DETAILED ACTION
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Applicant’s Response to Election/Restriction Filed, Amendment, and Arguments/Remarks, filed 15 September 2025, have been entered. Claims 1-39, 41-43, 69, 75, 77, 79, and 83 are currently pending. Claims 1, 39, 41, 43, 69, 75, 77, 79, and 83 are independent claims.
Applicant’s election without traverse of the invention of Group I, drawn to a non-human animal, a non-human animal cell, a nucleic acid, a targeting vector for generating a humanized endogenous TTR locus, a method of assessing the activity of a human-TTR-targeting reagent in vivo, and a method of optimizing the activity of a human-TTR-targeting reagent in vivo, is acknowledged.
Additionally, Applicant’s election of the following species:
TTR 3’ UTRs: a. human
Signal peptides: a. human
TTR locus sequences: a. SEQ ID NO: 24
without traverse in a reply filed 15 September 2025 is acknowledged. Upon further consideration, Examiner has withdrawn the election requirement for 1) TTR 3’ UTRs as examination of all species together does not represent undue burden.
Claims 75, 77, 79, and 83 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Claims 10 and 17-22 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected species, there being no allowable generic or linking claim. The requirement is still deemed proper and is therefore made FINAL.
Claims 1-9, 11-16, 23-39, 41-43, and 69 are currently pending in the application and under examination to which the following grounds of rejection are applicable. Claim 16 is examined to the extent that it reads on the elected species. An action on the merits follows.
Priority
The present application is a 35 U.S.C. 371 national stage filing of International Application No. PCT/US2021/023674, filed 23 March 2021, which claims priority to U.S. Provisional Application No. 62/993,289, filed 23 March 2020.
Thus, the earliest possible priority for the instant application is 23 March 2020.
Information Disclosure Statement
The information disclosure statements filed 22 February 2023 and 15 September 2025 have been considered by the Examiner. Examiner notes the filing of IDS Size Fee assertions for the IDS filed 15 September 2025, as required under 37 CFR 1.98, indicating that no IDS size fee is required under 37 CFR 1.17(v) at this time.
Claim Objections
Claims 1-9, 11, 13-16, 23-25, 29, 36-39, 41-43, and 69 are objected to because of the following informalities: claims 1-9, 11, 13-16, 23-25, 29, 36-37, 39, 41-43, and 69 each recite the abbreviation “TTR” without first writing out the term for which “TTR” is an abbreviation. Additionally, claim 38 recites the abbreviations “VP64” and “HSF1” without first writing out the term for which “VP64” and “HSF1” is an abbreviation. Appropriate correction is required.
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, 13, 16, 29, 33-34, 36-37, 38, and 69 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.
Claim 8 is indefinite in its recitation of “wherein the endogenous TTR 5’ untranslated region has not been deleted and replaced with the corresponding human TTR sequence” because t is unclear if the corresponding human TTR sequence the same corresponding human TTR sequence of claim 1 or whether the corresponding human TTR sequence is the 5’ UTR of the human TTR gene. As such, the metes and bounds of the claim cannot be determined.
Claim 13 is indefinite in its recitation of “the entire TTR coding sequence of the endogenous TTR locus has been deleted and replaced with the corresponding human TTR sequence”, because it is unclear if the corresponding human TTR sequence is also the entire coding sequence or a domain of said sequence. The metes and bounds of the claim are indefinite.
Claim 16 recites the limitation "the human TTR sequence" in line 3. There is insufficient antecedent basis for this limitation in the claim. Claim 1 recited “the corresponding human TTR sequence”. As such, the metes and bounds of the claim cannot be determined.
Claim 29 recites the limitation "the humanized endogenous TTR" in lines 2-3. There is insufficient antecedent basis for this limitation in the claim. As such, the metes and bounds of the claim cannot be determined. In the interest of compact prosecution, claim 29 has been interpreted to refer to “the humanized endogenous TTR locus” recited in claim 1.
Claim 33 recites the limitation "the liver, the lung, the heart, the spleen, the kidney, or any combination thereof of the non-human animal" in lines 2-3. There is insufficient antecedent basis for this limitation in the claim. Livers, lung, heart, spleens, kidneys, or combinations thereof are not necessarily inherent parts of any or all non-human animals. As such, the metes and bounds of the claim cannot be determined.
Claims 36 and 37 are vague and indefinite in the recitation of “…capable of…” in lines 5 and 5, respectively, since this phrase refers to a latent ability, and it is unknown whether the ability is expressed or observed in the invention. Note, it has been held that the recitation that an element is “capable of” performing a function is not a positive limitation, but only requires the ability to so perform. It does not constitute a limitation in any patentable sense. In re Hutchinson, 69 USPQ 138. As such, the metes and bounds of the claims cannot be determined.
Claim 38 has multiple issues of indefiniteness. Claim 38 recites the limitation "the first expression cassette" in line 2. Claim 35 recites “expression cassette”. There is insufficient antecedent basis for this limitation in the claim.
