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 .
Claims 1-15 are pending and under consideration.
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.
Instant claims 1-2, 4-8, 10, and 12-14 are entitled to the effective filing date of 03/24/2021, which is the filing date of provisional application 63/165,487.
The subject matter of instant claims 3, 9, 11, and 15 (drawn to insertions of up to 60 bp and treatment of Huntington’s Disease) is not disclosed in provisional application 63/165,487; thus, instant claims 3, 9, 11, and 15 are entitled to the effective filing date of 03/15/2022, which is the filing date of PCT/US/20392.
Information Disclosure Statement
Receipt of an information disclosure statement on 09/21/2023 is acknowledged. The signed and initialed PTO-1449 has been mailed with this action.
The information disclosure statement filed 09/21/2023 fails to comply with 37 CFR 1.98(a)(2), which requires a legible copy of each cited foreign patent document; each non-patent literature publication or that portion which caused it to be listed; and all other information or that portion which caused it to be listed. It has been placed in the application file, but the information referred to therein has not been considered, as indicated on the signed and initialed PTO-1449 that has been mailed with this action.
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 - The Incorporation by Reference paragraph required by 37 CFR 1.821(c)(1) is missing or incomplete. See item 1) a) or 1) b) above.
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 incorporation-by-reference paragraph, 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.
►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). Sequences are disclosed at Table 1, Table 2, and Table 3 that are not appropriately identified by sequence identifiers in accordance with 37 CFR 1.821(d). It would be remedial to clearly identify each disclosed sequence with its unique sequence identifier.
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.
►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. Sequences are disclosed at Figure 3, Figure 8, and Figure 9 that are not appropriately identified by sequence identifiers in accordance with 37 CFR 1.821(d). It would be remedial to clearly identify each disclosed sequence with its unique sequence identifier.
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.
Drawings
The drawings are objected to because:
Regarding Figures 2a, 3c, 3e, 4d, 4f, 5b, 6a-h, 7a-e, 8b-f, 9a-g, and 12c-e, the image quality is insufficient to be clearly and readily interpretable. It would be remedial to increase the image quality such that it is clearly and readily interpretable.
Regarding Figure 10b, the key distinguishing mice 1-4 is difficult to interpret in the current grayscale scheme. To clearly distinguish between mice, it would be remedial to ensure that the key is readily interpretable.
37 CFR 1.84 (u)(1) states “View numbers must be preceded by the abbreviation "FIG."” In the current case, the view numbers for Figures 1-12 are preceded by the word "Figure" instead of the abbreviation "FIG.". It would be remedial to replace the view numbers with the abbreviation “FIG.” in place of “Figure.”
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
Claims 1, 2, 6, 7, 10, and 12 are objected to because of the following informalities:
Claims 1, 7, and 12 all recite a first and second prime editor guide RNA (pegRNA), wherein the first recited prime editor guide RNA is indicated to correspond to the acronym “pegRNA.” However, this acronym is not used when reciting the second pegRNA within the same claim. For purposes of internal consistency and to comport with standard linguistic conventions, it would be remedial to use the defined acronym “pegRNA” when referring to subsequent pegRNAs following the recitation of the “first prime editor guide RNA (pegRNA).”
Claims 2 and 10 both recite a range of 1kb up to 10kb. However, the form of this recitation differs between both claims. While claim 2 recites “1kb to 10 kb” (bolded and underlined emphasis added), claim 10 recites “1kb-10kb” (bolded and underlined emphasis added). For purposes of internal consistency, it would be remedial to recite the claimed range in a consistent fashion throughout the instant claim set.
Claim 6 recites “the method of Claim 4, said target nucleotide sequence comprises a FahΔExon 5 mutation,” which does not comport with standard grammatical and/or linguistic conventions. A transitional word such as “wherein” is required to precede the additional claim recitations for proper sentence structure. It would be remedial to amend the instant claim language to comport with standard grammatical and/or linguistic conventions, for example by reciting “the method of Claim 4, wherein said target nucleotide sequence…”. This is merely an example set forth by the Examiner and are not intended to be limiting. Furthermore, the article “a” is grammatically improper when preceding words articulated with an initial vowel sound. While “FahΔExon 5” is spelled such that it recites a consonant letter “F,” the pronunciation of the “F” in “FahΔExon 5” nonetheless begins with a vowel sound. It would be remedial to update the instant claim language to be grammatically proper.
Appropriate correction is required.
Claim Rejections - 35 USC § 112(a)
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 7, 8, 10, and 11 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 treating FahΔExon5 mice mimicking tyrosinemia type I and Huntington disease in mice via administration of PE-Cas9 and paired pegRNAs, does not reasonably provide enablement for treating tyrosinemia type I or Huntington disease in non-mouse patients (including human patients) via the same methods, nor does it reasonably provide enablement for treating any other genetic disease in any patient. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to use the invention commensurate in scope with these claims.
