# US 12,110,502 B2 · Models of tauopathy

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- Number: US 12,110,502 B2
- Document ID: US12110502B2
- Status: Granted
- Kind: B2
- Application number: 18/502,516
- Filed: 2023-11-06
- Published: 2024-03-07
- Granted: 2024-10-08
- Earliest claimed: 2019-06-14
- Assignees: Regeneron Pharmaceuticals, Inc.
- Applicants: Regeneron Pharmaceuticals, Inc.
- Inventors: Marine Prissette; Matthew Koss; Mathieu Desclaux; John McWhirter; Arijit Bhowmick; David Frendewey; Brian Zambrowicz; Claudia Racioppi
- Web page: https://patentgrep.com/patent/US12110502B2

## CPC

- A01K 67/027: New or modified breeds of vertebrates
- C12N 5/0619: {Neurons}
- A61K 9/0019: {Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner (non-active ingredients are additionally classified in A61K47/00)}
- A61K 48/0058: {Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct}
- C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- C12N 15/8509: {for producing genetically modified animals, e.g. transgenic}
- A01K 2207/05: Animals modified by non-integrating nucleic acids, e.g. antisense, RNAi, morpholino, episomal vector, for non-therapeutic purpose
- A01K 2227/105: Murine
- A01K 2267/0312: Animal model for Alzheimer's disease
- A01K 2267/0318: Animal model for neurodegenerative disease, e.g. non- Alzheimer's
- C12N 2015/8527: {for producing animal models, e.g. for tests or diseases}
- C12N 2310/11: Antisense
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2310/341: Gapmers, i.e. of the type ===---===
- C12N 2310/346: having a combination of backbone and sugar modifications
- C12N 2320/11: for the determination of target sites, i.e. of active nucleic acids
- C12N 2320/12: in functional genomics, i.e. for the determination of gene function
- C12N 2740/16043: viral genome or elements thereof as genetic vector
- A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)

## Abstract

BANF1, PPP2CA, and ANKLE2 were identified as genes that promote tau aggregation when disrupted. Improved tauopathy models such as cells, tissues, or animals having mutations in or inhibition of expression of BANF1 and/or PPP2CA and/or ANKLE2 are provided. Methods of using such improved tauopathy models for assessing therapeutic candidates for the treatment of a tauopathy, methods of making the improved tauopathy models, and methods of accelerating or exacerbating tau aggregation in a tauopathy model are also provided.

## Claims

### Claim 1

A non-human animal comprising:
(a) a microtubule-associated protein tau coding sequence in one or more cells; and
(b) (i) a genetic modification in one or more or all of BANF1, PPP2CA, and ANKLE2 that reduces expression of the one or more or all of BANF1, PPP2CA, and ANKLE2, respectively, in the one or more cells and/or (ii) one or more agents that reduce expression of one or more or all of BANF1, PPP2CA, and ANKLE2 in the one or more cells,
wherein the microtubule-associated protein tau coding sequence is an exogenous human microtubule-associated protein tau coding sequence and/or wherein the microtubule-associated protein tau comprises a tauopathy-associated mutation.

### Claim 2 (depends on 1)

The non-human animal of claim 1, wherein the one or more cells are neuronal cells.

### Claim 3 (depends on 1)

The non-human animal of claim 1, wherein the microtubule-associated protein tau coding sequence is an exogenous human microtubule-associated protein tau coding sequence.

### Claim 4 (depends on 3)

The non-human animal of claim 3, wherein the exogenous human microtubule-associated protein tau coding sequence is genomically integrated.

### Claim 5 (depends on 3)

The non-human animal of claim 3, wherein the exogenous human microtubule-associated protein tau coding sequence comprises a complementary DNA (cDNA) sequence.

### Claim 6 (depends on 3)

The non-human animal of claim 3, wherein the exogenous human microtubule-associated protein tau coding sequence is codon-optimized for expression in the non-human animal.

### Claim 7 (depends on 3)

The non-human animal of claim 3, wherein the exogenous human microtubule-associated protein tau coding sequence is operably linked to a heterologous promoter.

### Claim 8 (depends on 7)

The non-human animal of claim 7, wherein the heterologous promoter is a mouse prion protein promoter.

### Claim 9 (depends on 7)

The non-human animal of claim 7, wherein the heterologous promoter is a neuron-specific promoter.

### Claim 10 (depends on 9)

The non-human animal of claim 9, wherein the neuron-specific promoter is a synapsin-1 promoter.

