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 .
Application Status
This action is written in response to applicant’s correspondence received 3/12/2026. Claims 1-14,16-19,22-23, filed 3/12/2026, are under examination in this Office action and claims 15, and 20-21 are cancelled.
On 3/12/2026, Applicant elected Group I (claims 1-14) with traverse citing that WO2018154412 does not disclose the claimed composition or a nucleic acid integrated within the cell’s genome, said nucleic acid comprising a transgene encoding laminin-alpha2 protein flanked by operational sequences for integrates and/or transposase mediated gene insertion, therefore does not anticipate claim 1 or 16. Applicant reserves right to request rejoinder of non-elected groups. The restriction is withdrawn and claims 1-14,16-19,22-23 are pending and under review in this Office action.
The species election for promoter is EalbAAT promoter of SEQ ID NO: 80 without traverse and gRNA SEQ ID NO: 90 without traverse (cl 9) and PiggyBac (cl 10).
Priority
This application claims priority to EP21209721.6 (11/22/2021) and EP2021469.6 (12/16/20). Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55 for EP21209721.6 (11/22/2021) and EP2021469.6 (12/16/20). This application is a 371 of PCT/EP2021/086333 (12/16/2021).
Specification
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code. Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01. Browser executable code is found on Pg 8 line 3, twice.
The use of the term PiggyBac, mincircle and doggybone, JetPei, PowerUP and all other registered marks which are trade names or marks used in commerce, have been noted in this application. The terms should be accompanied by the generic terminology; furthermore the terms should be capitalized wherever they appear or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term. These terms are found at least at Pg 3 (line 13), Page 4,64,71,78, and many other additional locations throughout the Specification.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
Claim Objections
Claim 8, 10 are objected to because of the following informalities:
Claim 8 recites “of cell” which is improper grammar. Using “of a cell” is acceptable.
Claim 10 recites “Sleepy Beauty” transposase, which is not a transposase name.
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.
Claims 1-14, 22-23 are 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 pre-AIA the applicant, regards as the invention.
Claim 1 is indefinite in the recitation, found in part c) of “or a functional variant or fragment thereof”. It is unclear whether the term functional is intended to refer to the variant only, or to the variant as well as the fragment, or whether the fragment may be non-functional, impacting the metes and bounds of the claim. Claims 2-14, 22, 23 depend from claim 1 and are rejected for the same reason.
Claim 7 contains the trademarks/trade names minicircle and doggybone. Claims 10-12 contains the trademark/trade name PiggyBac. Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph. See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademark/trade names minicircle and doggybone are used to identify vectors and piggybac describes an engineered transposase, accordingly, the identification/description is indefinite.
Claim 10 is indefinite in the recitation of in the rectitation of “said transposase is a modified hyperactive Piggybaac transposase or Sleepy Beauty transposase” because it is unclear whether the “modified hyperactive” is intended to refer only to the Piggybac or to both types of transposase.
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 1-14,16-19,22-23 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification does not reasonably provide enablement for using any site-specific binding protein capable of binding and cleaving target sequence. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the invention commensurate in scope with these claims. This is a scope of enablement rejection.
The test of enablement is whether one skilled in the art could make and use the claimed invention from the disclosure in the specification alone or in combination with information known in the art, without undue experimentation (see MPEP 2164.01(a)), (United States v. Telectronics., 8 USPQ2d 1217 (Fed. Cir. 1988)). Whether undue experimentation is needed is not based upon a single factor but rather is a conclusion reached by weighing many factors. These factors were outlined in Ex parte Forman, 230 USPQ 546 (Bd. Pat. App. & Inter. 1986) and again in: In re Wands, 8 USPQ2d 1400 (Fed. Cir. 1988). The most relevant factors are addressed below:
Nature of the Invention
The claims are directed to a composition of any site-specific binding protein that binds and cleaves, and any transposase, or constructs thereof, and a transgene encoding laiminin -alpha2 or variant/fragment thereof.