Claim 38 recites “optimally aligned” The term " optimally " in claim 38 is a relative term which renders the claim indefinite. The term " optimally " 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. Appropriate action is required.
Claim 38 recites “specifically bind” in line 14. The term " specifically " in claim 38 is a relative term which renders the claim indefinite. The term " specifically " 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. Appropriate action is required.
Additionally, claim 38 recites, “the adaptor protein comprises an MS2 coat protein or a functional fragment or variant thereof”. It is unclear what is encompassed by a “functional fragment or variant thereof”. The specification does not provide a standard for ascertaining the scope of a “functional fragment or variant thereof”. The specification recites:
The term "functional" refers to the innate ability of a protein or nucleic acid (or a fragment or variant thereof) to exhibit a biological activity or function. The biological functions of functional fragments or variants may be the same or may in fact be changed (e.g., with respect to their specificity or selectivity or efficacy) in comparison to the original molecule, but with retention of the molecule's basic biological function. [0097] The term "variant" refers to a nucleotide sequence differing from the sequence most prevalent in a population (e.g., by one nucleotide) or a protein sequence different from the sequence most prevalent in a population (e.g., by one amino acid). [0098] The term "fragment," when referring to a protein, means a protein that is shorter or has fewer amino acids than the full-length protein. The term "fragment," when referring to a nucleic acid, means a nucleic acid that is shorter or has fewer nucleotides than the full-length nucleic acid. [0096-0098, emphasis added].
However, neither the specification nor the claims provide teachings which would indicate the metes and bounds of a fragments or variants of the MS2 coat protein which would retain the basic biological function of an adaptor protein.
Claim 38 further recites the limitations "the target sequence” and “the transcription start site" in lines 18, 18-19, and 19. There is insufficient antecedent basis for these limitations in the claim.
As such, the metes and bounds of claim 38 cannot be determined.
Claim 69 recites, “performing the method of step (I) a second time”, which is indefinite because the method of step (I) is performing the method of claim 43 a first time, and so it is unclear how one can perform a method a first time for a second time. As such, the metes and bounds of the claim cannot be determined.
Claim 34 is included in this rejection due to its dependence on claim 33.
Claim Interpretation
Claim 12 recites the term “optionally” in line 3, which has been afforded its broadest reasonable interpretation such that the limitations following the term “optionally” are not required limitations of the claim.
Also note that the term “corresponding human TTR sequence” has been afforded its broadest reasonable interpretation to include any sequence of the human TTR gene locus which otherwise meets the explicit limitations of the claim (e.g., comprising both a TTR exonic sequence and a TTR intronic sequence as recited in claim 1).
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.
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 1-9, 11-16, 23-39, 41-43, and 69 are rejected under 35 U.S.C. 103 as being unpatentable over Zhao et al. [2008, Genes to Cells, 13, 1257-1268, IDS]; in view of Devoy et al. [2012, Nature Reviews Genetics, 13, 14-20]; Yu et al. [US20130316366A1, published 28 November 2013]; NCBI [2002, Homo sapiens BAC clone RP11-549B18 from 18, complete sequence, GenBank: AC017100.4, retrieved on 04 January 2026 from: <https://www.ncbi.nlm.nih.gov/nuccore/AC017100.4>, updated 25 January 2002]; Nagata et al. [1995, Journal of Biochemistry, 117(1), 169-175, IDS]; Saelices et al. [2018, Proceedings of the National Academy of Sciences, 115(29), E6741-6750, IDS]; Wei et al. [2004, Amyloid: The Journal of Protein Folding Disorders, 11, 113-120, IDS]; Butler et al. [2016, Amyloid: The Journal of Protein Folding Disorders, 23(2), 109-118]; Konermann et al. [2015, Nature, 517, 583-588]; and Chu et al. [2016, BMC Biotechnology, 16(4), 1-15].
Note that, as discussed above, “corresponding human TTR sequence” has been afforded its broadest reasonable interpretation to include any sequence of the human TTR gene locus which otherwise meets the explicit limitations of the claim (e.g., comprising both a TTR exonic sequence and a TTR intronic sequence as recited in claim 1).
Regarding independent claim 1, Zhao teaches a non-human animal comprising in its genome a humanized endogenous TTR locus in which a region of the endogenous TTR locus comprising a TTR exonic sequence has been deleted and replaced with a corresponding human TTR sequence comprising a TTR exonic sequence, wherein the humanized endogenous TTR locus comprises a V30M mutation [abstract, Figure 1].
Zhao does not teach that the region of the endogenous TTR locus which has been deleted comprises a mouse TTR intronic sequence nor that the corresponding human TTR sequence comprises a human TTR intronic sequence.