### Claim 11 (depends on 1)

The non-human animal of claim 1, wherein the microtubule-associated protein tau comprises a tauopathy-associated mutation.

### Claim 12 (depends on 11)

The non-human animal of claim 11, wherein the tauopathy-associated mutation comprises a P301S mutation.

### Claim 13 (depends on 11)

The non-human animal of claim 11, wherein the microtubule-associated protein tau comprises the sequence set forth in SEQ ID NO: 98.

### Claim 14 (depends on 11)

The non-human animal of claim 11, wherein the tauopathy-associated mutation comprises an A152T/P301L/S320F triple mutation.

### Claim 15 (depends on 11)

The non-human animal of claim 11, wherein the microtubule-associated protein tau coding sequence comprises the sequence set forth in SEQ ID NO: 83 or the microtubule-associated protein tau comprises the sequence set forth in SEQ ID NO: 84.

### Claim 16 (depends on 1)

The non-human animal of claim 1, wherein the non-human animal comprises the genetic modification in the one or more or all of BANF1, PPP2CA, and ANKLE2 that reduces expression of the one or more or all of BANF1, PPP2CA, and ANKLE2, respectively, in the one or more cells.

### Claim 17 (depends on 1)

The non-human animal of claim 1, wherein the non-human animal comprises the one or more agents that reduce expression of the one or more or all of BANF1, PPP2CA, and ANKLE2 in the one or more cells.

### Claim 18 (depends on 17)

The non-human animal of claim 17, wherein the one or more agents comprise a nuclease agent targeting BANF1, PPP2CA, or ANKLE2 or a nucleic acid encoding the nuclease agent.

### Claim 19 (depends on 18)

The non-human animal of claim 18, wherein the nuclease agent is a Zinc Finger Nuclease (ZFN), a Transcription Activator-Like Effector Nuclease (TALEN), or a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (Cas) protein and a guide RNA.

### Claim 20 (depends on 19)

The non-human animal of claim 19, wherein the nuclease agent is the Cas protein and the guide RNA.

### Claim 21 (depends on 20)

The non-human animal of claim 20, wherein the Cas protein is a Cas9 protein.

### Claim 22 (depends on 20)

The non-human animal of claim 20, wherein the Cas protein is a catalytically active Cas protein.

### Claim 23 (depends on 17)

The non-human animal of claim 17, wherein the one or more agents comprise a transcriptional repressor targeting BANF1, PPP2CA, or ANKLE2 or a nucleic acid encoding the transcriptional repressor.

### Claim 24 (depends on 23)

The non-human animal of claim 23, wherein the transcriptional repressor comprises a guide RNA and a catalytically inactive Cas protein fused to a transcriptional repressor domain, wherein the transcriptional repressor domain is a Krüppel associated box (KRAB) domain.

### Claim 25 (depends on 20)

The non-human animal of claim 20, wherein the guide RNA targets mouse Banf1 and comprises any one of the sequences set forth in SEQ ID NOS: 44-46 or the guide RNA targets human BANF1 and comprises any one of the sequences set forth in SEQ ID NOS: 27-30.

### Claim 26 (depends on 20)

The non-human animal of claim 20, wherein the guide RNA targets mouse Ppp2ca and comprises any one of the sequences set forth in SEQ ID NOS: 47-49 or the guide RNA targets human PPP2CA and comprises any one of the sequences set forth in SEQ ID NOS: 31-32.

### Claim 27 (depends on 20)

The non-human animal of claim 20, wherein the guide RNA targets mouse Ankle2 and comprises any one of the sequences set forth in SEQ ID NOS: 50-52 or the guide RNA targets human ANKLE2 and comprises the sequence set forth in SEQ ID NO: 38.

### Claim 28 (depends on 17)

The non-human animal of claim 17, wherein the one or more agents comprise an antisense oligonucleotide or an RNAi agent targeting BANF1, PPP2CA, or ANKLE2 or a nucleic acid encoding the antisense oligonucleotide or the RNAi agent.

### Claim 29 (depends on 28)

The non-human animal of claim 28, wherein the antisense oligonucleotide comprises the sequence set forth in any one of SEQ ID NOS: 105-324 or a modified version thereof.