Enablement is considered in view of the Wands factors (MPEP 2164.01(A)). These include: the breadth of the claims, the nature of the invention, the state of the prior art, the level of one of ordinary skill, the level of predictability in the art, the amount of direction provided by the inventor, the existence of working examples, and the quantity of experimentation needed to make or use the invention. All of the Wands factors have been considered with regard to the instant claims, with the most relevant factors discussed below.
Nature of the invention: Claims 7, 8, 10, and 11 are drawn to a method of treating a patient exhibiting at least one symptom of a genetic disease such as tyrosinemia (as recited at instant claim 8), said method comprising providing a composition comprising a catalytically active Cas9 protein fused to a reverse transcriptase, a first prime editor guide RNA (pegRNA) molecule conjugated to a first reverse transcriptase DNA insertion template and a second pegRNA molecule conjugated to a second reverse transcriptase DNA insertion template, wherein said first and second reverse transcriptase DNA templates are complementary (hereinafter referred to as the PEDAR system-see page 21, line 30-page 22, line 7 of the instant specification). The nature of the invention is complex in that one must be able to design gRNAs that are both compatible with the unique requirements of prime editing-based systems as well as effective in specifically targeting the loci implicated in the instantly claimed genetic disease (i.e. the gRNA must not elicit off-target effects). All of these requirements must be considered in the context of in vivo usage, as claimed, especially in human subjects, as envisioned by the instant specification (page 7, lines 29-31).
Breadth of the claims: The claims broadly encompass the treatment of any genetic disease via providing the PEDAR system to the patient in need thereof. While dependent claims 8 and 9 respectively limit the treated genetic disease to tyrosinemia or Huntington disease, these dependent limitations recite only a fraction of the subject matter broadly encompassed by the claims, as set forth above. Furthermore, while dependent claims 10 and 11 limit the size of the gene mutation insertion associated with the treated genetic disease and the size of the corrective insertion, these dependent limitations do not limit the breadth of genetic diseases claimed to be treatable via the PEDAR system. The complex nature of the subject matter of this invention is greatly exacerbated by the breadth of the claims.
Guidance of the specification and existence of working examples: While the specification envisions treatment of any genetic disease in human and patients in need thereof via administration of the PEDAR system (see page 7, lines 29-31; page 2, line 28-page 3, line 8; page 21, lines 20-22; page 22, lines 8-10; section: PEDAR System Therapeutic Applications), with specific regard to tyrosinemia (section: PEDAR System Therapeutic Applications) and Huntington disease (section: Implications of PEDAR In Correcting Genomic Duplications), it only teaches in vivo treatment of FahΔExon5 mice mimicking tyrosinemia via administration of PE-Cas9 and paired pegRNAs (see section: PEDAR System Therapeutic Applications; Example III). The data disclosed in the instant specification demonstrates that the PEDAR system is capable of treating FahΔExon5 mice mimicking tyrosinemia. However, as set forth below, in vivo administration of CRISPR-based machinery to mice is markedly different from administering the same to human patients, which is encompassed by the instant set of the claims, both under broadest reasonable interpretation, and as supported by the instant specification (see page 7, lines 29-31).
Predictability and state of the art: Administering CRISPR machinery to human patients (i.e. in prime editing or in other forms) is an underdeveloped and unpredictable topic in the current state of the art. Kantor et al., 2020 (hereinafter Kantor) emphasizes that the additional research required for utilizing base editing and prime editing systems in human clinical applications is still underway. Per Kantor, prime editors and similar CRISPR-based machinery are faced with the same predominant issue encountered in the art of genome editing: delivery. While both viral and non-viral delivery systems have been developed and are being optimized in the field, each approach has its own associated advantage as well as disadvantages. Regarding viral vectors, retroviral vectors are prone to random integration and require mitotic cell division for transduction, while adenoviral vectors are known to risk eliciting an immune response in the subject, and lentiviral vectors may be oncogenic (see section “5. Delivery Systems”). Although adeno-associated viral vectors (AAV) are the leading platform for in vivo gene therapy delivery, they have a limited DNA packaging capacity and require ongoing optimization to address these limitations (see section “5. Delivery Systems”). Additionally, AAV vectors are known to be immunogenic in both human and non-human primates, due to exposure to wild-type AAV resulting in anti-capsid neutralizing antibodies that can cross-react and prevent AAV (re)-administration (Mingozzi, 2018; page 2335, column 1, paragraphs 1 and 2). The instant application is silent as to any proposals to overcome any of these known hurdles to in vivo administration of prime editors and similar CRISPR-based machinery to humans, and as of the time of filing, in vivo CRISPR-based editing of humans or human somatic cells was not publicly disclosed or otherwise known.