Devoy teaches that the most precise method of humanization of mice is by targeted integration of a human sequence into the equivalent region of the mouse genome in ESCs, enabling a single copy of human sequence to reside at a natural site for its expression while simultaneously replacing the corresponding mouse sequence [column 4 ¶ 1]. Devoy additionally teaches that targeted genomic replacement traditionally has involved small size changes to replace some or all of the exons and introns of a mouse gene with the corresponding human sequence, allowing the human sequence to remain under the control of mouse transcriptional regulatory sequences [column 4 ¶ 1-2]. Devoy further teaches that replacements are now possible in which an entire mouse locus, including non-coding upstream and downstream sequences, is substituted by equivalent human sequences [column 4 ¶ 2]. Devoy also teaches that genomic humanization is necessary to investigate the functional importance of non-coding regions and therefore to model aneuploidy fully, to study disorders in which species-specific splicing patterns have a role, or to determine the functions of untranslated sequences; and that the non-coding genome must be taken into account in studying gene function such that genomic humanization will be essential to create an optimal set of models of human disease for both understanding pathogenic processes and for developing therapies such as gene therapies [column 11 ¶ 1, Box 1 ¶ 5-7].
Therefore, given the teachings of Devoy to replace some or all of the mouse exons and intron, up to the full genomic locus, and the teachings of Devoy of the importance of including the non-coding sequences (e.g., introns) along with the coding sequences in the humanization of mouse genomic loci, an ordinarily skilled artisan at the time of filing the instant application would have been motivated to replace exonic and intronic sequences of the mouse TTR gene locus with exonic and intronic human sequence from the human TTR gene in view of the benefits of generating non-human animals comprising human non-coding sequences in studying gene function, such as to generate appropriate splicing patterns or to study to role of noncoding sequences.
Regarding claim 2, Zhao teaches that the human TTR sequences comprises the V30M mutation [abstract, column 4 ¶ 3, Figure 1].
Regarding claim 3, Zhao teaches wherein the humanized endogenous TTR locus comprises an endogenous TTR promoter, wherein the human TTR sequence is operably linked to the endogenous TTR promoter [column 15 ¶ 1, Figure 1].
Regarding claim 4, Zhao and Devoy teach a non-human animal according to claim 1. As discussed above, Devoy teaches the replacement of some of all of the exons and introns of a mouse gene, up to an entire genomic locus, for the humanization of mouse genes to create optimal models of human disease [column 4 ¶ 2, Box 1 ¶ 5-7].
Regarding claims 5-7, Zhao teaches wherein the humanized endogenous TTR locus comprises a human TTR 3’ untranslated region (e.g., the full human cDNA sequence) and an endogenous mouse TTR 3’ untranslated region (e.g., all of exons 2-4) [column 17 ¶ 2, Figure 1].
Regarding claim 8, Zhao teaches wherein the endogenous TTR 5’ untranslated region has not been deleted and replaced with the corresponding human TTR sequence [column 17 ¶ 1, Figure 1].
Regarding claim 9, Zhao teaches wherein the humanized endogenous TTR locus encodes a transthyretin precursor protein comprising a human mature transthyretin protein sequence (e.g., comprises the full human cDNA sequence), [column 17 ¶ 2, Figure 1, 3, 4].
Regarding claim 11, Zhao does not explicitly teach wherein the humanized endogenous TTR locus encodes a transthyretin precursor protein comprising a human transthyretin signal peptide sequence. However, Zhao implicitly teaches wherein the humanized endogenous TTR locus encodes a transthyretin precursor protein comprising a human transthyretin signal peptide sequence by teaching that the humanized endogenous TTR locus comprises the full human cDNA sequence. Zhao further supports the implicit teaching that the humanized endogenous TTR locus encodes a transthyretin precursor protein comprising a human transthyretin signal peptide sequence by teaching the efficient trafficking of the hTTR through the normal protein secretory pathway (e.g., through the ER and Golgi) [column 9 ¶ 2- column 10 ¶ 1, column 17 ¶ 2, Figure 1].
Regarding claim 12, Zhao and Devoy teach a non-human animal according to claim 1, including that the humanized endogenous TTR locus encodes the complete coding sequence for a human transthyretin V30M protein. As discussed above, Zhao implicitly teaches that the complete coding sequence for the human transthyretin V30M protein includes the human signal peptide sequence.
Zhao and Devoy do not explicitly teach wherein the human transthyretin signal peptide sequence comprises the sequence set forth in instant SEQ ID NO: 3.
However, Yu teaches the sequence of a native transthyretin signal sequence, which is 100% identical to the sequence set forth in instant SEQ ID NO: 3 [SEQ ID NO: 324, 0039, Table 3]:
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Therefore, by teaching that the humanized endogenous TTR locus comprises the entire hTTR cDNA encoding the complete coding sequence for a human transthyretin V30M protein, Zhao is teaching that the humanized endogenous TTR locus comprises a signal peptide sequence as set forth in instant SEQ ID NO: 3. Additionally, Yu further teaches that the presence of a signal sequence on a polypeptide can mediate the translocation of the polypeptide to the cell’s endoplasmic reticulum and subsequent transport through the Golgi to the cell membrane/ extracellular space [0006]. As such, given the teachings of Zhao to express the full cDNA sequence of human TTR V30M, the teachings of Zhao that the hTTR V30M protein is secreted from the cell, the teachings of Devoy to replace some or all of the mouse gene with the full human gene to humanize the mouse locus and maintain the human regulatory sequences, and the teachings of Yu that the signal peptide facilitates the transport of the protein through the secretory pathway, an ordinarily skilled artisan would have been motivated to include the native hTTR signal peptide sequence according to instant SEQ ID NO: 3 (having 100% identity to the signal peptide of SEQ ID NO: 324 taught by Yu) in a humanized endogenous TTR locus to retain the human regulatory sequences.
