DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Priority
Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged.
Information Disclosure Statement
The information disclosure statements (IDSs) submitted on 04/25/2025, 09/22/, 2025, and 06/12/2026 are in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statements are being considered by the examiner.
Status of Claims
Claims 1-6, 9-20, 22, and 29 are pending and are under examination in this office action. This is the first office action on the merits.
Drawings
The drawings are objected to because FIG. 21 (pg. 46 of replacement sheet) is missing a figure label. 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.
Nucleotide and/or Amino Acid Sequence Disclosures
REQUIREMENTS FOR PATENT APPLICATIONS CONTAINING NUCLEOTIDE AND/OR AMINO ACID SEQUENCE DISCLOSURES
Items 1) and 2) provide general guidance related to requirements for sequence disclosures.
37 CFR 1.821(c) requires that patent applications which contain disclosures of nucleotide and/or amino acid sequences that fall within the definitions of 37 CFR 1.821(a) must contain a "Sequence Listing," as a separate part of the disclosure, which presents the nucleotide and/or amino acid sequences and associated information using the symbols and format in accordance with the requirements of 37 CFR 1.821 - 1.825. This "Sequence Listing" part of the disclosure may be submitted:
In accordance with 37 CFR 1.821(c)(1) via the USPTO patent electronic filing system (see Section I.1 of the Legal Framework for Patent Electronic System (https://www.uspto.gov/PatentLegalFramework), hereinafter "Legal Framework") as an ASCII text file, together with an incorporation-by-reference of the material in the ASCII text file in a separate paragraph of the specification as required by 37 CFR 1.823(b)(1) identifying:
the name of the ASCII text file;
ii) the date of creation; and
iii) the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(1) on read-only optical disc(s) as permitted by 37 CFR 1.52(e)(1)(ii), labeled according to 37 CFR 1.52(e)(5), with an incorporation-by-reference of the material in the ASCII text file according to 37 CFR 1.52(e)(8) and 37 CFR 1.823(b)(1) in a separate paragraph of the specification identifying:
the name of the ASCII text file;
the date of creation; and
the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(2) via the USPTO patent electronic filing system as a PDF file (not recommended); or
In accordance with 37 CFR 1.821(c)(3) on physical sheets of paper (not recommended).
When a “Sequence Listing” has been submitted as a PDF file as in 1(c) above (37 CFR 1.821(c)(2)) or on physical sheets of paper as in 1(d) above (37 CFR 1.821(c)(3)), 37 CFR 1.821(e)(1) requires a computer readable form (CRF) of the “Sequence Listing” in accordance with the requirements of 37 CFR 1.824.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed via the USPTO patent electronic filing system as a PDF, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the PDF copy and the CRF copy (the ASCII text file copy) are identical.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed on paper or read-only optical disc, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the paper or read-only optical disc copy and the CRF are identical.
Specific deficiencies and the required response to this Office Action are as follows:
Specific deficiency – Nucleotide and/or amino acid sequences appearing in the drawings are not identified by sequence identifiers in accordance with 37 CFR 1.821(d). Sequence identifiers for nucleotide and/or amino acid sequences must appear either in the drawings or in the Brief Description of the Drawings.
Required response – Applicant must provide:
Replacement and annotated drawings in accordance with 37 CFR 1.121(d) inserting the required sequence identifiers;
AND/OR
A substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3) and 1.125 inserting the required sequence identifiers 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.
For example, Figures 10, 16-20, and 21 continued contain peptide sequences that are not fewer than four specifically defined amino acids and are not identified by SEQ ID NOs either in the figure itself or in the figure description within the specification. While these are the occurrences the examiner has identified, applicant is encouraged to identify each and every occurrence of peptide sequence that is four or more amino acids listed in the figure set without a SEQ ID NO identifier.
Specific deficiency – Nucleotide and/or amino acid sequences appearing in the specification are not identified by sequence identifiers in accordance with 37 CFR 1.821(d).
Required response – Applicant must provide:
A substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3) and 1.125 inserting the required sequence identifiers, 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.
For example, Table 4 on page 59 of the specification contains peptide sequences that are not fewer than four specifically defined amino acids and are not identified by SEQ ID NOs. While these are the occurrences the examiner has identified, applicant is encouraged to identify each and every occurrence of peptide sequence that is four or more amino acids listed in the specification without SEQ ID NO identifier.
Specification
The disclosure is objected to because of the following informalities: disclosing the incorrect name for the acronym, mTOR. Paragraphs [0015] and [0045] disclose mTOR as “muscle target of rapamycin,” whereas the correct name of mTOR is “mechanistic target of rapamycin” or in some cases an alias, “mammalian target of rapamycin,” is used, and paragraphs [0015] and [0046] recite “clotting factor VIII,” whereas the correct name is “coagulation factor VIII.” Appropriate correction is required.
