene coding sequence with respect to the retrotransposon RNA [Schumann, Figure 2A-B]. Similarly, Schumann teaches that when presented in the sense orientation, the promoter is upstream of the transgene coding sequence [Figure 3A]. Accordingly, at the time of filing, expressing a transgene from a sense orientation expression cassette ordered 5’- poly A-3’ UTR-transgene encoding sequence-5’ UTR-promoter-3’ was still considered unpredictable.
Thus, at the time of filing, the skilled artisan did not consider expression of a gene from a 5’- poly A-3’ UTR-transgene encoding sequence-5’ UTR-promoter-3’ oriented expression cassette to be predictable. Applicant’s disclosure does not overcome this art recognized unpredictability as the disclosure does not provide any guidance for using an expression cassette so structured.
Therefore, in view of the state of the art at the time of filing for the combination of an endonuclease-deficient ORF2p with a Cas9 nickase, the art recognized unpredictability of getting targeted genomic integration using a combination of an endonuclease-deficient ORF2p with a Cas9 nickase, the art recognized necessity for including an ORF1p to achieve genomic integration of a LINE1 mobile genetic element, the art recognized need for ordering an expression cassette according to the sense orientation as 5’-promoter-5’UTR-coding sequence-3’UTR-polyA-3’, the limitation of the working examples to teaching only LINE1 mobile elements, the limitations of the specification to only teaching a Cas nickase generically, the lack of working examples demonstrating any functionality of a combination of an endonuclease-deficient ORF2p with a Cas9 nickase, Applicant’s provided post-filing data demonstrating that an endonuclease-deficient ORF2p combined with a Cas9 nickase only works for an H840A nickase, Applicant’s provided post-filing data teaching the necessity of the ORF1p coding sequence, and Applicant’s lack of teachings of a sense strand orientation of an expression cassette having the order of elements recited in amended claim 22, and the breadth of the claims, it would have required undue experimentation to practice the method of expressing an exogenous human therapeutic polypeptide from a genomically integrated DNA sequence of a cell of a target human cell population as claimed without undue experimentation.
Claim Rejections - 35 USC § 103
The rejection of amended, previously presented, original, and cancelled claims 1-3, 5-10, 13-14, 16-24, 27, and 29-32 under 35 U.S.C. 103 as being unpatentable over Schumann et al. (US20110045591A1); Rubens et al. (US20200109398A1, priority to 08-28-2018 62/723,886); Fujiwara et al. (US20060183226A1); and Ade et al. 2018, Gene, Vol. 642, 188-198; in view of Fadloun (WO2016030501A1); Gerkhe et al. (WO2020150534A9, priority to 16 January 2019 62/793,277); and further in view of Spadafora et al. (US10214591B1) and Gassull Duro et al. (US20120045389A1), is withdrawn in view of Applicant’s amendment to independent claim 1 to specifically recite “a sequence encoding a Cas9 nickase” in line 22. Although Ade teaches a low level of genomic integration with a construct comprising a nuclease-deficient ORF2p protein fused with a Cas9D10A nickase, Ade teaches that no target-specific insertion was observed from blasticidin-resistant colonies generated by the nickase-ORF2endo- fusion [column 14 ¶ 2]. Further, Ade states, “Overall, these data demonstrate that using components of the CRISPR/Cas9 system is not an efficient approach for redirecting ORF2p-driven retrotransposition events to specific locations“ [column 15 ¶ 2] and that “our strategy to use the Cas9 to provide the cleavage instead of the ORF2 endonuclease also failed to support retrotransposition” [column 17 ¶ 4]. Therefore, Ade teaches away from the instantly claimed invention.
Conclusion
No claim is allowed.
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DR. KATIE L. PENNINGTON
Examiner
Art Unit 1634
/KATIE L PENNINGTON/Examiner, Art Unit 1634
Dr. A.M.S. Wehbé
/ANNE MARIE S WEHBE/Primary Examiner, Art Unit 1634
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