Breadth of the claims
The claims broadly recite any binding protein that has the functional features of binding and cleaving, or any rna guided Cas, andany transposase. Claim 1 is not limited to any particular binding protein, and therefore broadly encompasses the large genus of binding proteins that cleave and bind. Claim 2 is any binding protein that binds and cleaves and is fused with a transposase.
Per claim 8, the composition’s binding protein is an RNA guided nuclease comprising any Cas protein, which is also generic. Per claim 10, the binding protein is that of claim 1 and the transposase is selected from two genera, and per claim 11 the binding protein is that of claim 1 and the transposase has particular mutations.
Teachings of the Specification
The specification teaches both unpredictability and limited uses such as “The inventors used a fusion protein comprising a site-specific binding protein fused to a transposase to integrate LAMA2…”(Pg 11) which relates to claim 2, a fusion, but is less broad than the scope of claim 1, which does not require fusion. The transposons presented are largely mutant hyperactive PiggyBac (hyPB) transposons. Frog Prince, Sleeping Beauty and hyperactive Sleeping beauty, or other transposons as disclosed in the Specification, are not presented in any working examples. In generalized singular embodiments, of composition that comprises two proteins, parenthetically Cas9 is recited (Pg 59 recites four of these generalized, “In one emboidment” disclosures, Pg 60 recites two), which, when fused to particular transposons, aligns with working examples.
Regarding unpredictability, the inventors were able to use in vivo, plasmid DNA and mRNA for FiCAT, which specifically employed Cas9-hyPB fusion with three hyPB mutations, to conduct proof of concept gene delivery (Pg 71). However, to further test hyPB capactiy four substitutions were made for this Cas’, but of those four, one, Cas X, did not achieve any integration, which was confirmed in a second set of experiments (Pg 72). Meaning not all site specific binding proteins, in-fact, not even all Cas binding proteins were predictable in functionality, indicative of unpredictability in the genera and species of all site specific binding proteins presented. No guidance was provided in the Specification regarding the reasons for this failure, or how to avoid such failure.
Re: payload, size of ITRs of payload affected binding of fusion protein and genome transfer, where tests were performed using particular Cas variants fused to particular transposons. Three inter-ITR sizes were tested with FiCAT which specifically used Cas9 + particular mutant hyperactive PiggyBac, plus FiCAT dimer Cas9+dimer of particular mutated hyperactive PiggyBac. Two results were better than the third. No guidance was provided regarding what generates successes versus failures.
Zinc finger-PiggyBac mutant fusions had no or very low targeted insertion, indicative of further unpredictability in this work. Rescue of this targeted insertion problem occurred with the addition of double stranded breaks made by added gRNA-Cas9 or a Cas9 nickase, seemingly indicative of the need for the binding and cleaving properties of Cas.
State of the Art
The state of the art was such that a composition comprising any site-specific binding protein that binds and cleaves, in combination with a transposase and a nucleic acid construct comprising a transgene encoding lamininin alpha 2 or a portion thereof as individual entities was unknown.
What was known were a particular subset of binding proteins used in a fusion with a transposon, such as exemplified by Strecker (Strecker, J. et al., Science 2019, 365:48-53). Here, nickase Cas9(D10A) was tethered to the transposase TnpA, and in combination with an sgRNA, used to successfully generate targeted DNA insertions (Pg 2 para 2). Strecker further points out that the association between particular (Tn7-like) transposons and CRISPR-Cas systems suggesting the rationale that transposons may have hijacked CRISPR effectors to generate R loops in target sites and thus facilitate transposoon spread via plasmids and phages. (Pg2, para 4). Transposases, however are a superfamily, the most abundant genes in nature, found in all major taxonomic groups (Aziz, R., et al., Nucleic Acids Res, 2010, 38: 4207-4217; Pg 4212 left col, para 1, Abstract), with individual transposases having different characteristics/mechanisms, even just within the Type II DNA transposases that move DS DNA but that may or may not be associated with insertions sequences, and may or may not catalyze just their own mobilization (Pg 4212 righ col para 1-2). DDE transposases have the DDE conserved triad in common, unlike rolling circle transposases, and retrotransposases for example, which function quite differently. Retrotransposases move a different class of transposons (Type I) and function by reverse transcription, and it is Type III transposons that are linked to inverted repetitive elements. Further, many transposases are not fully classified yet (Pg 4212 right col para 1), suggestive that considering them all interchangeable/valuable in a composition as disclosed may result in unpredictability.