Regarding claims 13-14, Zhao and Devoy teach a non-human animal according to claim 1. As discussed above, Devoy teaches to replace the entire mouse locus for precise humanization to replace all of the exons and introns of a mouse gene with the corresponding human sequence [column 4 ¶ 1-2, Box 1 ¶ 5-7, column 11 ¶ 1]. Therefore, Devoy teaches to delete and replace the entire coding sequence of a mouse gene with the corresponding human gene sequence and to delete and replace a region of the endogenous moue locus from the start codon to the stop codon with the corresponding human gene sequence. As such, given the motivations taught by Devoy to humanize the mouse locus to both study the pathogenic mechanisms associated with the human gene as well as to develop therapies, an ordinarily skilled artisan would have been motivated to delete the full endogenous mouse TTR coding sequence, such as deleting a region of the endogenous mouse TTR locus from the TTR start codon to the TTR stop codon, and to replace it with the corresponding human TTR sequence.
Regarding claim 15, Zhao and Devoy teach a non-human animal according to claims 1 and 13-14. As discussed above, Zhao teaches that the human TTR sequence comprises the full hTTR V30M cDNA sequence, which necessarily includes the human TTR 3’ untranslated region (3’UTR). Additionally, as discussed above, Zhao teaches a humanized endogenous TTR locus wherein the endogenous TTR 5’UTR has not been deleted and replaced with the human TTR sequence, wherein the humanized endogenous TTR locus comprises an endogenous TTR promoter, and wherein the humanized endogenous TTR locus comprises an endogenous TTR promoter, wherein the human TTR sequence is operably linked to the endogenous TTR promoter [column 15 ¶ 1, Figure 1].
Regarding claim 16, Zhao and Devoy teach a non-human animal according to claim 1. Zhao teaches that the non-human animal comprises in its genome a humanized endogenous TTR locus, wherein the humanized endogenous TTR locus comprises the full cDNA sequence of human TTR V30M [Figure 1].
Zhao and Devoy do not explicitly teach wherein the human TTR sequence at the humanized TTR locus comprises a sequence at least 90% identical to the sequence set forth in instant SEQ ID NO: 24, as elected.
However, NCBI teaches a native human TTR genomic sequence (e.g., BAC clone RP11-549B18 from chromosome 18, sequence ID: AC017100.4, nucleotides 143,625-136,207) which is 99.99% identical to instant SEQ ID NO: 24, wherein the only mismatch between instant SEQ ID NO: 24 and the sequence disclosed by NCBI is an G to A mutation which encodes the V30M amino acid substitution:
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Therefore, given the teachings and motivation of Zhao and Devoy discussed above to construct a humanized endogenous TTR locus comprising a full human TTR genomic sequence, including both exons and introns, wherein the hTTR sequence encodes a human TTR comprising a V30M mutation, an ordinarily skilled artisan would have been motivated to use the native sequence taught by NCBI, modified to comprise the V30M mutation, as a human TTR sequence for humanizing a non-human animal endogenous TTR locus.
Regarding claim 23, Zhao and Devoy teach a non-human animal according to claim 1. Zhao teaches a humanized endogenous TTR locus comprising a selection cassette. Devoy teaches that a step to delete the selection maker is usually desirable in targeted genomic replacement for humanization of mouse genes [column 4 ¶ 3, Figure 1]. Therefore, given the teachings of Devoy that removal of selection markers is desirable, an ordinarily skilled artisan at the time of filing the instant application would have been motivated to remove a selection marker, thereby producing a humanized endogenous TTR locus lacking a selection cassette.
Regarding claim 24, Zhao teaches wherein the non-human animal is homozygous for the humanized endogenous TTR locus [column 7 ¶ 2, column 10 ¶ 2, Figure 3, 5].
Regarding claim 25, Zhao teaches wherein the non-human animal comprises the humanized endogenous TTR locus in its germline [column 5 ¶ 1].
Regarding claims 26-28, Zhao teaches wherein the non-human animal is a mammal, specifically a mouse [abstract].
Regarding claim 29, Zhao teaches relative serum levels for the human TTR Val30 mice [abstract, column 4 ¶ 2, Figure 3, 4], but not absolute serum levels.