Claim Objections
Claim 12 is objected to because of the following informalities: the incorrect name for the acronym "mTOR” and coagulation factor VIII. Claim 12 recites mTOR as “muscle target of rapamycin,” whereas the correct name of mTOR is “mechanistic target of rapamycin” or in some cases an alias, “mammalian target of rapamycin,” is used, and claim 12 recites “clotting factor VIII,” whereas the correct name is “coagulation factor VIII.” Appropriate correction is required.
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 1-6, 9-12, and 14-19 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for a method of inducing production of an exogenous polypeptide in a cell, the method comprising contacting the cell with two or more adeno-associated viral (AAV) particles, does not reasonably provide enablement for a method of inducing production of an exogenous polypeptide in a cell, the method comprising contacting a cell with a first AAV particle and a second AAV particle, wherein the exogenous polypeptide is selected from therapeutic polypeptides recited in claim 12. 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.
Claims 12 and 14-18 are dependent from and limited by independent claim 1 to a method requiring exactly “a first adeno-associated virus (AAV) vector particle” and “a second AAV vector particle” (i.e., a dual-vector system). However, claim 12 recites extra-large polypeptides including dystrophin, nebulin, titin, Syne-1, ryanodine receptor 1, and other proteins whose coding sequences cannot be accommodated within the claimed two-vector architecture. Claims 14-18 specifically require the therapeutic polypeptide to be dystrophin whose coding sequence cannot be accommodated by the two-vector system of claim 1.
The specification explicitly recognizes that a single wild-type AAV vector has a strict packaging capacity limit of “about 5 kb (the wild-type AAV genome is about 4.7 kb; larger genomes up to 5.5 kb or more can be packaged under certain conditions, but they do not efficiently infect and transduce target cells),” and further recognizes that after accounting for necessary AAV genome components (e.g., ITRs), that only “about 3.5 kb of DNA” is available “for the promoter, transgene coding region, polyadenylation sequence and other regulatory elements for a transgene construct to be carried by a single AAV vector particle,” see [0087]. The human dystrophin is defined by the specification to be 13.957 kb (including the untranslated regions). Dividing the ~14 kb CDS sequence across only two AAV vectors requires each vector to accommodate nearly 7 kb of cargo (aside from the ITRs), which physically exceeds the known packaging capacity of an in dividual AAV vector particle and results in truncated genomic packaging and deficient AAV vector particles. The specification fails to provide any working examples, data, or instructions on how a dual-AAV system can successfully package and express dystrophin or other extra-large proteins, for example, titin, whose coding sequence exceeds even the packaging limits of a three-vector system (see page 44 column 2 of Trinick J. Understanding the functions of titin and nebulin. FEBS Lett. 1992 Jul 27;307(1):44-8). Rather, the specification only demonstrates the production of dystrophin via a three-AAV vector system, see FIG. 6B, example 2, and claim 20.
Furthermore, the scope of enablement is mathematically demonstrated by the structural linkages at the specific target regions claimed in dependent claims 14-16. Claims 14-15 permit selecting a split site within or adjacent to dystrophin Hinge domains 1, 2, 3, or 4 to form the N-terminal portion of the dystrophin extein. According to the specification’s domain coordinates, hinge 1 resides near the N-terminus at nucleotides 965-1219 and hinge 4 resides near the C-terminus at nucleotides 9329-9544. Splitting at hinge 1 leaves a massive remaining C-terminal portion of the dystrophin extein, spanning from nucleotide ~1,220 to 13,957, which totals over 10,000 nucleotides (10 kb). Whereas, splitting at hinge 4, similarly leaves a massive N-terminal portion of the dystrophin extein spanning from nucleotide 1 to ~9,328, which totals over 9,000 nucleotides (9 kb). Because claims 14-15 depend from claim 1, the remaining portion of the coding sequence must be packaged as a single contiguous fragment within the designated AAV vector particle. A single cargo of >9 kb or 10 kb exceeds the ~5 kb packaging limit of a single AAV. A similar analysis can be applied to the claim 16, which permits selecting a split site within a loop domain of any one of the 24 spectrin-like repeat domains. According to the specification, these 24 repeats span nearly the entire length of the protein (from nucleotide 1,220 to 9,328), and splitting at either the first or last spectrin-like repeat, creates a fragment that in unmanageable for any one AAV vector of a dual-vector system. Claims 17-18 mirror this structural scope of enablement. Instead of focusing on the N-terminal portion of the dystrophin extein, these claims define the boundaries where the C-terminal portion joins the split intein within those same hinge or repeat loops. Mathematically, the result is identical, where a single portion is created that exceeds the packaging capacity of an AAV vector.