Furthermore, as set forth above, the instant claim set broadly encompasses the treatment of any genetic disease via providing the PEDAR system to the patient in need thereof. Tyrosinemia and Huntington disease (as recited at instant claims 8 and 9) are two specific genetic diseases, but they are by no means representative of any and all genetic diseases. For example, tyrosinemia itself presents as three different types: type I, type II, and type III (reviewed in Russo et al., 2001: see Table 1). Type I is the most well-characterized type and is associated with decreased levels of fumarylacetoacetate hydroxylase (FAH), as well as mutations to the same (reviewed in Russo et al., 2001: see page 214, column 1, paragraph 2 and section “POPULATION GENETICS AND MUTATIONS”). However, Huntington disease is an entirely different genetic disease that develops from trinucleotide CAG repeat expansion in the HTT gene per the instant specification (page 35, lines 1-5).
These are merely two genetic diseases that display different mechanisms and pathologies. When expanding out to consider all possible genetic diseases, the breadth of possible types of mutations or other causations is staggering, and advances towards treating one genetic disease would not be reasonably predicted to be applicable towards the treatment of any other genetic disease.
While the instant specification envisions treatment of tyrosinemia type I (page 2, line 26) and Huntington disease (page 3, line 5), the instant claim language does not limit the treated disease to tyrosinemia type I or Huntington disease specifically and therefore encompasses any genetic disease and any type of tyrosinemia.
Amount of experimentation necessary: The quantity of experimentation needed to carry out the full scope of the claimed method is large. One could not solely rely upon the guidance provided in the instant disclosure or prior art, especially in view of the design and delivery concerns regarding prime editors and other CRISPR-based therapeutics, as set forth above. One would be required to address and overcome at least each and every consideration noted above in applying prime editor and other CRISPR-based machinery to patients in vivo to ensure both the safety and efficacy of the claimed treatment, which has not yet been achieved in the field despite a large amount of effort from the research community, underscoring the large amount of experimentation (with no guarantee or reasonable expectation of success) required to produce a CRISPR-based system suitable to in vivo administration to patients.
In view of the breadth of the claims and the lack of guidance provided by the specification as well as the unpredictability of the art, the skilled artisan would have required an undue amount of experimentation to make and/or use the claimed invention. Therefore, claims 7-11 are not considered to be fully enabled by the instant disclosure.
Claim Rejections - 35 USC § 112(b)
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 8-11 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 applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claims 8 and 9 depend from claim 1 and recite the limitation "said genetic disease" in line 1 of each claim. There is insufficient antecedent basis for this limitation in the claim. For purposes of examination, the Examiner has interpreted instant claims 8 and 9 to depend from instant claim 7, which provides support for the recited claim limitations. It would be remedial to amend the instant claim language such that there is sufficient antecedent basis for every claim term.
Claim 10 depends from claim 1 and recites the limitation "said patient" in line 1. There is insufficient antecedent basis for this limitation in the claim. For purposes of examination, the Examiner has interpreted instant claim 10 to depend from instant claim 7, which provides support for the recited claim limitations. Furthermore, claim 11 depends from claim 10 and does not resolve the lack of antecedent basis set forth above and therefore inherits the rejection thereof. It would be remedial to amend the instant claim language such that there is sufficient antecedent basis for every claim term.
Claim Rejections - 35 USC § 103
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.
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 1-5 are rejected under 35 U.S.C. 103 as being unpatentable over WO 2021/178709 A1 (hereinafter Steinberg; effectively filed 06/05/2020) in view of Choi et al., 2021 (hereinafter Choi; as cited in the IDS filed 09/21/2023), Chen et al., 2014 (hereinafter Chen).
With regard to claim 1, which recites “a method, comprising:
providing;
a genomic DNA locus comprising a target nucleotide sequence; and
a composition comprising a catalytically active Cas9 protein fused to a reverse transcriptase, a first prime editor guide RNA (pegRNA) molecule conjugated to a first reverse transcriptase DNA insertion template and a second prime editor guide RNA molecule conjugated to a second reverse transcriptase DNA insertion template, wherein said first and second reverse transcriptase DNA templates are complementary;
contacting said catalytically active Cas9 protein with said target nucleotide sequence, wherein said first pegRNA molecule binds to a sense strand of said target nucleotide sequence and said second pegRNA molecule binds to an antisense strand of said target nucleotide sequence;
creating two double strand breaks in said target nucleotide sequence with said catalytically active Cas9 protein such that said target nucleotide sequence is deleted; and
incorporating a double stranded insertion nucleotide sequence encoded by said first and second reverse transcriptase insertion templates into said genomic DNA sequence,”
while no one source discloses the claimed method in its entirety, the components of this method were known in the art prior to the effective filing date of the instant application such that one of ordinary skill in the art would have been motivated to combine teachings to arrive at the instant invention prior to the effective filing date of the instant application, as set forth below.