Nagata teaches a mouse transgenic for human TTR V30M which exhibits quantitatively normal expression, wherein the serum levels of hTTR V30M ranged from 0 to 17.1 mg/dl (e.g., 0 to 171 µg/ml), with five of the 8 mouse lines exhibiting serum levels higher than the average level of 10 mg/dl (e.g., higher than 100 µg/ml) found in FAB patients, and wherein the serum level was not correlated to copy number [abstract, column 5 ¶ 1, Figure 2, 6]. Therefore, an ordinarily skilled artisan at the time of filing the instant application would expect to achieve a mouse with serum levels of hTTR V30M protein expressed from the humanized endogenous TTR to be at least about 20 µg/ml (e.g., higher than 100 µg/ml).
Regarding claims 30 and 32, Zhao and Devoy teach a non-human animal according to claim 1.
Zhao and Devoy do not teach wherein the non-human animal has been seeded with exogenous, pre-formed transthyretin aggregates or fibrils.
However, Saelices teaches that in vivo amyloid seeding has been proposed to be the mechanism of spreading from cell to cell of amyloid fibrils associated with neurodegenerative diseases, and that despite clinical evidence of amyloid seeding of TTR in vivo, demonstration of TTR amyloid seeding has remained elusive in the laboratory [column 9 ¶ 2- column 10 ¶ 2]. Saelices teaches the seeding of amyloid fibrils ex vivo using human patient-extracted TTR seeds such that once TTR-amyloid (ATTR) forms amyloid fibrils, the fibrils have the capacity to convert wild-type or variant TTR into additional amyloid fibrils upon TTR dissociation [column 11 ¶ 2]. Saelices also teaches that it is important to know if the potent seeding potential of patient-extracted seeds operates under physiological conditions that more accurately represent in vivo environments [column 11 ¶ 2]. Saelices further teaches that other laboratories have injected ex vivo ATTR fibrils into transgenic mouse models, but have not observed acceleration of TTR deposition in vivo [column 12 ¶ 1]. Saelices suggests that the reason for the lack of observed acceleration of TTR deposition is unknown, but may be related to those studies including only a limited number of animals and conditions, thereby suggesting the need for additional studies investigating the potential of ex vivo patient-extracted fibrils to seed TTR deposition in a mouse model.
Therefore, an ordinarily skilled artisan at the time of filing the instant application would have been motivated to seed an hTTR mouse model with pre-formed TTR aggregates and/or fibrils (e.g., ex vivo patient-derived TTR fibrils) to study the seeding of TTR amyloid fibrils and assess potential acceleration of TTR deposition.
Regarding claims 31 and 33-34, Zhao, Devoy, and Saelices teach a non-human animal according to claim 30.
Zhao, Devoy, and Saelices do not explicitly teach wherein the exogenous, pre-formed transthyretin aggregates or fibrils comprise a V30M mutation.
However, Saelices cites Wei as one of the other laboratories which injected ex vivo ATTR fibrils into transgenic mouse models [column 12 ¶ 1]. Wei teaches the administration of TTR-amyloid fibrils (ATTR) extracted from the heart of an FAB TTR V30M patient to transgenic mice expressing the human mutant TTR gene responsible for FAP TTR V30M [abstract, column 4 ¶ 1]. Wei also teaches that FAP is a fatal autosomal dominantly inherited amyloidosis characterized by systemic deposition of amyloid mainly composed of variant TTR with single amino acid substitutions, the most common of which is V30M [column 3 ¶ 3]. Wei further teaches that one possible mechanism responsible for differences in mean age at onset and anticipation in FAP may be administration of TTR-amyloid fibrils (ATTR) from affected mothers through breast-feeding, but that acceleration of ATTR deposition by ATTR had not been investigated in vivo; therefore, Wei teaches the motivation to administer ATTR V30M extracted from an FAP patient to transgenic mice carrying and expressing the human mutant TTR gene responsible for this form of FAP is to test whether such seeding would accelerate human ATTR V30M deposition [column 4 ¶ 1]. As such, an ordinarily skilled artisan would have been motivated to use exogenous, pre-formed transthyretin aggregates or fibrils which comprise a V30M mutation for seeding ATTR deposition in a humanized TTR V30M mouse model.
Wei additionally teaches amyloid deposits were observed in the various tissues (e.g., heart, liver, and small intestine) of all five transgenic mice injected with ATTR, whereas no deposits were detected in any of the five control transgenic littermates injected with distilled water, indicating that ATTR extracted from the heart of an FAP type I patient exerts amyloid-enhancing activity in vivo [column 9 ¶ 3, Table 1, Figure 2]. Although Wei teaches that the amyloid deposits reacted only with anti-mouse AApoAII and not anti-human TTR [column 9 ¶ 3, Figure 3, Table 1], the teaching that the seeded ATTR exerted amyloid-enhancing activity teaches that the ATTR seeds at least were present in the tissues comprising the observed amyloid deposits.