A Wands factor analysis indicates that undue experimentation would be required by a person of ordinary skill in the art to make and use a dual-AAV vector system to produce the full-length proteins of claims 12 and 14-18.
The breath of the claims: claims 12 and 14-18 are broad, encompassing many proteins, some of which are massive proteins with coding regions from about 14 kb to upwards of 100 kb. However, the claims depend from independent claim 1, which limits the split-intein system to two AAV vector particles.
Nature of the invention: the invention splits a large exogenous protein into two or three pieces, packages each piece on separate AAV vectors, and uses split inteins to splice the pieces back together inside the cell [0004]-[0005], [0021]-[0026]. This enables production of a protein larger than a single AAV can encode while preserving function [0024], [0078], [00111]. The use of muscle-specific expression cassettes focuses expression in muscle cells [0006], [0028], [00134]. The application also favors split sites and intein footprints that leave little or no extra amino acids in the final protein [0011]-[0013], [0063].
The state of the prior art: the prior art demonstrates that reconstitution of extra-large genes requires increasing the vector count beyond two. For instance, Lostal et al., (HUMAN GENE THERAPY 19:958–964, published 2008, provided in IDS) explicitly teach that because dystrophin CDS (~14 kb) drastically exceeds the 5 kb carrying capacity of a single AAV, a tri-AAV vector system splitting the CDS into three distinct fragments packaged into separate recombinant AAV vectors is required to successfully achieve reconstitution. The art does not teach or suggest that a two-vector system is capable of bypassing these physical packaging constraints for a 14 kb CDS.
The level of one of ordinary skill: an artisan of ordinary skill would hold a PhD with several years post-doctoral research experience.
The predictability of the art: the field of split-intein viral gene therapy is highly unpredictable. As taught by Auricchio A., et al., (WO2020079034A2, provided in IDS), finding viable split sites that allow proper protein splicing without disrupting native secondary/tertiary structure, avoiding cellular aggregation, and remaining within viral structural cargo limits requires highly tailored engineering, see page 4, lines 26-28, page 13 lines 18-22, and page 25 lines 21-22.
The amount of direction provided by the inventor: while the specification provides guidance to utilize at least a three-vector system to enable certain larger proteins, the specification fails to provide guidance for how to utilize a dual-vector system for the extra-large proteins (i.e., those with a CDS greater than 10 kb).
The existence of working examples: while the specification provides working examples of employing a dual-vector system to produce proteins with a CDS less than 10 kb, the specification fails to provide any working examples applying the dual-vector system to extra-large proteins. In fact, the specification explicitly identifies dystrophin as a protein that is too large for a two vector system and requires a three-vector system, which such a three-vector system enables the method for dystrophin and was reduced to practice.
The quantity of experimentation needed to make or use the invention based on the content of the disclosure: the quantity of experimentation to adapt a dual-AAV vector particle system to package a CDS that exceeds the known carrying capacity (i.e., greater than 10 kb split between two vectors) would most likely occupy an entire scientific career, if it is even feasible to achieve such a feat engaging in open-ended genetic engineering. The packaging capacity of AAV has been a known issue for decades and is an ongoing hurdle, which many artisan, such as the current inventors, are devising strategies to circumvent the constraint instead of taking on the challenge of increasing the packaging size of AAV.
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 6 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.
The term “substantially the same size” in claim 6 is a relative term which renders the claim indefinite. The term “substantially the same size” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. Specifically, the term leaves the relative size limitation between the first and second portions of the exogenous polypeptide unclear. The specification lacks guidelines, examples, or numerical thresholds to define the acceptable size variance encompasses by “substantially the same size.” Because exogenous polypeptides vary greatly in total length, a person of ordinary skill in the art cannot objectively determine what size difference qualifies as “substantially the same.”
Markush Grouping
Claim 12 is rejected on the basis that it contains an improper Markush grouping of alternatives. See In re Harnisch, 631 F.2d 716, 721-22 (CCPA 1980) and Ex parte Hozumi, 3 USPQ2d 1059, 1060 (Bd. Pat. App. & Int. 1984). A Markush grouping is proper if the alternatives defined by the Markush group (i.e., alternatives from which a selection is to be made in the context of a combination or process, or alternative chemical compounds as a whole) share a “single structural similarity” and a common use. A Markush grouping meets these requirements in two situations. First, a Markush grouping is proper if the alternatives are all members of the same recognized physical or chemical class or the same art-recognized class, and are disclosed in the specification or known in the art to be functionally equivalent and have a common use. Second, where a Markush grouping describes alternative chemical compounds, whether by words or chemical formulas, and the alternatives do not belong to a recognized class as set forth above, the members of the Markush grouping may be considered to share a “single structural similarity” and common use where the alternatives share both a substantial structural feature and a common use that flows from the substantial structural feature. See MPEP § 2117.
