Non-human animals having a humanized complement factor b gene

Abstract

Disclosed herein are rodents (such as mice and rats) genetically modified to comprise a humanized CFB gene. Also disclosed herein are compositions (e.g., targeting vectors) and methods for making such genetically modified rodents. Additionally disclosed herein are methods of using the genetically modified rodent for assessing candidate compounds directed to human CFB or another complement molecule for treating ocular complement diseases such as AMD.

Description

NON-HUMAN ANIMALS HAVING A HUMANIZED COMPLEMENT FACTOR B GENECROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority from U.S. Provisional Application No. 63 / 606,319, filed December 5, 2023, the entire contents of which is incorporated herein by reference.INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The Sequence Listing in the XML format, named as42821WO_11692WO01_SequenceListing.xml of 57,344 bytes, created on November 29, 2024 and submitted to the United States Patent and Trademark Office via Patent Center, is incorporated herein in its entirety by reference.BACKGROUND

[0003] Age-related macular degeneration (AMD) is a progressive macular disease and the primary cause of central vision impairment in individuals over 50 years old in developed countries. Recent genetic and pathophysiological studies suggest that AMD progression may be partially due to an inflammatory state maintained by abnormal activity of the complement alternative pathway (Bradley et al., Eye 2011, 25, 683-693).

[0004] Complement factor B (CFB), a glycoprotein necessary for the initiation and propagation of activation of the alternative pathway of the complement system, is predominantly produced in the liver and subsequently secreted into the blood. Patients with AMD have been reported to exhibit elevated plasma levels of CFB (Chu ct al., J. Ophthalmology 2024, article ID 6416773). However, production of CFB also occurs locally in both the mouse and human choroid.SUMMARY

[0005] One aspect of this disclosure is directed to a genetically modified rodent animal comprising a humanized Complement Factor B (CFB) gene in its genome.

[0006] In some embodiments, the humanized CFB gene comprises a rodent CFB nucleic acid sequence and a human CFB nucleic acid sequence.

[0007] In some embodiments, the humanized CFB gene encodes a polypeptide comprising a mature protein sequence that is substantially identical (e.g., at least 90% or at least 95% identical) to the mature protein sequence of a human CFB protein. In some embodiments, the humanized CFB gene encodes a polypeptide comprising a mature protein sequence that is identical to the mature protein sequence of a human CFB protein.

[0008] In some embodiments, the humanized CFB gene encodes a polypeptide comprising a signal peptide substantially identical (e.g., at least 90% identical) to the signal peptide of the human CFB protein or the signal peptide of a rodent CFB protein (e.g., an endogenous rodent CFB protein). In some embodiments, the humanized CFB gene encodes a polypeptide comprising a signal peptide identical to the signal peptide of the human CFB protein or the signal peptide of a rodent CFB protein (e.g., an endogenous rodent CFB protein).

[0009] In some embodiments, the humanized CFB gene encodes a polypeptide substantially identical (e.g., at least 90% or at least 95% identical) to a human CFB protein. In some embodiments, the humanized CFB gene encodes a polypeptide identical to a human CFB protein.

[0010] In some embodiments, the human CFB nucleic acid sequence comprises the coding sequence (i.e., from the start codon to the stop codon) of a human CFB gene. In some embodiments, the human CFB nucleic acid sequence further comprises the 5’ untranslated region (5’ UTR) of the human CFB gene. In some embodiments, the human CFB nucleic acid sequence further comprises a 5’ genomic sequence upstream of the 5’ UTR of the human CFB gene, wherein the 5 ’ genomic sequence comprises one or more transcription regulatory sequences (e.g., promoter and / or enhancer) of the human CFB gene.

[0011] In some embodiments, the rodent CFB nucleic acid sequence comprises a 5’ regulatory sequence (e.g., promoter, and / or enhancer) of a rodent CFB gene. In some embodiments, the rodent CFB nucleic acid sequence comprises a 3’ sequence (e.g., the 3’ UTR or a portion thereof, and / or a genomic sequence downstream of the 3’ UTR) of a rodent CFB gene. In some embodiments, the rodent CFB gene is an endogenous rodent CFB gene.

[0012] In some embodiments, the humanized CFB gene is at an endogenous CFB locus, e.g., as a result of a replacement of a rodent CFB genomic sequence with a human CFB nucleic acid sequence.

[0013] In some embodiments, a rodent animal is heterozygous for a humanized CFB gene. In some embodiments, a rodent animal is homozygous for a humanized CFB gene.

[0014] In some embodiments, a humanized CFB gene expresses a polypeptide, e.g., a human mature CFB polypeptide, in the serum of the rodent animal.

[0015] In some embodiments, a rodent animal further comprises a humanized Complement 3 (C3) gene in its genome, i.e., the rodent animal is a double humanized rodent (humanized for CFB and C3). In some embodiments, a human C3 genomic fragment containing 5’ regulatory elements and all the coding exons 1 through 41 of the human C3 gene has replaced a rodent (e.g., mouse) C3 genomic fragment at an endogenous rodent C3 locus spanning 5’ regulatory elements and all of the coding exons 1 through 1, so as to form a humanized C3 gene at the endogenous rodent C3 locus.