Regarding claim 35, Zhao and Devoy teach a non-human animal according to claim 1. Zhao further teaches wherein the non-human animal further comprises in its genome a genomically integrated expression cassette, wherein the genomically integrated expression cassette comprises a nucleic acid encoding a puromycin resistance gene. Additionally, Devoy teaches that additive multi-copy transgenics will continue to be necessary for modeling disorders because some late-onset neurodegenerative diseases require the expression of human sequences to attain a threshold level to manifest a phenotype [column 12 ¶ 1]. Therefore, Devoy is teaching a need for expression levels to be increased above what is obtained by humanization of the endogenous locus for some late-onset neurodegenerative diseases.
Zhao and Devoy do not explicitly teach a motivation for increasing the expression of the hTTR V30M from the humanized mouse locus.
However, Butler teaches that RNAi-mediated silencing of hepatic TTR expression inhibited both deposition and facilitated regression of existing TTR deposits in pathologically relevant tissues, such that the extent of TTR deposit regression correlated with the level of RNAi-mediated knockdown of TTR in a murine model of hereditary ATTR amyloidosis (e.g., hTTR V30M HSF1+/- mice) [abstract, column 5 ¶ 2-column 6 ¶ 2, column 8 ¶ 2-column 9 ¶ 1, Figure 3], thereby indicating that the level of TTR expression correlates with the disease burden in that higher levels of TTR expression result in higher levels of TTR deposits.
Given the teachings of Devoy that some late onset neurodegenerative diseases require expression levels above those obtained from humanized loci and the teachings of Butler that increased levels of hTTR V30M result in increased hTTR amyloid deposition, an ordinarily skilled artisan would have been motivated to increase the expression of the hTTR from the humanized TTR locus to increase the hTTR amyloid deposition in the humanized mouse to thereby increase the disease phenotype for modeling FAP.
Zhao, Devoy, and Butler do not teach wherein the genomically integrated expression cassette comprises: a) a nucleic acid encoding a chimeric Cas protein comprising a nuclease-inactive Cas protein fused to one or more transcriptional activation domains; and b) a nucleic acid encoding a chimeric adaptor protein comprising an adaptor protein fused to one or more transcriptional activation domains.
However, Konermann teaches cells with a genomically integrated expression cassette comprising both a) a nucleic acid encoding a chimeric Cas protein comprising a nuclease-inactive Cas protein fused to one or more transcriptional activation domains; and b) a nucleic acid encoding a chimeric adaptor protein comprising an adaptor protein fused to one or more transcriptional activation domains [column 11 ¶ 7, column 13 ¶ 4, Figure 1, 5, ED7]. Konermann further teaches that the combination of sgRNA-2xMS2, NLS-dCas9-VP64, and MS2-p65-HSF1, which they named “synergistic activation mediator (SAM)”, comprises the most effective transcription activation system among the many iterations they tested for activation of endogenous genomic loci [abstract, column 2 ¶ 2, column 4 ¶ 3].
Therefore, an ordinarily skilled artisan at the time of filing the instant application would have been motivated to use a genomically integrated expression cassette comprising a) a nucleic acid encoding a chimeric Cas protein comprising a nuclease-inactive Cas protein fused to one or more transcriptional activation domains (e.g., dCas9-VP64) and b) a nucleic acid encoding a chimeric adaptor protein comprising an adaptor protein fused to one or more transcriptional activation domains (e.g., MS2-p65-HSF1) to increase the expression of hTTR V30M from the humanized TTR locus to increase the hTTR amyloid deposition in the humanized mouse to thereby increase the disease phenotype for modeling FAP.
Regarding claim 36, Konermann further teaches that the SAM system for endogenous gene activation further comprises one or more guide RNAs or an expression cassette that encodes the one or more guide RNAs, each guide RNA comprising one or more adaptor-binding elements (e.g., MS2 stem loops) to which the chimeric adaptor protein (e.g., MS2 protein) can specifically bind, wherein each of the one or more guide RNAs is capable of forming a complex with the Cas protein and guiding it to a target sequence within a target gene [Figure 1, 5]. Konermann also teaches that sgRNAs are designed to target specific genes to direct dCas9 binding to target genes for the activation of those genes [Figure 2, 3]. Therefore, in using the SAM system to increase the hTTR V30M amyloid deposition in the humanized mouse, an ordinarily skilled artisan would necessarily design at least one of the guide RNAs to target the humanized endogenous TTR locus expressing the hTTR V30M for activation of the encoding gene.
Regarding claim 37, Zhao, Devoy, Butler, and Konermann teach the limitations of claims 35-36. Konermann also teaches wherein the expression cassette that encodes the one or more guide RNAs is genomically integrated (e.g., delivered via lentiviral delivery with puromycin or zeocin selection, which obtains cells wherein the lentiviral genome comprising the guide RNA expression cassette is integrated into the host cell genome) [Figure 4].