The Markush grouping of dystrophin, mini-dystrophin, utrophin, dysferlin, nebulin, titin, myosin, spectrin repeat containing nuclear envelope protein 1 (Syne-1), dystroglycan, ATP synthase, clotting factor VIII, lamin A/C, thyroglobulin, epidermal growth factor receptor (EGFR), alpha- and/or beta spectrin, muscle target of rapamycin (mTOR), and ryanodine receptor 1 is improper because the alternatives defined by the Markush grouping do not share both a single structural similarity and a common use for the following reasons:
The members of this group represent proteins with distinct primary, secondary, tertiary, and even quaternary structure in certain cases (e.g., ATP synthase). For example, an alignment of the dystroglycan primary amino acid sequence (NCBI Reference Sequence: NM_004393.6) to the EGFR primary amino acid sequence (NCBI Reference Sequence: NM_005228.5) results in a 1% sequence/structure identity (alignment provided below) with at most only five contiguous amino acids. Thus, there cannot be a common core sequence/structure among the members listed.
PNG
media_image1.png
200
619
media_image1.png
Greyscale
Furthermore, the members of this group represent vastly different types of proteins. For example, titin and nebulin are massive structural proteins that maintain the integrity of sarcomeres (see: Trinick J. Understanding the functions of titin and nebulin. FEBS Lett. 1992 Jul 27;307(1):44-8), while ATP Synthase is a multi-subunit transmembrane enzyme complex that is responsible for producing adenosine triphosphate (ATP) (see: Jasmine A. Nirody, et al. ATP synthase: Evolution, energetics, and membrane interactions. J Gen Physiol 2 November 2020; 152 (11): e201912475). A single utility or biological mechanism cannot flow from these disparate structures, as their physiological roles and biochemical pathways are unrelated. Thus, the members of this group do not share a single common use, nor are they functionally equivalent in the art.
To overcome this rejection, Applicant may set forth each alternative (or grouping of patentably indistinct alternatives) within an improper Markush grouping in a series of independent or dependent claims and/or present convincing arguments that the group members recited in the alternative within a single claim in fact share a single structural similarity as well as a common use.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
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.
Claims 1-6, 9-13, 19-20, 22, and 29 are rejected under 35 U.S.C. 103 as being unpatentable over Auricchio A., et al., (WO2020079034A2, provided in IDS) in view of Beyer HM, et. al. (FEBS J.;287(9):1886-1898, published 2020).
Regarding claims 1, 11, 19-20, and 22, Auricchio teaches that “delivery of multiple AAV vectors each encoding one of the fragments of either reporter or large therapeutic proteins flanked by short split-inteins results in protein trans-splicing and full-length protein reconstitution both in vitro and in vivo,” and that “vector genomes were properly packaged into AAV capsids,” see page 4 lines 28-29, page 5 lines 1-2, and example 9. Auricchio further teaches “when the first vector, the second vector and optionally the third vector are inserted in a cell, a least two fusion proteins or three fusion proteins are formed and when contacting said two fusion proteins or three fusion proteins, the protein product of the coding sequence is produced,” see page 9 lines 22-26 and 27-29. Thus, Auricchio teaches “cellular large protein reconstitution by providing to a target cell two or more fragments of said large protein fused to split inteins to promote intein-mediated trans-splicing and reconstitute the functional protein,” see page 6 lines 1-3.
Further regarding claims 1 and 22, Auricchio further teaches “a vector system to express the coding sequence of a gene of interest in a cell comprise two vectors, each vector comprising a portion of said coding sequence flanked by an intein sequence, wherein the 5' end of said coding sequence is flanked at the 3' terminus by the sequence of an N-intein, and the 3' end of the coding sequence of the gene of interest is flanked by the sequence of a C-lntein, such that when both vectors are expressed in a cell, two fusion proteins are produced and the full length protein of interest is generated as a result of a spontaneous trans-splicing reaction,” and that a host cell is transformed with the vector system and the vector system and/or transformed host cell is for medical use, see page 10 lines 3-10 and claims 1, 15, and 18-19.
Regarding claim 2, Auricchio teaches “preferred promoters are muscle-specific promoters including MCK, MYODI,” see page 13 lines 3-5.
Regarding claims 3, Auricchio teaches “ split inteins of the invention may be 100% identical, 98%, 80%, 75%, 70%, 65%, or 50% identical to naturally occurring inteins, wherein said inteins retain the ability to undergo trans-splicing reactions,” see page 51 lines 12-15.