[0016] In some embodiments, the rodent is a mouse or a rat.

[0017] Another aspect of this disclosure is directed to an isolated rodent tissue or cell, wherein the genome of the isolated rodent tissue or cell comprises a humanized CFB gene described herein. In some embodiments, an isolated rodent tissue or cell further comprises a humanized C3 gene in its genome. In some embodiments, the rodent cell is a rodent embryonic stem cell. In some embodiments, the rodent cell is an egg or a sperm. In some embodiments, an isolated rodent tissue or cell is a mouse tissue or mouse cell, or a rat tissue or rat cell.

[0018] Still another aspect of this disclosure is directed to a rodent embryo comprising a rodent embryonic stem cell which comprises a humanized CFB gene described herein. In some embodiments, a rodent embryo comprises a rodent embryonic stem cell comprising a humanized CFB gene described herein and a humanized C3 gene.

[0019] A further aspect is directed to a method of making a genetically modified rodent. In some embodiments, the method comprises modifying a rodent genome to comprise a humanized CFB gene described herein. In some embodiments, modifying a rodent genome comprises the steps of introducing a nucleic acid molecule comprising a human CFB nucleic acid sequence intothe genome of a rodent embryonic stem (ES) cell, obtaining a rodent ES cell in which the human CFB nucleic acid sequence has integrated into an endogenous CFB locus to replace a rodent CFB genomic DNA thereby forming a humanized CFB gene, and generating a rodent animal from the obtained rodent ES cell. In some embodiments, the method further comprises modifying the rodent genome to further comprise a humanized C3 gene. In some embodiments, the method comprises crossing a rodent comprising a humanized CFB gene with a rodent comprising a humanized C3 gene to obtain a double humanized rodent. In some embodiments of the method, the rodent is a mouse or a rat.

[0020] In some embodiments, disclosed herein is a targeting nucleic acid construct, comprising a human CFB nucleic acid sequence to be integrated into a rodent CFB gene at an endogenous rodent CFB locus, flanked by a 5’ nucleotide sequence and a 3’ nucleotide sequence that are homologous to nucleotide sequences at the rodent CFB locus, wherein integration of the human CFB nucleic acid sequence into the rodent CFB gene results in a replacement of a rodent CFB genomic DNA with the human CFB nucleic acid sequence, thereby forming a humanized CFB gene as described herein. In some embodiments, the human CFB nucleic acid sequence encodes a polypeptide comprising the mature sequence of a human CFB protein. In some embodiments, the human CFB nucleic acid sequence comprises the full coding sequence (from the start codon to the stop codon) of a human CFB gene. In some embodiments of a targeting nucleic acid, the rodent is a mouse or a rat.

[0021] In some embodiments, disclosed herein is an in vitro method for generating a genetically modified rodent cell, comprising introducing into a rodent cell a targeting vector comprising a human CFB nucleic sequence comprising the full coding sequence of human CFB gene, flanked by rodent homology arms that mediate integration of the human CFB nucleic acid sequence into an endogenous rodent CFB locus, which results in replacement of a rodent CFB genomic DNA with the human CFB nucleic acid sequence to form a humanized CFB gene as described herein, thereby generating a genetically modified rodent cell. In some embodiments, the rodent cell is mouse cell or a rat cell. In some embodiments, the rodent cell is a rodent ES cell, and the method generates a genetically modified rodent ES cell.

[0022] In some embodiments, disclosed herein is a method of assessing pharmacokinetic properties of a candidate compound, the method comprising administering the candidatecompound to a rodent animal described herein (e.g., a rodent comprising a humanized CFB gene, or comprising a humanized CFB gene and a humanized C3 gene), and performing one or more assays to determine the pharmacokinetic properties of the candidate compound in the rodent animal. In some embodiments, the candidate compound is an antibody that binds to human CFB. In some embodiments, the candidate compound is an inhibitory nucleic acid (such as siRNA or antisense nucleic acid) targeting a human CFB gene.

[0023] In some embodiments, disclosed herein is a method for screening or evaluating candidate compounds, the method comprising administering a candidate compound to a rodent animal described herein (e.g., a rodent comprising a humanized CFB gene, or comprising a humanized CFB gene and a humanized C3 gene), and performing one or more assays to determine whether the candidate drug has an effect on the rodent animal (e.g., on the complement pathway of the rodent animal). Candidate compounds can be a compound targeting human CFB or another complement molecule. In some embodiments, the candidate drug is an antibody. In some embodiments, the candidate drug is an inhibitory nucleic acid (such as siRNA or antisense nucleic acid).BRIEF DESCRIPTION OF DRAWINGS

[0024] FIG. 1A depicts an example of a targeting strategy for humanization of a mouse CFB gene. A mouse CFB genomic DNA was deleted starting at the 5’ untranslated region (UTR), through the full coding sequence and including 31bp of the mouse 3’ UTR. This was replaced with a human CFB genomic DNA, stalling upstream of the start of the human CFB 5’ UTR (e.g., starting at about 590 bp upstream of the stall of the human CFB 5’ UTR, or starting at about 717bp upstream of the stall codon), including the full human coding sequence, and ending at the human CFB stop codon.

