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EP-4737577-A1 - CFB GENE MODIFIED NON-HUMAN ANIMAL

EP4737577A1EP 4737577 A1EP4737577 A1EP 4737577A1EP-4737577-A1

Abstract

The disclosure relates to a non-human animal that express a human or chimeric (e.g., humanized) CFB protein and methods of use thereof. The disclosure further provides a non-human animal genome, a humanized CFB gene, and cells, tissues, organs, or non-human animals comprising the non-human animal genome or the humanized CFB gene. The non-human animals obtained in present disclosure successfully express human or humanized CFB protein, which can be cleaved by CFD, bind to C3b, and exert functions similar to those in the human. Moreover, the non-human animals obtained in present disclosure maintain intact renal function status and normal blood biochemical parameters. Notably, these non-human animals do not result in the potential pathologies observed in some other transgenic mice known in the art.

Inventors

  • ZHANG, SHUJIN
  • ZHOU, XIAOFEI
  • ZHANG, Zan

Assignees

  • Biocytogen Pharmaceuticals (Beijing) Co., Ltd.

Dates

Publication Date
20260506
Application Date
20240628

Claims (20)

  1. A genetically-modified, non-human animal whose genome comprises at least one chromosome comprising a sequence encoding a human or chimeric complement factor B (CFB).
  2. The animal of claim 1, wherein the sequence encoding the human or chimeric CFB is regulated by regulatory elements, and the regulatory element is an endogenous regulatory element or a human regulatory element.
  3. The animal of claim 1, wherein the chimeric CFB is a humanized CFB, and the humanized CFB comprises a portion of a human CFB; preferably, the portion of the human CFB comprises the Ba region and/or the Bb region; further preferably, the Bb region comprises a VWA domain; further preferably, the Bb region comprises von Willebrand factor and/or peptidase S1.
  4. The animal of claim 3, wherein the humanized CFB comprises at least 50 to 764 contiguous amino acid sequences that are identical to a portion of the human CFB.
  5. The animal of any one of claims 1-4, wherein the animal is a mammal, such as a monkey or a rodent (e.g., a mouse or a rat).
  6. The animal of any one of claims 1-5, wherein the human or chimeric CFB comprises an amino acid sequence set forth in SEQ ID NO: 2 or amino acids 26-764 of SEQ ID NO: 2, or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 2 or amino acids 26-764 of SEQ ID NO: 2, or an amino acid sequence that differs from SEQ ID NO: 2 or amino acids 26-764 of SEQ ID NO: 2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid, or an amino acid sequence comprising a substitution, a deletion and/or insertion of one or more amino acid residue set forth in SEQ ID NO: 2 or amino acids 26-764 of SEQ ID NO: 2.
  7. The animal of any one of claims 1-6, wherein the animal does not express endogenous CFB or expresses a decreased level of endogenous CFB as compared to CFB expression level in a wild-type animal.
  8. A genetically-modified, non-human animal, wherein at the endogenous CFB gene locus, the endogenous CFB gene nucleotide sequence is replaced with a nucleotide sequence comprising a nucleotide sequence of the human CFB gene.
  9. The animal of claim 8, wherein the expression of the nucleotide sequence of the human CFB gene is regulated by a regulatory element, and the regulatory element is an endogenous regulatory element or a human regulatory element; preferably, the animal has one or more cells expressing human or chimeric CFB.
  10. The animal of claim 8, wherein the animal does not express endogenous CFB or expresses a decreased level of endogenous CFB as compared to CFB expression level in a wild-type animal.
  11. The animal of any one of claims 8-10, wherein the nucleotide sequence of the human CFB gene comprises a portion of exon 1 to a portion of exon 18 of the human CFB gene; preferably, the nucleotide sequence of the human CFB gene comprises a nucleotide sequence of the coding region of the human CFB gene; further preferably, the nucleotide sequence of the human CFB gene comprises a nucleotide sequence from the start codon to the stop codon of the human CFB gene.
  12. The animal of claim 11, wherein the nucleotide sequence of human CFB gene further comprises the 5'UTR and/or the 3'UTR of human CFB gene, and further comprises at least a 50 bp contiguous nucleotide sequence upstream of the 5'UTR of human CFB gene and/or at least a 50 bp contiguous nucleotide sequence downstream of the 3'UTR of human CFB gene.
  13. The animal of any one of claims 8-12, wherein the nucleotide sequence of human CFB gene comprises the 5'UTR of human CFB and at least a 50 bp contiguous nucleotide sequence upstream thereof, a nucleotide sequence from the start codon to the stop codon of the human CFB gene, and the 3'UTR of human CFB and at least a 50 bp contiguous nucleotide sequence downstream thereof.
  14. The animal of any one of claims 8-13, wherein the nucleotide sequence of human CFB comprises a nucleotide sequence set forth in SEQ ID NO: 7 or 39, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 7 or 39, or a nucleotide sequence that differs from SEQ ID NO: 7 or 39 by no more than 50, 40, 30, 20, 10, 9, 6, 3, or 1 nucleotide, or c a nucleotide sequence comprising a substitution, a deletion and/or insertion of one or more nucleotides set forth in SEQ ID NO: 7 or 39.
  15. The animal of any one of claims 8-14, wherein the endogenous CFB gene exons 2 to 17 are replaced; preferably, the endogenous CFB gene further comprises a portion of exon 1, all of intron 1, and/or a portion of intron 17 are replaced; further preferably, the endogenous CFB gene further comprises a portion of exon 18 are replaced.
  16. The animal of any one of claims 8-14, wherein the endogenous CFB gene is replaced from the start codon to the stop codon thereof, or the endogenous CFB gene is replaced from a portion of exon 1 to a portion of intron 17, wherein the portion of exon 1 comprises up to 70 nucleotides at the 3' end of exon 1.
  17. The animal of any one of claims 8-16, wherein the animal is a mammal, such as a monkey or a rodent (e.g., a mouse or a rat).
  18. The animal of any one of claims 8-17, wherein the animal whose genome comprises a modified endogenous CFB nucleotide sequence that transcribes an mRNA comprising SEQ ID NO: 8 or 40, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 8 or 40, or a nucleotide sequence that differs from SEQ ID NO: 8 or 40 by no more than 50, 40, 30, 20, 10, 9, 6, 3, or 1 nucleotide, or a nucleotide sequence comprising a substitution, a deletion and/or insertion of one or more nucleotides set forth in SEQ ID NO: 8 or 40.
  19. The animal of claim 8, wherein the animal is homozygous or heterozygous with respect to the replacement at the endogenous CFB gene locus.
  20. The animal of any one of claims 1-19, wherein the animal expresses the human or chimeric CFB having at least one mouse CFB activity and/or at least one human CFB activity.