Regarding claims 4 and 5, Auricchio teaches “split inteins may be further improved in desirable characteristics including activity, efficiency, generality, and stability through site-directed mutagenesis or modifications of the intein sequences based on rational design, and/or through directed evolution using methods like functional selection, phage display, and ribosome display,” see page 48 lines 16-19.
Regarding claim 6, Auricchio teaches engineering inteins of several proteins for example: EGFP, ABCA4, CEP290, and F8. Among these working examples, Auricchio teaches that the first and second portions of the exogenous polypeptide are substantially the same size or wherein the first and second portions of the exogenous polypeptide differ in size by no more than 50 amino acids. For example, Auricchio teaches splitting the F8 gene in half, upon which creates two half that are substantially the same size, see Figure 24B, lane 5 (AAV2/8-3’ F8 (Cys 1312)) and lane 6 (AAV2/8-5’ F8 (Ser984)), see also plasmids of invention table. Furthermore, Auricchio teaches splitting the EGFP gene in half such that the resulting plasmids, comprising either 5’ EGFP or 3’ EGFR, differ no more than 150 nucleotides from ITR to ITR; thus, given that each amino acid is encoded by 3 nucleotides, plasmid vectors that differ less than 150 nucleotides will result in N-terminal and C-terminal portion that differ in size no more than 50 amino acids.
Regarding claims 11-13, Auricchio teaches that “several attempts have been made at exploiting intein-based protein splicing to reconstitute expression of therapeutic genes including the Factor VIII gene, wherein the Synechocystis sp (Ssp) DnaB intein-fused heavy and light chain genes of Factor VIII were demonstrated to lead to reconstitution of Factor VIII in cell culture and in animal models.” Auricchio further teaches that, “similarly, a highly functional form of the dystrophin gene was expressed in vitro and in vivo, wherein the 6.3- kb Becker dystrophin gene was split onto two AAV vectors and each half was fused to split inteins obtained from the Synechocystis sp. PCC 6803 (Ssp) DnaB intein or the Rhodothermus marinus (Rma) DnaB intein.” Auricchio further teaches, that Xiao et al. (US 6,544,786, provided in IDS), “reports the use of split inteins to deliver a dystrophin minigene,” see page 5 15-25. Auricchio further experimentally teaches in example 10 that “AAV intein vectors can be used to deliver the large F8 gene affected in Hemophilia A.” In this example 10, Auricchio teaches contacting cells in vivo via systemic injection of two AAV2/8 F8 intein vector particles each comprising one of the two halves of the large FVIII protein flanked by the split Npu DnaE inteins, see pages 145-146.
Further regarding claim 13, Auricchio teaches that the 6.3kb Becker mini-dystrophin gene was split onto two AAV vectors and each half was fused to split inteins , see page 5 lines 18-22 and 25. Thus, given that the gene is 6,300 bases, and 3 bases encode an amino aide, the Becker mini-dystrophin used is 2,100 amino acids. Calculating the kDa of a 2,100 amino acid polypeptide results in approximately 231 kDa: 2100 X 110 Da (the average Da of an amino acid) = 231,000 Da, which converts to 231,000/1,000 = 231 kDa.
Further regarding claim 20, Auricchio teaches a “vector system to express the coding sequence of a gene of interest in a cell comprises three vectors, each vector comprising a portion of said coding sequence flanked by an intein sequence, wherein the coding sequence is divided in three portions such that the 5'end of said coding sequence is flanked at the 3' terminus by the sequence of a first N-intein; the middle portion of said coding sequence is flanked at the 5' terminus by a first C-lntein, and at the 3' terminus with a second N-lntein; the 3' portion of said coding sequence is flanked at the 5' terminus by a second C-lntein, such that when all three vectors are expressed in a cell, three fusion proteins are produced, and the full length protein of interest is generated as a result of a spontaneous trans-splicing reaction wherein the first N-lntein reacts with the first C-lntein and the second N-lntein reacts with the second C-lntein,” and that a host cell is transformed with the vector system and the vector system and/or transformed host cell is for medical use, see page 10 lines 11-21 and claims 1, 15, and 18-19.
Regarding claim 29, Auricchio teaches using the vector system to prevent and/or treat “Duchenne muscular dystrophy, cystic fibrosis, hemophilia A, Wilson disease, Phenylketonuria, dysferlinopathies, Rett's syndrome, Polycystic kidney disease, Niemann-Pick type C, Huntington's disease,” see claim 22. Auricchio further teaches pharmaceutical composition comprising the vector system,” see claim 23.