[0025] FIG. IB depicts the humanized CFB allele based on the strategy depicted in FIG. 1A, with a self-deleting cassette containing a neomycin resistance gene placed in human intron 10. Top: cassette-containing humanized CFB allele; bottom: cassette-deleted humanized CFB allele.

[0026] FIGS. 2A-D present data showing CFB reduction in wild type CRISPR-edited mice. FIG. 2A: The Simple Western Automated Western Blot (WES) shows downregulation of CFB (FB) levels in the serum. Two guide RNAs (g2” and “g4”) targeting mouse CFB were used. FIG. 2B: Quantification of FIG. 2A reveals successful reduction of CFB in the serum of treatedgroups in C57BL / 6J mice (Naive =6, CRISPR treated: g2 n= 3, g4 n=3, g2+g4 n= 3). The relative expression is determined based on the amount of the pixels of each of FB band versus each ApoE band in FIG. 2A. FIG. 2C: Western blot reveals partial knockdown of ocular CFB in eye tissue (Naive n= 2, CRISPR treated: g2 n=4, g4 n= 6, g2+g4 n= 2). FIG. 2D: Quantification of FIG. 2C reveals presence of Complement Factor B protein in the eye, suggesting some local production of the protein in the eye. The percentages of reduction in the ocular CFB levels (or knock down (“KD”) percentages) were also indicated.

[0027] FIGS. 3A-C present data showing effective silencing of wild type CRISPR-edited mice using gRNAs. FIG. 3 A: Quantitative Polymerase Chain Reaction (qPCR) shows successful downregulation of CFB mRNA in the liver of C57BL / 6I mice, normalized to Gapdh and Naive group (Naive n= 5, CRISPR treated: g2 n= 5, g4 n= 5, g2+ g4 n= 5). FIG. 3B: NGS sequencing confirms 60% Indel events in CRISPR treated livers. FIG. 3C: qPCR results from eye tissue show no difference in expression of CFB mRNA (Naive n= 3, g2 n= 5, g4 n= 4, g2 +g4 n= 5). This suggests that the mRNA from the eye is locally produced and cannot be further downregulated from a systemic approach.

[0028] FIGS. 4A-C presents data showing silencing of CFB in double humanized C3 / CFB (“huC3 / huCFB”) mice. FIG. 4A: ELISA shows a 62% reduction of CFB protein in the liver of huC3 / huCFB mice (Naive n= 5, CRISPR treated n = 3. Note: 2 mice sacc’ed). FIG. 4B: ELISA also detected an 83% knockdown in serum levels of CFB in huC3 / huCFB mice (non treated n= 5, CRISPR n = 5). FIG. 4C: A 45% reduction in ocular CFB in CRISPR treated samples is observed (non treated n= 5, CRISPR n = 3). The eye was perfused prior to harvesting, ensuring any CFB from circulation was drained. This suggests that silencing CFB in the liver allowed for a 45% reduction in ocular CFB. The remaining CFB in the eye may be from local production,

[0029] FIGS. 5A-C present data showing correlation of CFB in the serum, liver, and eye of huC3 / huCFB mice. FIG. 5A: Significant correlation observed between serum & liver with a p value = 0.0069 in huC3 / huCFB mice (Naive n= 4, CRISPR treated n = 3) may suggest CFB found in the serum is originating from the liver. FIG. 5B: Significant correlation also observed in eye & serum with a p value = 0.0066 suggests CFB circulating from the serum may be contributing to the eye. FIG. 5C: Strong correlation observed between eye & liver with a p value = 0.047 suggests a ocular CFB may be originating from the liver.DETAILED DESCRIPTION

[0030] Disclosed herein are rodents (such as, but not limited to, mice and rats) genetically modified to comprise a humanized CFB gene. Also disclosed herein are compositions (e.g., targeting vectors) and methods for making such genetically modified rodents. The rodents disclosed herein can be used, e.g., but not limited to, as an in vivo system for evaluating candidate compounds directed to human CFB, such as anti-human CFB antibodies, for treating ocular complement diseases such as AMD. Accordingly, also disclosed herein are methods of using a genetically modified rodent for assessing candidate compounds directed to human CFB.Humanization of Rodent CFB

[0031] Complement, an essential component of the immune system, consists of more than 30 serum and cellular proteins that are involved in two linked biochemical cascades, the classical and alternative pathways. Complement functions to assist the immune system to destroy invading microorganisms and maintain tissue homeostasis. However, excessive or unregulated activation of complement contributes to tissue damage, and is associated with a variety of variety of human diseases, disorders and conditions that are associated with complement activation, for example, ocular inflammatory and retinal degenerative diseases {see Makrides (1998) Therapeutic inhibition of the complement system, Pharmacological Reviews 50(l):59-87; and Mollnes et al. (2006) Strategies of therapeutic complement inhibition, Molecular Immunology 43; 107- 121 ).