Some transposases (e.g. Tn7) including integrases (ICEBs1) used for genomic integration are complex and problematic to reprogram and as such, require the presence of particular attachment sites or their additional introduction into a genome (Vo, P. et al., online 23 Nov 2020, Vol 39 Apr 2021, 480-489) (Pg 480 right col, para 2)
However, the most widely used transposases for transfer of therapeutic transgenes are a small subset of transposases, Sleeping Beauty (SB), piggyBac (PB) and Tol2 (Sandoval-Villegas, N. et al., 2021, Int J Mol Sci 22: 5084, Abstract) that interact with binding sites in terminal inverted repeats, inducing double-stranded breaks, and catalyzing excision and integration (Fig 1). Sleeping beauty transposases have subdomains important to DNA binding and core recognition sequences, and preference to integrate at TA dinucleotides, with close to unbiased, random integration that benefits safety, and the mutations in hyperactive SB result in much higher transposition rates than non mutated SB, and with particular other modifications, larger transgenes may be moved (e.g. to 7kb, or 100 kb in some circumstances), which differs from Tol2 capacity (e.g. 11-12kb to mainitain transposition efficiency, most useful in fish) and PB (>100kb) indicative of less predictability with many transposases relative to others when cargo is large (Pg 5 para 2, Pg 6 para 3 and 4, Pg 8 para 2, Pg 11, para 2, Pg 12-13 final to first para, Pg 18, para 4). PB in contrast, integrates at TAA sequences often near transcription start sites, beneficially absent a footprint at the excision site, with some mutant PB functioning as excision-only transposases (Pg 6 para 4). While SB has a favorable safety profile, PB presents risk of disrupting transcriptional regulation of endogenous genes (Pg 5, para 1, Pg 6 para 4) and as transposases, SB is considered safer than (the more biased) PB and Tol2 for therapeutic applications given integration locations, which may differ in utility in a fusion-based system with different integration (Pg 16 final para).
CRISPR/Cas9 nucleases’ precise targeting is valuable, but homology directed repair efficiency goes down with larger insert size, again making fusion functionally different in specificity and integration capacity than isolated (nuclease and transposase) proteins (Pg 18, para 4). Even in fusions, systems matter for predictability of insertion. Tn7-like transposons can use CRISPR/dCas systems with near 100% integratoin in bacteria, though integration rates in vertebrates is unknown and unpredictable. In contrast, in Cas9 mediated methods (HITI) using SB vectors, when Cas9 was fused with SB transposase, a different rate of success occurred with unpredictable off-target risks, as well (Pg 18 final para to 19 top para). This also differs in predictability of success from the strategy of the use of transposases fused to dCas9 with sgRNA use, where cargo is directed to a particular target sequence but targeting efficiency is poor, and thus unpredictable (Pg 19, para 1)
Amount of Experimentation
Given the limited breadth of teachings in the specification, focused on particular Cas fusions and hyperactive PiggyBac transposases, with both success and failure (e.g. CasX and zinc-fingers) in use, though no guidance on how to avoid failure, combined with the absence of art on non-fusion based compositions of proteins and transgene relevant here, in addition to the variability and unpredictability in both the specifcation and the art regarding functionality (or lack of functionality) for particular site-specific binding proteins in composition with particular transposases, making and using the full scope of the claimed invention, inclusive of any site specific binding proteins, and all types of transposases, including hyperactive or not, would be time-consuming and unpredictable and would require substantive, not routine optimization. Taking into consideration the relevant Wands factors, an undue amount of experimentation would be required to practice the invention as broadly as claimed.
Claim Interpretation
In evaluating the patentability of the claims presented in this application, the claims will be given their broadest reasonable interpretation using the plain meaning of the claim language in light of the specification, as it would be understood by one of ordinary skill in the art, and as set forth at MPEP§ 2111.