Regarding the footprint limitation of claims 1, 9, 20, and 22, Auricchio teaches that “intein activity is context-dependent, with certain peptide sequences surrounding their ligation junction (called N- and C-exteins) that are required for efficient trans-splicing to occur, of which the most important is an amino acid containing a thiol or hydroxyl group (i.e., Cys, Ser or Thr) as first residue in the C-extein,” see page 54 lines 24-27. Auricchio further teaches that inteins “self-excise similarly to a protein intron, without leaving amino acid modifications in the final protein product,” see page 5 lines 4-6. Auricchio further teaches that “the coding sequence is split into the first portion, the second portion and optionally the third portion, at a position consisting of a nucleophile amino acid which does not fall within a structural domain or a functional domain of the encoded protein product, wherein the nucleophile amino acid is selected from serine, threonine, or cysteine,” see claim 8. Auricchio reduces this to practice across multiple exogenous polypeptides: the ABCA4 coding sequence split at native Ser1168 or Ser1090 residues (in addition to Cys1150), see page 15 lines 15-18; the CEP290 coding sequence is split at a native Ser453 residue (in addition to Cys1076/Cys929/Cys1474), see page 15 lines 19-23); and the F8 coding sequence is split at a native Ser984 residue (split set 2) in addition to Cys1312 (split set 1), see page 18 lines 10-12. In each instance, the native Ser or Cys residue already present in the exogenous polypeptide’s sequence serves as the required +1 C-extein nucleophile required for trans-splicing, such that the reconstituted full-length protein contains no additional amino acids contributed by the split intein at the junction site. Thus, Auricchio teaches selecting a native split site bearing the intein’s required nucleophile residue (Cys, Ser, or Thr) such that the residue is contributed by the exogenous polypeptide’s own native sequence rather than being an additional residue introduced by the intein, resulting in a spliced exogenous polypeptide having a minimal footprint of intein-derived amino acids.
Auricchio does not explicitly characterize the spliced exogenous polypeptide produced from its native Cys or Ser split sites as having a footprint of less than four amino acids from the split intein, 3 or fewer amino acids from the split intein, or without extra amino acids from the split intein.
Beyer teaches “ever since the discovery of protein splicing by inteins, engineering of inteins toward high performance, high tolerance of junction sequences, and smaller variants have been an ongoing quest,” see introduction second paragraph. Beyer further teaches the resolved crystal structure of the “naturally split gp41-1 intein,” and that based on the crystal structure, grafting “the structural features of naturally split inteins onto a cis-splicing intein to develop novel natural-like split inteins,” successfully engineered “orthogonal split intein fragments from a cis-splicing intein,” see introduction last paragraph. Beyer further teaches that an advantage of this engineering approach starting from inteins such as gp41-1, is that it “makes use of Ser as the catalytic residue at the +1 position, the first extein residue following the intein sequence,” and that because of “the much higher frequency of Ser over Cys within pro- and eukaryotic proteins, inteins with +1 Ser allow a broader spectrum of possible insertion sites for scar-less protein ligation than naturally split inteins with Cys at the +1 position, thereby expanding potential applications,” see introduction third paragraph. Based on this, Beyer further teaches the generation of “split inteins engineered from Npu DnaB mini-intein,” and that they “contribute to overcoming the junction sequence and extein dependencies that currently complicate protein trans-splicing applications.” Thus, Beyer teaches that “engineered inteins expand the applicability of protein trans-splicing in protein engineering, chemical biology, and synthetic biology, in particular when applications require scar-less protein ligation,” see discussion last paragraph.
It would have been obvious to a person having ordinary skill in the art (PHOSITA) before the effective filing date to recognize that Auricchio’s split-intein system employing a native Ser residue at the +1 C-extein position results in a spliced exogenous polypeptide having a footprint of less than four amino acids from the split intein, 3 or fewer amino acids from the split intein, or without extra amino acids from the split intein. A PHOSITA would have been motivated to do so because Beyer confirms that split inteins employing a native Ser residue at the +1 position achieve scar-less protein ligation (i.e., reconstitution of the exogenous polypeptide without additional amino acids contributed by the intein) consistent with and confirming the result that necessarily follows from Auricchio’s teaching of splitting the coding sequence at a native serine residue serving as the required C-extein nucleophile. A PHOSITA would have had a reasonable expectation of success because both Auricchio and Beyer successfully use the same trans-splicing chemistry of a nucleophilic residue (e.g., Ser) at the +1 C-extein position, while Beyer confirms that splitting a target protein at a native Ser residue, as Auricchio does for ABCA4, CEP290, and F8, predictably and reliably yields a reconstituted scar-less protein.
Claims 14-18 are rejected under 35 U.S.C. 103 as being unpatentable over Auricchio A., et al., (WO2020079034A2, provided in IDS) in view of Beyer HM, et. al. (FEBS J.;287(9):1886-1898, published 2020) as applied to claims 1-6, 9-13, 19-20, 22, and 29 above, and further in view of Xiao X., et al., (US6544786B1, provided in IDS), Li J., et al., (Hum Gene Ther.;19:958–964, published 2008, provided in IDS), and Lostal W., et al., (Hum Gene Ther.;25(6):552-62, published 2014, provide in IDS) as evidenced by Kawecka K. et al., (Current Gene Therapy, 15, 395-415, published 2015).