Steinberg discloses a genome editing system, said system comprising a nuclease active Cas9 linked to a reverse transcriptase (Figure 5) guided to a target genomic locus (page 593, lines 12-13) by a reverse transcriptase DNA insertion template coupled to a pegRNA (page 585, lines 22-36; page 589, lines 14-15), wherein the heterologous object sequence encoded within said insertion template is reverse transcribed to insert the sequence of interest at the targeted locus (page 586, lines 26-27; page 724, lines 13-15). The inserted heterologous object sequence is disclosed to be 10-20, 20-30, 30-40, 40-50, or 50-60 nucleotides in length (page 585, lines 31-33). Per Steinberg, the system taught therein is capable of treating genetic diseases such as tyrosinemia type I (Table 10A) and repeat expansion diseases such as Huntington disease (Table 10C; Table 26). Thus, while Steinberg discloses a genomic DNA locus comprising a target nucleotide sequence, as well as a composition comprising a catalytically active Cas9 protein fused to a reverse transcriptase and a pegRNA conjugated to a reverse transcriptase DNA insertion template, wherein the reverse transcriptase DNA insertion template is reverse transcribed for insertion into the genome, thereby treating diseases such as tyrosinemia and Huntington disease, Steinberg does not disclose dual pegRNA molecules, nor do they explicitly address any other of the instant claim limitations. These deficiencies are cured by Choi and Chen, as set forth below.
Choi discloses concurrent programming of deletion and insertion using PRIME-Del, wherein a desired short insertion (up to 30 bp) is encoded into a pair of pegRNAs (targeting opposite strands, as instantly claimed) in a reverse complementary manner such that following deletion of the targeted genomic region by Cas9 nicking, the encoded insertion is reverse transcribed into the genome, resulting in a double stranded insertion (lines 153-182; Figure 2), as is also disclosed in Steinberg. Thus, Choi discloses the utility of a first and second pegRNA molecule targeting opposite strands for simultaneous gene deletion and insertion, wherein the templates for insertion are complementary to each other. However, Choi explicitly discloses that these insertions are small (i.e. up to 30 nucleotides), as compared to the large insertions facilitated by the system of Steinberg set forth above.
Regarding the claimed deletion, Chen discloses that Cas9 nucleases, when targeted to specified loci via dual gRNAs, are capable of deleting large segments of genomic DNA, as shown in Figure 1, which depicts deletion of 1,200 bases of genomic sequence (mutant #14 of Figure 1).
Thus, Steinberg discloses insertion of large nucleotide sequences via a nuclease-active Cas9 linked to a reverse transcriptase guided to a target genomic locus by a reverse transcriptase DNA insertion template coupled to a pegRNA, wherein the heterologous object sequence encoded within said insertion template is reverse transcribed to insert the sequence of interest at the targeted locus, Choi discloses simultaneous genomic insertion and deletion via a Cas9 nickase and paired pegRNAs with complementary insertion templates, and Chen discloses deletion of large segments of the genome via dual gRNAs targeting Cas9 nuclease to a specified genomic locus.
Therefore, given that:
Steinberg discloses insertion of large nucleotide sequences via a nuclease-active Cas9 linked to a reverse transcriptase guided to a target genomic locus by a reverse transcriptase DNA insertion template coupled to a pegRNA;
Choi discloses simultaneous genomic insertion and deletion via a Cas9 nickase and paired pegRNAs with complementary insertion templates; and
Chen discloses deletion of large segments of the genome via dual gRNAs targeting Cas9 nuclease to a specified genomic locus,
it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to modify the system of Steinberg by substituting the single pegRNA-conjugated reverse transcriptase insertion template taught therein for dual pegRNA-conjugated reverse transcriptase insertion templates, wherein said insertion templates are complementary to each other to predictably facilitate simultaneous insertion and deletion (as disclosed in Choi), wherein said insertion is large (as disclosed in Steinberg) and said deletion is large (as disclosed in Chen). One would have been motivated to make such a modification in order to receive the expected benefit of simultaneously inserting and deleting large sequences into a targeted genomic locus.
Thus, it is considered that Steinberg, Choi, and Chen collectively disclose each and every limitation of instant claim 1.
With regard to claim 2, which recites “said target nucleotide sequence [of the method of claim 1] ranges between 1kb to 10kb,” as set forth above, Chen discloses that Cas9 nucleases, when targeted to specified loci via dual gRNAs, are capable of deleting large segments of genomic DNA, as shown in Figure 1, which depicts deletion of 1,200 bases of genomic sequence (mutant #14 of Figure 1). Thus, it is considered that Chen discloses each and every additional limitation of instant claim 2.