[0032] Complement factor B (CFB), also known as “factor B” (FB), is a 93-kDa single chain glycoprotein necessary for the initiation and propagation of the alternative pathway activation. When CFB associates with a cleavage product of complement protein C3 (C3b), it is cleaved by factor D to release an N-terminal fragment (Ba) and the carboxyl-terminal fragment (Bb). Bb remains bound to C3b and this complex is also known as C3-convertase. Upon the addition of properdin (a serum protein), a stable complex (C3bBbP) is formed and can bind an additional C3b to form alternative pathway C5-convertase. See, e.g., Janeway, Charles A. (2005). “The complement system and innate immunity”. Immunobiology : the immune system in health and disease (5th ed.). New York: Garland Science. ISBN 978-0-8153-4101-7. Bb has been reported to be involved in the proliferation of preactivated B lymphocytes, while Ba inhibits theirproliferation (Ambrus et al., Journal of Immunology. 144 (5): 1549-53. 1990). CFB is predominantly produced in the liver and subsequently secreted into the blood.

[0033] Example CFB sequences, including nucleic acid and protein sequences for human, mouse, and rat CFB molecules as described herein, are disclosed in the Sequence Listing and summarized in Tables 1-3 below.Table 1. Human CFBTable 2. Mouse CFB (minus strand)Table 3. Rat CFB (minus strand)

[0034] In some embodiments, a rodent disclosed herein comprises a humanized CFB gene in the germline.

[0035] In some embodiments, a rodent disclosed herein comprises a humanized CFB gene in its genome that includes a nucleotide sequence of a rodent CFB gene (e.g., an endogenous rodent CFB gene) and a nucleotide sequence of a human CFB gene. As used herein, “a nucleotide sequence of a gene” can be a genomic sequence, an mRNA or cDNA sequence, in full or in part of the gene. A “part” of a gene includes one or more or all of the 5’ transcription regulatory sequences (e.g., promoter, enhancer), the 5’ untranslated region (5’ UTR), the coding sequence from the ATG start to the stop codon (including exonic and intronic sequences), and the 3’ UTR. As a non-limiting example, a nucleotide sequence of a human CFB gene can be a genomic sequence, an mRNA or cDNA sequence, in full or in part of the human CFB gene. The nucleotide sequence of the rodent CFB gene and the nucleotide sequence of the human CFBgene are operably linked to each other such that the humanized CFB gene in the rodent genome encodes and expresses a protein that maintains the structure and function of a CFB protein.

[0036] “Human CFB” gene and protein, as used herein, refers to CFB gene and protein of the human origin. In some embodiments, a human CFB protein comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, a human CFB protein comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 6. In some embodiments, a human CFB protein comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 6. In some embodiments, a human CFB protein comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 6. In some embodiments, a human CFB protein comprises an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 6 by 1-3 amino acids as a result of polymoiphism.

[0037] “Rodent CFB” gene and protein, as used herein, refers to CFB gene and protein of a rodent (e.g., mouse or rat) origin. In some embodiments, a mouse CFB protein comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, a mouse CFB protein comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 12. In some embodiments, a mouse CFB protein comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 12. In some embodiments, a mouse CFB protein comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 12. In some embodiments, a mouse CFB protein comprises an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 12 by 1-3 amino acids as a result of polymorphism. In some embodiments, a rat CFB protein comprises the amino acid sequence of SEQ ID NO: 17. In some embodiments, a rat CFB protein comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17. In some embodiments, a rat CFB protein comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 17. In some embodiments, a rat CFB protein comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17. In some embodiments, a rat CFB protein comprises an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 17 by 1-3 amino acids as a result of polymorphism.

[0038] In some embodiments, a genetically modified rodent comprises a humanized CFB gene in its genome, wherein the humanized CFB gene encodes a protein having a mature protein sequence that is substantially identical with the mature sequence of a human CFB protein. A mature protein sequence that is “substantially identical with the mature sequence of a human CFB protein” includes a sequence that is at least 95% identical with the mature sequence of a human CFB protein, a sequence that is at least 98% identical with the mature sequence of a human CFB protein, or a sequence that is at least 99% identical with the mature sequence of a human CFB protein. A sequence that is “substantially identical with the mature sequence of a human CFB protein” can be a sequence that is 100% identical with the mature sequence of a human CFB protein. Alternatively or additionally, a sequence that is “substantially identical with the mature sequence of a human CFB protein” can be a sequence that differs from the mature sequence of a human CFB protein by not more than 5 amino acids, a sequence that differs from the mature sequence of a human CFB protein by not more than 4 amino acids, a sequence that differs from the mature sequence of a human CFB protein by not more than 3 amino acids, a sequence that differs from the mature sequence of a human CFB protein by not more than 2 amino acids, or a sequence that differs from the mature sequence of a human CFB protein by not more than 1 amino acid. Alternatively or additionally, a sequence that is “substantially identical with the mature sequence of a human CFB protein” can be a sequence that differs from the mature sequence of a human CFB protein only at the N- or C- terminal portion of the sequence, e.g., by having addition, deletion and / or substitution of amino acids at the N- and / or C- terminal portion of the sequence (i.e., within 5-10 amino acids from the N or C terminus of the sequence). Alternatively or additionally, a sequence that is “substantially identical with the mature sequence of a human CFB protein” can be a sequence that has one or more of the features delineated in above, e.g., a sequence that is at least 95% identical in sequence with the mature sequence of a human CFB protein and differs from the mature sequence of a human CFB protein only at the N- or C- terminal portion by not more than 5 amino acids, or a sequence that is at least 98% identical in sequence with the mature sequence of a human CFB protein and differs from the mature sequence of a human CFB protein only at the N- or C- terminal portion of the sequence by not more than 3 amino acids. In some embodiments, a human CFB protein comprises the amino acid sequence as set forth in SEQ ID NO: 6, and its mature protein sequence is composed of amino acids 26-764 of SEQ ID NO: 6. In some embodiments, a humanized CFB geneencodes a protein whose mature protein sequence is substantially identical with the mature protein sequence of the human CFB protein as set forth in SEQ ID NO: 6, i.c., substantially identical with amino acids 26-764 of SEQ ID NO: 6. For example, the humanized CFB gene encodes a protein having a mature protein sequence that comprises amino acids 26-764, 27-764 or 28-764 of SEQ ID NO: 6. In some embodiments, the humanized CFB gene encodes a protein having a mature protein sequence that comprises amino acids 26-764 of SEQ ID NO: 6.