The teachings of Auricchio and Beyer are incorporated herein by reference to the preceding 103 rejection.
Regarding claims 14-18, Auricchio teaches “the coding sequence is split into the first portion, the second portion and optionally the third portion, at a position consisting of a nucleophile amino acid which does not fall within a structural domain or a functional domain of the encoded protein product, wherein the nucleophile amino acid is selected from serine, threonine, or cysteine,” see page 13 lines 18-22.
Neither Auricchio or Beyer teach the N-terminal portion of the dystrophin extein is joined to the N-terminal portion of a split intein within or adjacent to a dystrophin hinge domain; the N-terminal portion of the dystrophin extein is joined to a loop domain joining helix b to helix c, or helix c to helix a' within one of the 24 dystrophin spectrin-like repeat domains; the C-terminal portion of the dystrophin extein is joined to the C-terminal portion of the split intein within or adjacent to a dystrophin hinge domain or to a loop domain joining helix b to helix c, or helix c to helix a' within one of the 24 dystrophin spectrin-like repeat domains; and wherein the hinge domain comprises hinge 1, 2, 3, or 4 of dystrophin.
Xiao teaches “a method for preparing a trans-spliced peptide in vitro, comprising the steps of: a. providing (i) a first nucleic acid encoding and capable of expressing a first peptide in a suitable host cell, the first peptide having at its C-terminal end an N-terminal portion of a split intein and (ii) a second nucleic acid encoding and capable of expressing a second peptide in the host cell, the second peptide having at its N-terminal end a C-terminal portion of the same split intein, wherein the first and second nucleic acids are provided on the same or on different nucleic acid molecules; and b. producing the trans-spliced peptide by transferring the first nucleic acid and the second nucleic acid into the host cell, wherein at least one of the first nucleic acid and the second nucleic acid includes nucleotide sequences which allow for packaging of the nucleic acid into a recombinant Adeno-associated virus particle,” and “wherein the trans-spliced peptide is dystrophin, or a functional derivative thereof,” see claims 1 and 2. Xiao further teaches that “three or more protein or peptide sequences can be joined at two or more junction sites,” see column 6 lines 45-46.
Xiao further teaches “the central region is a long and rod-like domain that consists of 24 repeats of a triple helical coiled-coil, or of 9 repeats in the smaller, but still functional, Becker form,” and that “each individual repeat is believed to fold independently into a structural module, and neighboring repeats are connected by a short, flexible linker sequence, see column 3 lines 1-12.
Xiao further teaches “Dystrophin, because of its structure, described above, is particularly suited for production by protein trans-splicing. One consideration in producing a protein through protein trans-splicing is avoiding misfolding of the half-proteins prior to splicing. The dystrophin gene can be split into two half-genes at locations corresponding to flexible linker sequences in the central rod domain. The dystrophin structure predicts that each of the two half-proteins will fold independently and properly, like they normally do in a complete dystrophin. This minimizes the risk of mis-folding of the half-proteins before protein trans-splicing, which ensures correct structure and function of the dystrophin after its formation by trans-splicing. Also, the flexible linker sequences have several nucleophilic amino acid residues (Ser, Cys, or Thr) that are needed at the intein insertion site,” see column 11 lines 52-67 and column 7 lines 1-4.
Similar to Xiao, Li also teaches employing the split intein strategy to reconstitute mini-dystrophin. Li teaches the split intein is inserted in the loop portion connecting helix 2 (i.e., helix b) to helix 3 (i.e., helix c), see FIG. 2B and reproduced below for convenience:
PNG
media_image2.png
192
557
media_image2.png
Greyscale
Thus, Li teaches the N-terminal portion and the C-terminal portion of the dystrophin extein is joined to a loop domain joining helix b to helix c, within one of the 24 dystrophin spectrin-like repeat domains. Furthermore, Li teaches selecting a Ser +1 residue in the loop domain to serve as the nucleophile of the C-extein, see FIG. 2B above.
While Xiao and Li teach splitting mini-dystrophin in the spectrin-like repeat domains (and specifically between helix b and c), and Xiao teaches that the split intein method can be adapted to reconstitute larger proteins that are split into three or more segments, neither Xiao or Li explicitly teach splitting full-length dystrophin, nor do they address splitting within or adjacent to dystrophin’s hinge domains specifically.