With regard to claim 3, which recites “said insertion nucleotide sequence [of the method of claim 1] has a length of up to 60 bp,” as set forth above, the inserted heterologous object sequence of Steinberg is disclosed to be 10-20, 20-30, 30-40, 40-50, or 50-60 nucleotides in length (page 585, lines 31-33). Thus, it is considered that Steinberg discloses each and every additional limitation of instant claim 3.
With regard to claim 4, which recites “said target nucleotide sequence [of the method of claim 1] is linked to a genetic disease,” as set forth above, per Steinberg, the system taught therein is capable of treating genetic diseases such as tyrosinemia type I (Table 10A) and repeat expansion diseases such as Huntington disease (Table 10C; Table 26). Such treatment is achieved by targeting the FAH gene and CAG repeats within the HTT gene, which are linked to tyrosinemia type I and Huntington disease, respectively (Table 10A; Table 26). Thus, it is considered that Steinberg discloses each and every additional limitation of instant claim 4.
With regard to claim 5, which recites “said genetic disease [of the method of claim 4] is tyrosinemia I,” as set forth above, per Steinberg, the system taught therein is capable of treating genetic diseases such as tyrosinemia type I (Table 10A) by targeting the FAH gene (Table 10A). Thus, it is considered that Steinberg discloses each and every additional limitation of instant claim 5.
Given that Steinberg, Choi, and Chen collectively disclose the method of claim 1, as set forth above, and that Steinberg further discloses that the system taught therein may insert a nucleotide sequence up to 60 nucleotides and may treat genetic diseases such as tyrosinemia I or Huntington disease by targeting the affected loci, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to apply the method collectively disclosed by Steinberg, Choi, and Chen to target loci linked to genetic diseases such as tyrosinemia I and Huntington disease to predictably treat such genetic diseases, as disclosed in Steinberg. One would have been motivated to make such a modification in order to receive the expected benefit of treating genetic diseases such as tyrosinemia I and Huntington disease.
Claims 7-9 and 12-15 are rejected under 35 U.S.C. 103 as being unpatentable over WO 2021/178709 A1 (hereinafter Steinberg; effectively filed 06/05/2020) in view of Choi et al., 2021 (hereinafter Choi; as cited in the IDS filed 09/21/2023).
With regard to claim 7, which recites “a method, comprising:
providing;
a patient exhibiting at least one symptom of a genetic disease; and
a composition comprising a catalytically active Cas9 protein fused to a reverse transcriptase, a first prime editor guide RNA (pegRNA) molecule conjugated to a first reverse transcriptase DNA insertion template and a second prime editor guide RNA molecule conjugated to a second reverse transcriptase DNA insertion template, wherein said first and second reverse transcriptase DNA templates are complementary;
administering said composition to said patient such that said at least one symptom of said genetic disease is reduced,”
while no one source discloses the claimed method in its entirety, the components of this method were known in the art in the art prior to the effective filing date of the instant application such that one of ordinary skill in the art would have been motivated to combine teachings to arrive at the instant invention prior to the effective filing date of the instant application, as set forth below.
Steinberg discloses a genome editing system, said system comprising a nuclease active Cas9 linked to a reverse transcriptase (Figure 5) guided to a target genomic locus (page 593, lines 12-13) by a reverse transcriptase DNA insertion template coupled to a pegRNA (page 585, lines 22-36; page 589, lines 14-15), wherein the heterologous object sequence encoded within said insertion template is reverse transcribed to insert the sequence of interest at the targeted locus (page 586, lines 26-27; page 724, lines 13-15). The inserted heterologous object sequence is disclosed to be 10-20, 20-30, 30-40, 40-50, or 50-60 nucleotides in length (page 585, lines 31-33). Per Steinberg, the system taught therein is capable of treating genetic diseases such as tyrosinemia type I (Table 10A) and repeat expansion diseases such as Huntington disease (Table 10C; Table 26) by administering said system to patients in need thereof to reduce symptom severity (page 722, line 1-page 724, line 25). Thus, while Steinberg discloses a genomic DNA locus comprising a target nucleotide sequence, as well as a composition comprising a catalytically active Cas9 protein fused to a reverse transcriptase and a pegRNA conjugated to a reverse transcriptase DNA insertion template, wherein the reverse transcriptase DNA insertion template is reverse transcribed for insertion into the genome, thereby treating diseases such as tyrosinemia and Huntington disease, Steinberg does not disclose dual pegRNA molecules, nor do they explicitly address any other of the instant claim limitations. These deficiencies are cured by Choi and Chen, as set forth below.