For clarity of the record, a definition of cleavage provided in the Specification refers to breaking covalent backbone of a DNA molecule, ….with both single stranded and double stranded cleavage possible. Oxford English Dictionary (www.oed.com) refers to cleaving as meaning to part or divide by cutting. More relevant to the art, Ma (Ma, E et al., 2015, Molec Cell, 60:398-407) uses the term cleaving to reference double stranded breaks (double stranded cleavage) and single-stranded breaks (Summary, Pg 398 right col, first and final para), akin to the breadth of the definition in the specification.
Claim Rejections - 35 USC § 103
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 1, 2, 7, 8, and 23 are rejected under 35 U.S.C. 103 as being unpatentable over Strecker (Strecker et al., Science, 365, 48-53, 2019, 1-6) in view of Lundberg (US2020/0095579 A1; published Mar 26, 2020).
Regarding claim 1, Strecker disclosed a site-specific binding protein capable of binding and cleaving a target sequence (where the site specific binding protein nickase Cas9(D10A) was tethered to the ssDNA transposase TnpA, and combined with a Cas9 sgRNA, resulted in targeted DNA insertions (Pg 2, para 2; Fig 1 depicts binding of Cas9(D10A) and the cleaved nucleic acid, and nickase references the ability to cleave). Re claim 2, wherein the proteins are fused, Strecker FIG S1 depicts the Cas9-TnpA fusion).
Strecker did not disclose LAMA2.
Lundberg discloses LAMA2 (laminin alpha2) as a therapeutic gene and provides materials and compositions for genome engineering [00077], including for treating a patient with disorders associated with LAMA2, such as muscular dystrophy (Abstract). This work addressed altering or entirely replacing wild-type sequence, for theapeutic purposes (Abstract), where it was recognized that a full-length cDNA could be used for knock-in’s ([000249]-[000250]).
Prior to the effective filing date, it would have been prima facie obvious to one of ordinary skill in the art to have simply substituted the known LAMA2 sequence of ‘579 into the composition of Strecker since precision integration of targeted DNA insertions was the fundamental goal of Strecker (Abstract) and insertion of this medically important transgene would have been beneficial for therapeutic advances in muscular dystrophy treatment. Strecker had disclosed how mutations, sometimes many and in varied positions, in LAMA2 were associated with muscular dystrophy, and a construct with the LAMA2 transgene in this particular composition would allow specifically for highly beneficial targeted integration, valuable for an insert intended to be only medically beneficial and that would not contribute any additional off-target problematic health issues when introduced. Strekcer does consider using safe harbor sites for insertions (Pg 6 left col para 1) and Lundberg had suggested them as a viable location for integration, too.
Re: claim 7, Strecker disclosed the composition wherein the nucleic acid construct is comprised in a vector that is a plasmid vector (Fig S1 A-B).
Re: claim 8, Strecker disclosed the composition wherein the site specific binding protein (Cas9(D10A)) is RNA guided nuclease comprising Cas protein (with a Cas9 sgRNA), for integrating in a specific site in a genome (targeted DNA insertions Pg 2, para 2; Fig 1, Abstract).
Strecker did not disclose the composition further comprising a gRNA with a complementary sequence to a target nucleic acid sequence for integrating said LAMA2 gene
Lundberg disclosed a gRNA including complementary sequence to a target nucleic acid sequence for integrating said LAMA2 transgene (one or more gRNA comprises a spacer that is complementary to a DNA sequence within or near one of the introns or exons of LAMA2 [000449]).
Prior to effective filing date it would have been prima facie obvious to one of ordinary skill in the art to have used the gRNA of Lundberg in the method of Strecker for precision integration of the LAMA2 gene with the simple substitution of the gRNA of Lundberg since it would have been particularly and specifically designed to be complementary to the LAMA2 gene being integrated.
Re: claim 23, a method for treating MDC1A comprising administering to subject a therapeutically effected amount of the composition of claim 1,
The composition has been addressed.