[0039] In some embodiments, a genetically modified rodent comprises a humanized CFB gene in its genome, wherein the humanized CFB gene encodes a protein having a signal peptide that is substantially identical with the signal peptide of a human CFB protein. A signal peptide that is “substantially identical with the signal peptide of a human CFB protein” includes a signal peptide that is at least 90% identical in sequence with the signal peptide of a human CFB protein, or a signal peptide that is at least 95% identical in sequence with the signal peptide of a human CFB protein. A signal peptide that is “substantially identical with the signal peptide of a human CFB protein” can be a signal peptide that is 100% identical in sequence with the signal peptide of a human CFB protein. Alternatively or additionally, a signal peptide that is “substantially identical with the signal peptide of a human CFB protein” can be a signal peptide that differs from the signal peptide of a human CFB protein by not more than 3 amino acids, a signal peptide that differs from the signal peptide of a human CFB protein by not more than 2 amino acids, or a signal peptide that differs from the signal peptide of a human CFB protein by not more than 1 amino acid. Alternatively or additionally, a signal peptide that is “substantially identical with the signal peptide of a human CFB protein” can be a signal peptide that differs from the signal peptide of a human CFB protein only at the N- or C- terminal portion of the protein, e.g., by having addition, deletion and / or substitution of amino acids at the N- and / or C- terminal portion of the signal peptide (i.e., within 2-4 amino acids from the N or C terminus of the signal peptide). Alternatively or additionally, a signal peptide that is “substantially identical with the signal peptide of a human CFB protein” can be a signal peptide that has one or more of the features delineated in above, e.g., a signal peptide that is at least 90% identical in sequence with the signal peptide of a human CFB protein and differs from the signal peptide of a human CFB protein only at the N- or C- terminal portion of the signal peptide by not more than 3 amino acids. In some embodiments, a human CFB protein comprises the amino acid sequence as set forth in SEQ ID NO: 6, and its signal peptide is composed of amino acids 1-25 of SEQ ID NO:6. In some embodiments, a humanized CFB gene encodes a protein having a signal peptide substantially identical with the signal peptide of the human CFB protein as set forth in SEQ ID NO: 6, i.e., substantially identical with amino acids 1-25 of SEQ ID NO: 6. For example, the humanized CFB gene encodes a protein having a signal peptide that comprises amino acids 1-25, 2-25, or 3-25 of SEQ ID NO: 6. In some embodiments, the humanized CFB gene encodes a protein having a signal peptide that comprises amino acids 1-25 of SEQ ID NO: 6.