Description

CROSS-REFERENCES OF RELATED APPLICATIONS This application claims the benefit of Chinese Patent Application App. No. 202310778275.7, filed on June 28, 2023; Chinese invention Chinese Patent Application App. No. 202410169335.X filed on February 6, 2024; Chinese Patent Application App. No. 202410462699.7, filed on April 17, 2024 and Chinese Patent Application App. No. 202410555788.6 filed on May 7, 2024. The entire contents of the foregoing applications are incorporated herein by reference. TECHNICAL FIELD This disclosure relates to genetically modified animals expressing human or chimeric (e.g., humanized) CFB protein, and methods of use thereof. BACKGROUND The traditional drug research and development typically use in vitro screening approaches. However, these screening approaches cannot provide the body environment (such as tumor microenvironment, stromal cells, extracellular matrix components and immune cell interaction, etc.), resulting in a higher rate of failure in drug development. In addition, in view of the differences between humans and animals, the test results obtained from the use of conventional experimental animals for in vivo pharmacological test may not reflect the real disease state and the interaction at the targeting sites, resulting in that the results in many clinical trials are significantly different from the animal experimental results. Therefore, the development of humanized animal models that are suitable for human antibody screening and evaluation will significantly improve the efficiency of new drug development and reduce the cost for drug research and development. However, the construction of humanized animal models remains highly challenging. For instance, the non-patent literature "Generation and utility of genetically humanized mouse models" (Scheer N, Snaith M, Wolf CR, Seibler J., Drug Discovery Today; 18(23-24):1200-11, 2013) points out that even with meticulously designed strategies, there is no guarantee of achieving proper expression and functionality of the human gene in animals. SUMMARY In view of the deficiencies in the prior art, disclosure is related to a genetically modified non-human animal. This animal expresses human CFB protein normally in vivo, which can be cleaved by CFD, binds to C3b, and exerts functions similar to those in humans. Importantly, the non-human animal obtained in this application exhibits intact renal function and normal blood biochemical parameters. Even more notably, it does not develop the potential diseases that are known in the art to occur in certain other transgenic mouse models. This disclosure is related to an animal model with human or chimeric CFB. The animal model can express human or chimeric CFB (e.g., humanized CFB). It can be used in the studies on the function of CFB genes, and can be used in the screening and evaluation of CFB signaling pathway modulators (e.g., anti-human CFB antibodies, oligonucleotide drugs, and/or polypeptides drugs). In addition, the animal models prepared by the methods described herein can be used in drug screening, pharmacodynamics studies, treatments for immune-related diseases, and diseases therapy for human CFB target site; they can also be used to facilitate the development and design of new drugs, and save time and cost. In summary, this disclosure provides a powerful tool for studying the function of CFB protein and a platform for screening drugs. In one aspect, the disclosure is related to a genetically-modified, non-human animal whose genome comprises at least one chromosome comprising a sequence encoding a human or chimeric complement factor B (CFB). In some embodiments, the sequence encoding the human or chimeric CFB is regulated by regulatory elements (e.g., an endogenous regulatory element or a human regulatory element; preferably, the regulatory element is the 5'UTR and/or the 3'UTR). In some embodiments, the chimeric CFB is a humanized CFB, and the humanized CFB comprises a portion of a human CFB. In some embodiments, the portion of the human CFB comprises the Ba region and/or the Bb region; preferably, the Bb region comprises a VWA domain; further preferably, the Bb region comprises von Willebrand factor and/or peptidase S1. In some embodiments, the humanized CFB comprises at least 50 to 764 contiguous amino acid sequences that are identical to a portion of the human CFB. In some embodiments, the animal is a mammal, such as a monkey or a rodent (e.g., a mouse or a rat). In some embodiments, the human or chimeric CFB comprises an amino acid sequence set forth in SEQ ID NO: 2 or amino acids 26-764 of SEQ ID NO: 2, or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 2 or amino acids 26-764 of SEQ ID NO: 2, or an amino acid sequence that differs from SEQ ID NO: 2 or amino acids 26-764 of SEQ ID NO: 2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid, or an amino acid sequence comprising a substitution, a deletion and/or insertion