Regarding claim 38, as discussed above, Konermann teaches wherein the Cas protein is a dCas9 protein comprising D10A and N863A mutations [column 2 ¶ 3], the one or more transcriptional activator domains in the chimeric Cas protein comprises VP64 [column 4 ¶ 3, Figure 1], the adaptor protein comprises an MS2 coat protein [column 4 ¶ 3, Figure 1], the one or more transcriptional activator domains in the chimeric adaptor protein comprise p65 and HSF1 [column 4 ¶ 3, Figure 1], the non-human animal further comprises one or more guide RNAs or an expression cassette that encodes the one or more guide RNAs [column 4 ¶ 3, Figure 1], and wherein each of the one or more guide RNAs comprises two adaptor-binding elements to which the chimeric adaptor protein can specifically bind [column 4 ¶ 3, Figure 1]. Konermann further teaches wherein the two adaptor-binding elements comprise a first adaptor binding element within a first loop (e.g., tetraloop) of each of the one or more guide RNAs and a second adaptor binding element within a second loop (e.g. stem loop 2) of each of the one or more guide RNAs [Figure 1]. Konermann also teaches wherein the target sequence is within a region 200 base pairs upstream of the transcription start site and 1 base pair downstream of the transcription start site of each target gene, which region was previously determined to provide more efficient activation [column 7 ¶ 3].
Additionally, Devoy teaches the knock-in of genes into the murine Rosa26 locus, and that the murine Rosa26 locus is a ubiquitously expressed locus [column 11 ¶ 1].
Further, Chu teaches that the Rosa26 locus on chromosome 6 is frequently used for the integration of transgene constructs to achieve ubiquitous or conditional gene expression in mice for inserting single transgene copies in a standardized configuration into the mouse genome [column 1 ¶ 1-2]. Therefore, an ordinarily skilled artisan at the time of filing the instant application would have been motivated to insert an expression cassette into the Rosa26 locus for inserting single transgene copies in a standardized configuration into the mouse genome.
Regarding claim 39, Zhao and Devoy teach a non-human animal according to claim 1. Additionally, Zhao teaches a non-human animal cell comprising in its genome the humanized endogenous TTR locus [column 7 ¶ 1, column 17 ¶ 3-4, Figure 3].
Regarding claim 41, Zhao and Devoy teach a non-human animal according to claim 1. Additionally, Zhao teaches a nucleic acid comprising the humanized non-human animal TTR gene described above, e.g., the genome of a cell of the non-human animal comprising in its genome the humanized endogenous TTR locus [Figure 1].
Regarding claim 42, Zhao and Devoy teach a non-human animal according to claim 1. Zhao further teaches a targeting vector for generating the humanized endogenous TTR locus wherein the targeting vector comprises an insert sequence (e.g., PGK-neo) flanked by a 5’ homology arm targeting a 5’ target sequence at the endogenous TTR locus and a 3’ homology arm targeting a 3’ target sequence at the endogenous TTR locus [Figure 1]. Zhao also teaches a replacement vector comprising an insert nucleic acid comprising the V30M mutation and the corresponding human TTR sequence for replacing the PGK-neo insert sequence with the human TTR V30M sequence [Figure 1]. Zhao does not specifically teach that the 5’ homology arm and the 3’ homology arm flank the corresponding human TTR V30M sequence. However, Devoy teaches humanization of a mouse locus using BAC vector homologous recombination for targeted genomic replacement, wherein genomic replacements can be achieved by homologous recombination with a hybrid BAC vector, such that long regions of mouse genomic sequence flanking the insert sequence for homology arms, allowing for efficient homologous recombination in ESCs for targeted integration of the insert sequence into the genomic target site [column 4 ¶ 3]. Devoy further teaches that the advantage of this strategy is that it can be directly applied to unmodified ESCs and is essentially a single-step procedure [column 4 ¶ 3]. Therefore, an ordinarily skilled artisan at the time of filing the instant application would have been motivated to use 5’ and 3’ homology arms flanking a human TTR V30M insertion sequence for the efficient targeted integration of the human TTR V30M into a specific mouse locus (e.g., the mouse TTR locus).
Regarding claim 43, Zhao and Devoy teach the non-human animal of claim 1. Butler teaches that RNAi is a clinically validated technology that may be a promising approach to the treatment of ATTR amyloidosis [abstract]. Butler teaches a method of assessing the activity of a human TTR-targeting reagent in vivo, comprising a) administering the human-TTR-targeting reagent (e.g., anti-human TTR siRNA) to a non-human animal which is a murine model of hereditary ATTR amyloidosis, wherein the non-human animal is an hTTR V30M HSF1+/- mouse which expresses the pathogenic human Val30Met TTR variant [column 7 ¶ 3], and b) assessing the activity of the human-TTR-targeting regent in the non-human animal [column 7 ¶ 3, Figure 2, 3]. Butler also teaches that their data support the therapeutic hypothesis behind TTR lowering and highlight the potential of RNAi in the treatment of patients afflicted with ATTR amyloidosis. Therefore, and ordinarily skilled artisan at the time of filing the instant application would have been motivated to assess the activity of a human-TTR-targeting reagent in vivo to assess the therapeutic potential of the reagent.