Lundberg described treatment for MDC1A (aka merosin deficient congenital muscular dsytrophy) by administration to a subject of a therapeutically effective amount of a composition, or an effective amount of myogenic progenitor cells, administered via systematic route of administration to prevent dystrophy (Abstract, [000356], [00359]). An effective amount would be used or a sufficient amount of the composition to provide the desired effect, e.g. treatment [00360]. Lundberg disclosed providing materials, and compositions for genome engineering [00077], including for treating a patient with disorders associated with LAMA2, such as MDC1A (Abstract), altering or replacing wild-type sequence for therapeutic purposes (Abstract). Lundberg acknowledged the importance of precision delivery, providing reasons recited previously for the composition of claim to be generated by Strecker in view of Lundberg, as previously described.
Claims 16 and 19 are rejected under 35 U.S.C. 103 as being unpatentable over Calos (US9932607 B2; published Apr 3, 2018) in view of Lundberg (US2020/0095579 A1; published Mar 26, 2020).
Regarding claim 16, Calos disclosed an engineered cell, wherein the engineered cell comprises a nucleic acid integrated within its genome (insertion and integration of a transgene or polynucleotide sequence into the genome of a human cell) (Abstract). The donor cassette (with nucleic acid to be integrated) is flanked (placed between recombination sites ФC31 and Bxb1) and the genome contains the related flanking operational sequences (attachment sites; attB, attP sites, Fig 1A) recognized by integrase for the (ФC31 and Bxb1) integrase- mediated cassette insertion (Fig 1A, Col 2).
Calos does not teach LAMA2 as the transgene.
Lundberg discloses LAMA2 (laminin alpha2) as a therapeutic gene and treatment of MDC1A (merosin deficient muscular dystrophy). Methods may edit/correct one or both LAMA2 alleles [000248] in patients, or in a cell, with genome engineering, and via different correction strategies, where a full-length cDNA can be knocked into any safe harbor locus ([000249]-[000250]). Lundberg continues, for example, if there are small/large deletions or multiple mutations, with genome engineering strategies, a wild-type LAMA2 gene or a cDNA or a minigene (one or more exons and introns) can be knocked into the gene locus or a safe harbor locus ([000250],[000253]). A LAMA2 gene or cDNA can be inserted to the locus of a corresponding gene to replace the mutant gene, or (can be) knocked-in to a safe harbor locus [000262], as a transgene [00079], including as part of or the entire LAMA2 gene or cDNA [000263]), (satisfying claim 19, encoding the full length lamininin alpha 2 protein)
Prior to the effective filing date, it would have been prima facie obvious to one of ordinary skill in the art to have substituted the known LAMA2 sequence of ‘579 into the expression cassette of Calos since Calos teaches stable genomic integration of exogenous genes in mammalian cells using site-specific integrase systems. Lundberg teaches expression of LAMA2 in mammalian cells is valuable as a treatment for muscular dystrophy
A person of ordinary skill would have been motivated to use Calos’ functional integrase-mediated stable genomic insertion methods in mammalian cells, to integrate the known coding sequence of the therapeutic LAMA2 (transgene) of Lundberg, to generate a long-term expression system of LAMA2 in mammalian cells for treatment of muscular dystrophy, given Calos’ system was generally applicable to exogenous nucleic acids, and there was value to performing this insertion with a gene of medical therapeutic use. One of ordinary skill would have a reasonable expectation of success generating a mammalian cell expressing LAMA2 post integrase mediated integration, given that this combination would require a modification involving a transgene insertion, a routine modification in the art, replacing one gene for another in the integration system, and given the prior discussion of knock-in of LAMA2 to treat muscular dystrophy.
Conclusion
Claims 1-14,16-19,22-23 are rejected, claims 8 and 10 are objected to.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Lisa Horth whose telephone number is (703)756-4557. The examiner can normally be reached Monday-Friday 8:30-4:30 EST.
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/LISA HORTH/Examiner, Art Unit 1636
/NEIL P HAMMELL/Supervisory Patent Examiner, Art Unit 1636
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