[0040] In some embodiments, a genetically modified rodent comprises a humanized CFB gene in its genome, wherein the humanized CFB gene encodes a protein having a signal peptide that is substantially identical with the signal peptide of a rodent CFB protein (e.g., a mouse or rat CFB protein, such as an endogenous mouse or rat CFB protein). A signal peptide that is “substantially identical with the signal peptide of a rodent CFB protein” includes a signal peptide that is at least 90% identical in sequence with the signal peptide of a rodent CFB protein, or a signal peptide that is at least 95% identical in sequence with the signal peptide of a rodent CFB protein. A signal peptide that is “substantially identical with the signal peptide of a rodent CFB protein” can be a signal peptide that is 100% identical in sequence with the signal peptide of a rodent CFB protein. Alternatively or additionally, a signal peptide that is “substantially identical with the signal peptide of a rodent CFB protein” can be a signal peptide that differs from the signal peptide of a rodent CFB protein by not more than 3 amino acids, a signal peptide that differs from the signal peptide of a rodent CFB protein by not more than 2 amino acids, or a signal peptide that differs from the signal peptide of a rodent CFB protein by not more than 1 amino acid. Alternatively or additionally, a signal peptide that is “substantially identical with the signal peptide of a rodent CFB protein” can be a signal peptide that differs from the signal peptide of a rodent CFB protein only at the N- or C- terminal portion of the protein, e.g., by having addition, deletion and / or substitution of amino acids at the N- and / or C- terminal portion of the signal peptide (i.e., within 2-4 amino acids from the N or C terminus of the signal peptide).Alternatively or additionally, a signal peptide that is “substantially identical with the signal peptide of a rodent CFB protein” can be a signal peptide that has one or more of the features delineated in above, e.g., a signal peptide that is at least 90% identical in sequence with the signal peptide of a rodent CFB protein and differs from the signal peptide of a rodent CFB protein only at the N- or C- terminal portion of the signal peptide by not more than 3 amino acids. In some embodiments, a rodent CFB protein is a mouse CFB protein which comprises theamino acid sequence as set forth in SEQ TD NO: 12, and its signal peptide is composed of amino acids 1-24 of SEQ ID NO: 12. In some embodiments, a humanized CFB gene encodes a protein having a signal peptide substantially identical with the signal peptide of the mouse CFB protein as set forth in SEQ ID NO: 12, i.e., substantially identical with amino acids 1-24 of SEQ ID NO: 12. For example, the humanized CFB gene encodes a protein having a signal peptide that comprises amino acids 1-24, 1-23, or 1-22 of SEQ ID NO: 12. In some embodiments, the humanized CFB gene encodes a protein having a signal peptide that comprises amino acids 1-24 of SEQ ID NO: 12. In some embodiments, a rodent CFB protein is a rat CFB protein which comprises the amino acid sequence as set forth in SEQ ID NO: 17, and its signal peptide is composed of amino acids 1-24 of SEQ ID NO: 17. In some embodiments, a humanized CFB gene encodes a protein having a signal peptide substantially identical with the signal peptide of the rat CFB protein as set forth in SEQ ID NO: 17, i.e., substantially identical with amino acids 1-24 of SEQ ID NO: 17. For example, the humanized CFB gene encodes a protein having a signal peptide that comprises amino acids 1-24, 1-23, or 1-22 of SEQ ID NO: 17. In some embodiments, the humanized CFB gene encodes a protein having a signal peptide that comprises amino acids 1-24 of SEQ ID NO: 17.

[0041] In some embodiments, a genetically modified rodent comprises a humanized CFB gene in its genome, wherein the humanized CFB gene encodes a protein that is substantially identical with a human CFB protein. A protein that is “substantially identical with a human CFB protein” includes a polypeptide that is at least 95% identical in sequence with the human CFB protein, a polypeptide that is at least 98% identical in sequence with the human CFB protein, or a polypeptide that is at least 99% identical in sequence with the human CFB protein. A protein that is “substantially identical with a human CFB protein” can be a polypeptide that is 100% identical in sequence with the human CFB protein. Alternatively or additionally, a protein that is “substantially identical with a human CFB protein” can be a polypeptide that differs from the human CFB protein by not more than 5 amino acids, a polypeptide that differs from the human CFB protein by not more than 4 amino acids, a polypeptide that differs from the human CFB protein by not more than 3 amino acids, a polypeptide that differs from the human CFB protein by not more than 2 amino acids, or a polypeptide that differs from the human CFB protein by not more than 1 amino acid. Alternatively or additionally, a protein that is “substantially identical with a human CFB protein” can be a polypeptide that differs from the human CFB protein onlyat the N- or C- terminal portion of the sequence, e.g., by having addition, deletion and / or substitution of amino acids at the N- and / or C- terminal portion of the sequence (i.c., within 5-10 amino acids from the N or C terminus of the sequence). Alternatively or additionally, a protein that is “substantially identical with a human CFB protein” can be a polypeptide that has one or more of the features delineated in above, e.g., a polypeptide that is at least 95% identical in sequence with the human CFB protein and differs from the human CFB protein only at the N- or C- terminal portion by not more than 5 amino acids, or a polypeptide that is at least 98% identical in sequence with the human CFB protein and differs from the a human CFB protein only at the N- or C- terminal portion by not more than 3 amino acids. In some embodiments, a human CFB protein comprises the amino acid sequence as set forth in SEQ ID NO: 6, and a humanized CFB gene encodes a protein substantially identical with the human CFB protein as set forth in SEQ ID NO: 6. In some embodiments, the humanized CFB gene encodes a protein identical in sequence with the human CFB protein.

[0042] In some embodiments, the humanized CFB gene in the genome of a genetically modified rodent includes a nucleotide sequence of a human CFB gene (“a human CFB nucleotide sequence”) and a nucleotide sequence of a rodent CFB gene (“a rodent CFB nucleotide sequence”, such as an endogenous rodent CFB nucleotide sequence), wherein the human CFB nucleotide sequence encodes at least a substantial portion of the mature protein of a human CFB protein. Examples of a substantial portion of the mature protein of a human CFB can include amino acids 26-764, 27-764, or 28-764 of SEQ ID NO: 6. In some embodiments, a substantial portion of the mature protein of a human CFB protein is the full mature protein as set forth in amino acids 26-764 of SEQ ID NO: 6. In some embodiments, the human CFB nucleotide sequence is a cDNA sequence. In some embodiments, the human CFB nucleotide sequence in a humanized CFB gene encodes the mature protein of a human CFB protein (e.g., a human CFB protein as defined in SEQ ID NO: 6). In some embodiments, the human CFB nucleotide sequence is a genomic fragment of a human CFB gene. In some embodiments, the human CFB nucleotide sequence is a genomic fragment of a human CFB gene comprising the start codon in exon 1 through the stop codon in exon 18.