Regarding claim 69, Zhao, Devoy, and Butler teach the method of claim 43. Butler further teaches that the method was used for optimizing the activity of a TTR-targeting reagent in vivo (e.g., siRNA targeting hTTR) by (i) performing the method of assessing the activity of a human-TTR-targeting reagent in vivo a first time in a first non-human animal; (ii) changing a variable (e.g., lipid nanoparticle (LNP)-facilitated delivery vs. N-acetylgalactosamine (GalNAc) ligand-facilitated delivery) and performing the method of assessing a second time with the changed variable in a second non-human animal; and (iii) comparing the activity of the human-TTR-targeting reagent in step (i) with the activity of the human-TTR-targeting reagent in step (ii), and selecting the method resulting in the higher activity [column 7 ¶ 2-column 8 ¶ 3, Figure 2, 3]. Butler teaches that the method performed with both variable conditions resulted in robust (>95%) and sustained knockdown of serum TTR protein relative to control and statistically significant reductions in TTR deposition [column 9 ¶ 1-column 11 ¶ 1, Figure 3].
Butler additionally teaches the variation of the amount of reagent administered (e.g., 1, 2.5, or 25 mg/kg of siTTR2), wherein less TTR deposit reduction was observed with lower amounts of siTTR2 administered [column 11 ¶ 12, Figure 4]. Butler further varied the dose frequency of TTR-targeted siRNA (e.g., patisiran) and varied the dose levels of another TTR-targeted siRNA (e.g., revusiran), and compared the activity of the TTR-targeting reagents under each varied condition with the activity of the other varied conditions to determine the method resulting in the higher targeting activity, wherein more frequent administration of patisiran and higher doses of revusiran resulted in lower serum TTR protein concentrations [column 14 ¶ 2-column 15 ¶ 1]. Therefore, given the extensive teachings of Butler of optimizing the activity of TTR-targeting reagents in vivo to determine the conditions with the highest TTR-targeting activity, such that variables of dose, frequency, and delivery were evaluated for effects on the TTR-targeting activity of the reagents, wherein some conditions resulted in higher activity than others, an ordinarily skilled artisan at the time of filing the instant application would have been motivated to optimize the activity of a human-TTR-targeting reagent in vivo in the development and assessment of potential therapeutic reagents.
Given the motivation taught by Devoy to replace exonic and intronic sequences of the mouse TTR gene locus with exonic and intronic human sequence from the human TTR gene; the motivation taught by Zhao, Devoy, and NCBI to use the native sequence taught by NCBI, modified to comprise the V30M mutation, as a human TTR sequence for humanizing a non-human animal endogenous TTR locus; the additional motivation taught by Devoy to remove a selection marker, thereby producing a humanized endogenous TTR locus lacking a selection cassette; the motivation taught by Saelices and Wei to seed an hTTR mouse model with pre-formed TTR V30M aggregates and/or fibrils (e.g., ex vivo patient-derived TTR fibrils) to study the seeding of TTR amyloid fibrils and assess potential acceleration of TTR deposition; the motivation taught by Devoy and Butler to increase the expression of the hTTR from the humanized TTR locus to increase the hTTR amyloid deposition in the humanized mouse to thereby increase the disease phenotype for modeling FAP; the motivation taught by Konermann to use a genomically integrated expression cassette comprising a) dCas9-VP64 and b) MS2-p65-HSF1 to increase the expression of hTTR V30M from the humanized TTR locus to increase the hTTR amyloid deposition in the humanized mouse to thereby increase the disease phenotype for modeling FAP; the motivation taught by Devoy and Chu to insert an expression cassette into the Rosa26 locus for inserting single transgene copies in a standardized configuration into the mouse genome; the further motivation taught by Devoy to use 5’ and 3’ homology arms flanking a human TTR V30M insertion sequence for the efficient targeted integration of the human TTR V30M into a mouse TTR locus; the motivation taught by Butler to assess the activity of a human-TTR-targeting reagent in vivo to assess the therapeutic potential of the reagent; and the further motivation taught by Butler optimize the activity of a human-TTR-targeting reagent in vivo in the development and assessment of potential therapeutic reagents; it would have been prima facie obvious to an ordinarily skilled artisan at the time of filing the instant application to modify the non-human animal and methods of Zhao to replace both exons and introns of the mouse TTR locus with both exons and introns of the human TTR V30M gene (such as the TTR gene sequence according to instant SEQ ID NO: 24) to produce a humanized endogenous TTR locus lacking a selection cassette, to seed the development of hTTR deposits to accelerate hTTR amyloid formation, and to include expression cassettes for a SAM system to increase the expression of hTTR to further advance hTTR amyloid formation in the generation of a humanized model of TTR V30M FAP; and to use the humanized model in method assessing and optimizing the activity of human-TTR-targeted reagents with a reasonable expectation of success.
Conclusion
No claim is allowed.
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DR. KATIE L. PENNINGTON
Examiner
Art Unit 1634
/KATIE L PENNINGTON/Examiner, Art Unit 1634
/MARIA G LEAVITT/Supervisory Patent Examiner, Art Unit 1634