Lostal teaches reconstitution of full-length dystrophin employing three AAV vectors each comprising a segment of dystrophin amounting to roughly a third of the protein. Specifically, Lostal teaches the N-terminal 1/3 head portion comprises exons 1-26, the body 1/3 portion comprises exons 27-middle of 53, and the C-terminal 1/3 tail portion comprises exons 50-79, see FIG 2, FIG. 2 legend, title, and abstract. As evidenced by Kawecka’s teaching of the dystrophin protein structure (reproduced below for convenience) showing hinge (H) domains, spectrin-like repeat rod domains (R), and exon structure, the first fragmentation boundary taught by Lostal, at the exon 26/27 region, falls within the region of dystrophins spectrin-like repeat domains corresponding to repeats R7/R8, and the second fragmentation boundary taught by Lostal, at exon 50, falls within the spectrin-like repeat domain corresponding to R19, which directly abuts H3. Lostal’s selection of these specific boundaries for fragmentating full-length dystrophin into AAV-packageable portions demonstrates that the R7/R8 and R19/H3 regions are art-recognized working locations for dividing the dystrophin coding sequence.
PNG
media_image3.png
288
581
media_image3.png
Greyscale
It would have been obvious to a person having ordinary skill in the art (PHOSITA) before the effective filing date to adapt Auricchio’s split-intein three AAV vector system to full-length dystrophin by selecting split-intein sites within or adjacent to a dystrophin hinge domain (such as H3) and/or within a loop domain connection helix b to helix c, or helix c to helix a’, within one of the dystrophin spectrin-like repeat domains (per Xiao and Li), such that the N- and C-split-inteins are joined to the dystrophin N- and C-exteins at the respective sites.
A PHOSITA would have been motivated to do so because in order to package full-length dystrophin into AAV vectors, it is necessary to fragment dystrophin into at least three segments at structurally tolerant boundaries. Lostal demonstrates that a tri-AAV vector system, which successfully packages a fragmented dystrophin at boundaries corresponding to one of the spectrin-like repeat domains (i.e., R7/R8) and adjacent to a hinge domain (i.e., R19 adjacent to H3), successfully produces full-length dystrophin. A PHOSITA would have therefore been motivated to examine the spectrin-like repeat rod domains R7/R8 at the first fragmentation regions and the spectrin-like repeat rod domains R19/R20 adjacent to the hinge 3 domain for suitable C-extein nucleophiles (i.e., Ser, Cys, or Thr) as these regions are art-recognized fragmentation points for full-length dystrophin. Separately, Xiao motivates to look at the spectrin-like repeat rod domains by expressly teaching that linker loops between each helix are “particularly suited for production by protein trans-splicing” because splitting at these flexible linker sequences “minimize the risk of mis-folding of the half-proteins” while ensuring “correct structure and function of the dystrophin after its formation by trans-splicing,” and further teaches that these linker sequences contain the nucleophilic Ser, Cys, or Thr residues required at the intein insertion site, directly satisfying Auricchio’s general teaching of selecting split sites at native nucleophile residues outside structural or functional domains. Li corroborates this teaching by demonstrating a working dystrophin split-intein in which a split intein is inserted specifically within the spectrin-repeat loop connecting helix b to helix c. A PHOSITA combining Auricchio’s general split-intein AAV platform with the art-recognized fragmentation points near R7/R8 and R19/H3/R20 and clear guidance to find C-extein nucleophiles in the loop linkers, would have been motivated to optimize dystrophin split sites at one or both of these locations when designing a multi-AAV split-intein dystrophin system.
A PHOSITA would have had a reasonable expectation of success because Xiao and Li each reduced to practice split-intein mediated dystrophin reconstitution at the spectrin-like repeat linker loop locations, and Lostal reduced to practice fragmenting full-length dystrophin at regions coinciding with spectrin-like repeat domains, one oof which is adjacent to the H3 domain and with this fragmentation strategy, successfully delivered each fragment with AAV to reconstitute full-length dystrophin in vivo. Given that Auricchio established that split-intein trans-splicing reliably reconstitutes larger exogenous polypeptides via three AAV vectors when split at a native nucleophilic residue outside a functional domain, and that Xiao, Li and Lostal collectively confirm that both the spectrin-like repeat linker loops of dystrophin tolerate fragmentation, a PHOSITA would have reasonably expected that selecting dystrophin split sites at these art-recognized regions combined with selection of a native Ser, Cys, or Thr residue at these locations, would predictably reconstitute a properly folded and functional full-length dystrophin.
Conclusion
No claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to COREY LANE BRETZ whose telephone number is (571)272-7299. The examiner can normally be reached M-F 7:30am - 6:30pm.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Ram Shukla can be reached at (571) 272-0735. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/COREY LANE BRETZ/Patent Examiner, 1635
/RAM R SHUKLA/Supervisory Patent Examiner, Art Unit 1635
Prosecution Insights