Choi discloses concurrent programming of deletion and insertion using PRIME-Del, wherein a desired short insertion (up to 30 bp) is encoded into a pair of pegRNAs (targeting opposite strands, as instantly claimed) in a reverse complementary manner such that following deletion of the targeted genomic region by Cas9 nicking, the encoded insertion is reverse transcribed into the genome, resulting in a double stranded insertion (lines 153-182; Figure 2), as is also disclosed in Steinberg. Thus, Choi discloses the utility of a first and second pegRNA molecule targeting opposite strands for simultaneous gene deletion and insertion, wherein the templates for insertion are complementary to each other. However, Choi explicitly discloses that these insertions are small (i.e. up to 30 nucleotides), as compared to the large insertions facilitated by the system of Steinberg set forth above.
Thus, Steinberg discloses insertion of large nucleotide sequences via a nuclease-active Cas9 linked to a reverse transcriptase guided to a target genomic locus by a reverse transcriptase DNA insertion template coupled to a pegRNA, wherein the heterologous object sequence encoded within said insertion template is reverse transcribed to insert the sequence of interest at the targeted locus, Choi discloses simultaneous genomic insertion and deletion via a Cas9 nickase and paired pegRNAs with complementary insertion templates, and Chen discloses deletion of large segments of the genome via dual gRNAs targeting Cas9 nuclease to a specified genomic locus.
Thus, it is considered that Steinberg, Choi, and Chen collectively disclose each and every limitation of instant claim 7.
With regard to claims 8 and 9, which respectively recite “said genetic disease [of the method of claim 7-see section Claim Rejections - 35 USC § 112(b)) is tyrosinemia” or “Huntington disease,” as set forth above, per Steinberg, the system taught therein is capable of treating genetic diseases such as tyrosinemia type I (Table 10A) and repeat expansion diseases such as Huntington disease (Table 10C; Table 26). Thus, it is considered that Steinberg discloses each and every additional limitation of instant claims 8 and 9.
Therefore, given that:
Steinberg discloses insertion of large nucleotide sequences via a nuclease-active Cas9 linked to a reverse transcriptase guided to a target genomic locus by a reverse transcriptase DNA insertion template coupled to a pegRNA;
Choi discloses simultaneous genomic insertion and deletion via a Cas9 nickase and paired pegRNAs with complementary insertion templates; and
Steinberg further discloses that the compositions taught therein may be applied to the treatment of genetic diseases such as tyrosinemia I and Huntington disease,
it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to modify the system of Steinberg by substituting the single pegRNA-conjugated reverse transcriptase insertion template taught therein for dual pegRNA-conjugated reverse transcriptase insertion templates, wherein said insertion templates are complementary to each other to predictably facilitate simultaneous insertion and deletion (as disclosed in Choi), for purposes of treating a genetic disease as taught in Steinberg. One would have been motivated to make such a modification in order to receive the expected benefit of treating a genetic disease as taught in Steinberg.
With regard to claim 12, which recites “a composition comprising a catalytically active Cas9 protein fused to a reverse transcriptase, a first prime editor guide RNA (pegRNA) molecule conjugated to a first reverse transcriptase DNA insertion template and a second prime editor guide RNA molecule conjugated to a second reverse transcriptase DNA insertion template, wherein said first and second reverse transcriptase DNA templates are complementary,”
while no one source discloses the claimed composition in its entirety, the components of this composition were known in the art in the art prior to the effective filing date of the instant application such that one of ordinary skill in the art would have been motivated to combine teachings to arrive at the instant invention prior to the effective filing date of the instant application, as set forth below.
Steinberg discloses a genome editing system, said system comprising a nuclease active Cas9 linked to a reverse transcriptase (Figure 5) guided to a target genomic locus (page 593, lines 12-13) by a reverse transcriptase DNA insertion template coupled to a pegRNA (page 585, lines 22-36; page 589, lines 14-15), wherein the heterologous object sequence encoded within said insertion template is reverse transcribed to insert the sequence of interest at the targeted locus (page 586, lines 26-27; page 724, lines 13-15). The inserted heterologous object sequence is disclosed to be 10-20, 20-30, 30-40, 40-50, or 50-60 nucleotides in length (page 585, lines 31-33). Per Steinberg, the system taught therein is capable of treating genetic diseases such as tyrosinemia type I (Table 10A) and repeat expansion diseases such as Huntington disease (Table 10C; Table 26) by administering said system to patients in need thereof to reduce symptom severity (page 722, line 1-page 724, line 25). Thus, while Steinberg discloses a genomic DNA locus comprising a target nucleotide sequence, as well as a composition comprising a catalytically active Cas9 protein fused to a reverse transcriptase and a pegRNA conjugated to a reverse transcriptase DNA insertion template, wherein the reverse transcriptase DNA insertion template is reverse transcribed for insertion into the genome, thereby treating diseases such as tyrosinemia and Huntington disease, Steinberg does not disclose dual pegRNA molecules, nor do they explicitly address any other of the instant claim limitations. These deficiencies are cured by Choi and Chen, as set forth below.