[0043] In some embodiments, the human CFB nucleotide sequence is a genomic fragment of a human CFB gene comprising the 5’ untranslated region (UTR) in exon 1 of the human CFB gene. In some embodiments, the 5’ UTR of a human CFB gene is not included in a humanizedCFB gene. Tn some embodiments, the genomic fragment of a human CFB gene included in the humanization comprises, in addition to the 5’ UTR of the human CFB gene, a 5’ genomic sequence upstream of the 5’ UTR of the human CFB gene. In some embodiments, the 5’ genomic sequence contains one or more transcriptional regulatory sequences, such as promoter and enhancer. In some embodiments, the 5’ genomic sequence and the 5’ UTR contain one or more or all of the transcriptional regulatory sequences of the human CFB gene, such as the promoter and enhancer(s). In some embodiments, the 5’ genomic sequence upstream of the 5’ UTR of the human CFB gene is of a length of at least 100-200 bp. In some embodiments, the 5’ genomic sequence upstream of the 5’ UTR of the human CFB gene is of a length of at least 200- 400 bp. In some embodiments, the 5’ genomic sequence upstream of the 5’ UTR of the human CFB gene is of a length of at least 500 bp. In some embodiments, the 5’ genomic sequence upstream of the 5’ UTR of the human CFB gene is of a length of at least 1000 bp. In an embodiment, the 5’ genomic sequence upstream of the 5’ UTR of the human CFB gene is of a length of about 204 bp. In another embodiment, the 5’ genomic sequence upstream of the 5’ UTR of the human CFB gene is of a length of about 590 bp (or beginning at about 717bp upstream of the start codon).

[0044] In some embodiments, the human CFB nucleotide sequence is a genomic fragment of a human CFB gene comprising the 3’ UTR, in full or in part, (in exon 18) of the human CFB gene. In some embodiments, the human CFB nucleotide sequence is a genomic fragment of a human CFB gene comprising a 5’ portion of the 3’ UTR of the human CFB gene. The 5’ portion of the 3’ UTR of the human CFB gene may be about 5-20 bp. In some embodiments, the full length of the 3’ UTR of a human CFB gene can be included, e.g., the 3’ UTR of 54 bp of the human CFB gene as set forth in SEQ ID NO: 1. In some embodiments, the 3’ UTR of a human CFB gene is not included in a humanized CFB gene.

[0045] In some embodiments, the humanized CFB gene in the genome of a genetically modified rodent includes a rodent CFB nucleotide sequence and a human CFB nucleotide sequence, wherein the rodent CFB nucleotide sequence comprises a 5’ genomic sequence upstream of the 5’ UTR of a rodent CFB gene (e.g., an endogenous rodent CFB gene). In some embodiments, the 5’ genomic sequence includes one or more 5’ transcription regulatory sequences, such as a rodent CFB promoter or an enhancer. In some embodiments, the rodent CFB nucleotide sequence includes the 5’ UTR in full or in part of a rodent CFB gene. In some embodiments, therodent CFB nucleotide sequence includes the 3’ UTR in full or in part of a rodent CFB gene. In some embodiments, the rodent CFB nucleotide sequence includes a 3’ portion of the 3’ UTR of a rodent CFB gene, e.g., a 3’ portion of about 10-45 bp, or 15-30bp, or 15-25 bp. In an embodiment, the rodent CFB nucleotide sequence is a mouse CFB nucleotide sequence which includes a 3’ portion of about 22 bp of the 3’ UTR of a mouse CFB gene (e.g., the mouse CFB gene as defined in SEQ ID NO: 7).

[0046] In some embodiments, a humanized CFB gene comprises a human CFB genomic DNA which includes a 5’ genomic sequence upstream of the 5’ UTR, the 5’ UTR, and the start codon through the stop codon of the human CFB gene, wherein the human CFB genomic DNA is followed by and operably linked to a rodent CFB 3’ UTR or a 3’ portion thereof. In some embodiments, the 5’ genomic sequence upstream of the 5’ UTR, and the 5’ UTR, in combination, include one or more or all of the transcription regulatory sequences (e.g., promoter and / or enhancer) of the human CFB gene. In some embodiments, the humanized CFB gene also comprises a 5’ genomic sequence of a rodent CFB gene, operably linked to the human CFB genomic DNA, wherein the 5’ genomic sequence of the rodent CFB gene includes one or more of the native 5’ transcription regulatory sequences of the rodent CFB gene.