### Claim 30 (depends on 29)

The non-human animal of claim 29, wherein the antisense oligonucleotide comprises the sequence set forth in any one of SEQ ID NOS: 105, 106, 110-113, 115, 120-122, 124, 125, 130 133, 136, 137, 150, 152, 153, 155, 158-160, 162, 165, 166, 169, 171-173, 175, 177, 181-184, 187, 194, 197, 211, 213, 215, 216, 220-223, 225, 230-232, 234, 235, 240, 243, 246, 247, 260 262, 263, 265, 268-270, 272, 275, 276, 279, 281-283, 285, 287, 291-294, 297, 304, 307, 321, and 323 or a modified version thereof.

### Claim 31 (depends on 29)

The non-human animal of claim 29, wherein the antisense oligonucleotide comprises one or more phosphorothioate linkages and/or one or more 2′-methoxyethyl modified bases.

### Claim 32 (depends on 31)

The non-human animal of claim 31, wherein the antisense oligonucleotide is a 5-10-5 gapmer comprising a phosphorothioate backbone, a 5′ wing of 2′-methoxyethyl modified bases, a central 10-nucleotide core of DNA, and a 3′ wing of 2′-methoxyethyl modified bases.

### Claim 33 (depends on 1)

The non-human animal of claim 1, wherein at least one sign or symptom of tauopathy is increased in the non-human animal relative to a non-human animal that does not comprise the genetic modification in the one or more or all of BANF1, PPP2CA, and ANKLE2 or does not comprise the one or more agents that reduce expression of one or more or all of BANF1, PPP2CA, and ANKLE2.

### Claim 34 (depends on 33)

The non-human animal of claim 33, wherein the at least one sign or symptom comprises tau hyperphosphorylation or tau aggregation.

### Claim 35 (depends on 33)

The non-human animal of claim 33, wherein the at least one sign or symptom comprises increased tau and/or phospho-tau in an insoluble fraction following cell fractionation, increased phospho-tau in the somatodendritic compartment of neurons, increased phospho-tau in the perinuclear region of neurons, decreased nuclear pore complex protein Nup98-Nup96 (Nup98) nuclear-to-cytoplasmic ration in neurons, decreased GTP-binding nuclear protein Ran (Ran) nuclear-to-cytoplasmic ratio in neurons, or decreased Ran GTPase-activating protein 1 (RanGAP1) nuclear-to-cytoplasmic ratio in neurons.

### Claim 36 (depends on 1)

The non-human animal of claim 1, wherein the genetic modification in one or more or all of BANF1, PPP2CA, and ANKLE2 is in one or more neuronal cells and/or wherein the one or more agents that reduce expression of one or more or all of BANF1, PPP2CA, and ANKLE2 are in one or more neuronal cells, wherein the one or more neuronal cells are in the hippocampus.

### Claim 37 (depends on 1)

The non-human animal of claim 1, wherein the non-human animal is a mouse or a rat.

### Claim 38 (depends on 37)

The non-human animal of claim 37, wherein the non-human animal is the mouse.

### Claim 39 (depends on 38)

The non-human animal of claim 38, wherein the mouse is a PS19 transgenic mouse further comprising the genetic modification in the one or more or all of BANF1, PPP2CA, and ANKLE2 that reduces expression of the one or more or all of BANF1, PPP2CA, and ANKLE2, respectively, in the one or more cells and/or further comprising the one or more agents that reduce expression of one or more or all of BANF1, PPP2CA, and ANKLE2 in the one or more cells.

### Claim 40 (depends on 1)

A method for assessing a therapeutic candidate for the treatment of a tauopathy, comprising:
(a) administering a candidate agent to the non-human animal of claim 1;
(b) performing one or more assays to determine if the candidate agent has an effect on one or more signs or symptoms associated with the tauopathy; and
(c) identifying the candidate agent that has an effect on the one or more signs or symptoms associated with the tauopathy as a therapeutic candidate.

### Claim 41 (depends on 1)

A method of making the non-human animal of claim 1, comprising:
(a) introducing the one or more agents that reduce expression of one or more or all of BANF1, PPP2CA, and ANKLE2 into a non-human animal that comprises the microtubule-associated protein tau coding sequence; and
(b) screening the non-human animal to confirm the presence of the one or more agents.

### Claim 42 (depends on 1)

A method of making the non-human animal of claim 1, comprising:
(a) introducing into a non-human animal:
(i) an exogenous human microtubule-associated protein tau coding sequence; and
(ii) the one or more agents that reduce expression of one or more or all of BANF1, PPP2CA, and ANKLE2; and
(b) screening the non-human animal to confirm the presence of the one or more agents and the exogenous human microtubule-associated protein tau coding sequence.