Choi discloses concurrent programming of deletion and insertion using the PRIME-Del system, wherein a desired short insertion (up to 30 bp) is encoded into a pair of pegRNAs (targeting opposite strands, as instantly claimed) in a reverse complementary manner such that following deletion of the targeted genomic region by Cas9 nicking, the encoded insertion is reverse transcribed into the genome, resulting in a double stranded insertion (lines 153-182; Figure 2), as is also disclosed in Steinberg. Thus, Choi discloses the utility of a first and second pegRNA molecule targeting opposite strands for simultaneous gene deletion and insertion, wherein the templates for insertion are complementary to each other. However, Choi explicitly discloses that these insertions are small (i.e. up to 30 nucleotides), as compared to the large insertions facilitated by the system of Steinberg set forth above.
Thus, Steinberg discloses insertion of large nucleotide sequences via a nuclease-active Cas9 linked to a reverse transcriptase guided to a target genomic locus by a reverse transcriptase DNA insertion template coupled to a pegRNA, wherein the heterologous object sequence encoded within said insertion template is reverse transcribed to insert the sequence of interest at the targeted locus, while Choi discloses simultaneous genomic insertion and deletion via a Cas9 nickase and paired pegRNAs with complementary insertion templates.
Thus, it is considered that Steinberg and Choi collectively disclose each and every limitation of instant claim 12.
With regard to claims 13 and 14, which respectively recite “the first reverse transcriptase DNA template is conjugated as a 3’ extension to said first pegRNA molecule,” and “the second reverse transcriptase DNA template is conjugated as a 3’ extension to said first pegRNA molecule,” Steinberg further discloses that the gRNA (i.e. pegRNA per page 589, lines 14-19) of the template RNA taught therein is linked to the template RNA in the following order from 5’ to 3’: (1) a gRNA spacer; (2) a gRNA scaffold; (3) heterologous object sequence; (4) 3’ homology domain (page 585, lines 22-24). Therefore, the reverse transcriptase DNA templates (corresponds to the heterologous object sequence) of Steinberg are conjugated to the 3’ end of the disclosed gRNA (i.e. pegRNA), as instantly claimed. Thus, it is considered that Steinberg discloses each and every additional limitation of instant claims 13 and 14.
With regard to claim 15, which recites “said first and second reverse transcriptase DNA templates [of the composition of claim 12] have a length of up to 60 bp,” as set forth above, the inserted heterologous object sequence of Steinberg is disclosed to be 10-20, 20-30, 30-40, 40-50, or 50-60 nucleotides in length (page 585, lines 31-33). Thus, it is considered that Steinberg discloses each and every additional limitation of instant claim 15.
Therefore, given that:
Steinberg discloses a system for insertion of large nucleotide sequences via a nuclease-active Cas9 linked to a reverse transcriptase guided to a target genomic locus by a reverse transcriptase DNA insertion template coupled to a pegRNA;
Choi discloses a system for simultaneous genomic insertion and deletion via a Cas9 nickase and paired pegRNAs with complementary insertion templates; and
Steinberg further discloses that the compositions taught therein may be applied to the treatment of genetic diseases such as tyrosinemia I and Huntington disease,
it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to modify the system of Steinberg by substituting the single pegRNA-conjugated reverse transcriptase insertion template taught therein for dual pegRNA-conjugated reverse transcriptase insertion templates, wherein said insertion templates are complementary to each other to predictably facilitate simultaneous insertion and deletion (as disclosed in Choi), for purposes of treating a genetic disease as taught in Steinberg. One would have been motivated to make such a modification in order to receive the expected benefit of producing a system/composition that is capable of treating a genetic disease in patients in need thereof (as taught in Steinberg).
Claim 6 is rejected under 35 U.S.C. 103 as being unpatentable over WO 2021/178709 A1 (hereinafter Steinberg; effectively filed 06/05/2020) in view of Choi et al., 2021 (hereinafter Choi; as cited in the IDS filed 09/21/2023), Chen et al., 2014 (hereinafter Chen) as applied to claims 1 and 4 above, and further in view of Orejuela et al., 2008 (hereinafter Orejuela).
The combined disclosures of Steinberg, Choi, and Chen are described above and applied as before. However, these disclosures do not teach the FahΔExon 5 mutation of instant claim 6.
With regard to claim 6, which recites “said target nucleotide sequence [of the method of claim 4] comprises a FahΔExon 5 mutation,” as set forth above, per Steinberg, the system taught therein is capable of treating genetic diseases such as tyrosinemia type I (Table 10A) by targeting the FAH gene (Table 10A). However, Steinberg does not specify that the targeted FAH gene comprises an FahΔExon 5 mutation. This deficiency is cured by Orejuela.
Orejuela discloses a murine fah-/- knockout model of tyrosinemia type I, which is caused by a deficiency in fumarylacetoacetate hydrolase (FAH) (abstract; page 308, column 1, paragraph 1-page 308, column 2, paragraph 1). Said knockout model is homozygous FAH deficient mouse model FahΔExon 5, fah-/- (page 309, column 1, paragraph 2), which comprises an FahΔExon 5 mutation, as instantly claimed. Thus, it is considered that Orejuela discloses each and every additional limitation of instant claim 6.
Given that Steinberg, Choi, and Chen collectively disclose the method of instant claims 1 and 4, wherein the editing composition is targeted to a nucleotide sequence linked to a genetic disease such as tyrosinemia type I, and that Orejuela discloses a murine fah-/- knockout model of tyrosinemia type I comprising an FahΔExon 5 mutation, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to target the editing composition to the Fah locus to correct an FahΔExon 5 mutation causing tyrosinemia type I (as disclosed in Orejuela) to predictably treat tyrosinemia type I (as disclosed in Steinberg). One would have been motivated to make such a modification in order to receive the expected benefit of treating tyrosinemia type I.
Claims 10 and 11 are rejected under 35 U.S.C. 103 as being unpatentable over WO 2021/178709 A1 (hereinafter Steinberg; effectively filed 06/05/2020) in view of Choi et al., 2021 (hereinafter Choi; as cited in the IDS filed 09/21/2023) as applied to claim 7 above (see section Claim Rejections - 35 USC § 112(b)), and further in view of Chen et al., 2005 (hereinafter Jian-Min Chen; as cited in the IDS filed 09/21/2023), as evidenced by Chen et al., 2014 (hereinafter Chen).
The combined disclosures of Steinberg and Choi are described above and applied as before. However, these disclosures do not teach the gene mutation insertion and replacement thereof of instant claims 10 and 11.
With regard to claim 10, which recites “said patient [of the method of claim 7-see section Claim Rejections - 35 USC § 112(b)] further comprises a gene mutation insertion between 1kb-10kb,” as set forth above, per Steinberg, the system taught therein is capable of treating genetic diseases such as tyrosinemia type I (Table 10A) and repeat expansion diseases such as Huntington disease (Table 10C; Table 26). Such treatment is achieved by targeting the FAH gene and CAG repeats within the HTT gene, which are linked to tyrosinemia type I and Huntington disease, respectively (Table 10A; Table 26). Thus, while Steinberg discloses targeting genes and mutations thereof associated with diseases for purposes of treating said diseases (page 591, lines 25-29), Steinberg does not disclose that said mutations comprise an insertion between 1kb-10kb.
This deficiency is cured by Jian-Min Chen, which respectively discloses that LINE-1 retrotransposons mediate retrotranspositional events that cause human genetic disease (abstract; Table 2). As shown in Table 2 of Jian-Min Chen, mutative retrotranspositional events elicited by LINE-1 retrotransposons causing human genetic disease frequently insert sequences from 1kb-10kb (see disorders Choroideremia, CGD, DMD, Haemophilia A, β-Thalassemia), as instantly claimed. Thus, it is considered that Jian-Min Chen discloses each and every additional limitation of instant claim 10.
Furthermore, and as set forth above, Chen teaches that dual gRNAs are capable of deleting large segments of genomic DNA (Figure 1), including large chromosomal segments of at least 8.5 kb or 16.5 kb (Figure 4).
With regard to claim 11, which recites “said administering [of the method of claim 10] replaces said gene mutation insertion with an insertion nucleotide sequence that has a length of up to 60bp,” as set forth above, the inserted heterologous object sequence of Steinberg is disclosed to be 10-20, 20-30, 30-40, 40-50, or 50-60 nucleotides in length (page 585, lines 31-33). Thus, it is considered that Steinberg discloses each and every additional limitation of instant claim 11.
Given that Steinberg and Choi collectively disclose the method of instant claim 7, wherein the editing composition is provided to a patient exhibiting at least one symptom of a genetic disease and targeted to a nucleotide sequence linked to said genetic disease and that Jian-Min Chen discloses that LINE-1 retrotransposons causing human genetic disease frequently insert sequences from 1kb-10kb, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to target the editing composition to a LINE-1 retrotransposon disease-causing insertion to predictably simultaneously delete the insertion (as taught in Chen) and insert the correct sequence (as disclosed in Steinberg and Choi), thereby treating the LINE-1 retrotransposon disease. One would have been motivated to make such a modification in order to receive the expected benefit of treating the LINE-1 retrotransposon disease.
Conclusion
No claims are allowed.
Claims 1, 2, 6, 7, 10, and 12 are objected to.
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/SARAH E ALLEN/ Examiner, Art Unit 1637
/J. E. ANGELL/ Primary Examiner, Art Unit 1637
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