[0047] In some embodiments, the humanized CFB gene is at an endogenous rodent CFB locus. In some embodiments, the humanized CFB gene is at a locus other than an endogenous rodent CFB locus; e.g., as a result of random integration. In some embodiments, the humanized CFB gene is at a ROSA26 locus (which locus is as described by Zambrowicz et al., 1997, PNAS USA 94:3789-3794, which is incorporated herein by reference). In some embodiments where the humanized CFB gene is at a locus other than an endogenous rodent CFB locus, the rodents are incapable of expressing a rodent CFB protein, e.g., as a result of inactivation (e.g., deletion in full or in part) of the endogenous rodent CFB gene.

[0048] In some embodiments where a humanized CFB gene is at an endogenous rodent CFB locus, the humanized CFB gene results from a replacement of a nucleotide sequence of an endogenous rodent CFB gene at the endogenous rodent CFB locus with a nucleotide sequence of a human CFB gene.

[0049] In some embodiments, the nucleotide sequence of an endogenous rodent CFB gene at an endogenous rodent CFB locus that is being replaced is a genomic fragment of an endogenousrodent CFB gene that encodes at least a substantial portion of the mature protein of the rodent CFB protein. In some embodiments, the rodent is a mouse, and the mouse CFB genomic fragment being replaced encodes at least a substantial portion of the mature protein of the endogenous mouse CFB protein. For example, in the case of a mouse CFB protein of SEQ ID NO: 12, examples of a substantial portion of the mature protein can include amino acids 25-763, 26-763, or 27-763 of SEQ ID NO: 12. In some embodiments, a substantial portion of the mature protein of a mouse CFB protein comprises the full mature protein as set forth in amino acids 25- 763 of SEQ ID: 12. In some embodiments, the genomic fragment of an endogenous rodent CFB gene being replaced encodes the full rodent CFB protein, i.e., including the rodent CFB signal peptide and mature protein. In some embodiments, the genomic fragment of an endogenous rodent CFB gene being replaced also includes the 5’ UTR in full or in pail, and / or the 3’ UTR in full or in part. In some embodiments, the genomic fragment of an endogenous rodent CFB gene being replaced also includes the 5’ UTR in full and a 5’ portion of the 3’ UTR (e.g., 25-35 bp or about 31 bp at the 5’ portion of the 3’ UTR). In some embodiments, the genomic fragment of an endogenous rodent CFB gene being replaced also includes a 5’ genomic sequence upstream of the 5’ UTR, which may include one or more endogenous rodent transcription regulatory sequences.

[0050] In some embodiments, the nucleotide sequence of a human CFB gene that replaces a genomic fragment of a rodent CFB gene at an endogenous rodent CFB locus is a cDNA sequence. In some embodiments, the human CFB nucleotide sequence that replaces a genomic fragment of a rodent CFB gene at an endogenous rodent CFB locus is a genomic fragment of a human CFB gene. In some embodiments, a genomic fragment of a human CFB gene that replaces a genomic fragment of a rodent CFB gene at an endogenous rodent CFB locus includes exons, in full or in part, of a human CFB gene, that encode at least a substantial portion of the mature protein of the human CFB protein. Examples of a substantial portion of the mature protein of a human CFB have been described above, e.g., amino acids 26-764, 27-764, or 28-764 of SEQ ID NO: 6. In some embodiments, a substantial portion of the mature protein of a human CFB protein comprises the full mature sequence as set forth in amino acids 26-764 of SEQ ID NO: 6. In some embodiments, a genomic fragment of a human CFB gene that replaces a genomic fragment of a rodent CFB gene at an endogenous rodent CFB locus includes exons, in full or in part, of a human CFB gene, that encode the human CFB protein, i.e., including thehuman CFB signal peptide and mature protein sequence. In some embodiments, a genomic fragment of a human CFB gene that replaces a genomic fragment of a rodent CFB gene at an endogenous rodent CFB locus also includes the 5’ UTR in full or in part, and / or the 3’ UTR in full or in part. In some embodiments, a genomic fragment of a human CFB gene that replaces the genomic fragment of an endogenous rodent CFB gene also includes a 5’ genomic sequence upstream of the 5’ UTR, which may include one or more transcription regulatory sequences (such as promoter and / or enhancer) of the human CFB gene. In some embodiments, the 5’ genomic sequence upstream of the 5’ UTR of the human CFB gene which is included in the human genomic fragment is of a length of at least 100-200 bp. In some embodiments, the 5’ genomic sequence upstream of the 5’ UTR of the human CFB gene is of a length of at least 200- 400 bp. In some embodiments, the 5’ genomic sequence upstream of the 5’ UTR of the human CFB gene is of a length of at least 500 bp. In some embodiments, the 5’ genomic sequence upstream of the 5’ UTR of the human CFB gene is of a length of at least 1000 bp. In an embodiment, the 5’ genomic sequence upstream of the 5’ UTR of the human CFB gene is of a length of about 204 bp. In another embodiment, the 5’ genomic sequence upstream of the 5’ UTR of the human CFB gene is of a length of about 590 bp (or beginning at about 717bp upstream of the start codon).

[0051] In some embodiments, the rodent is a mouse, and a genomic fragment of an endogenous mouse CFB gene comprising exon 1 (including the 5’ UTR) through the stop codon in exon 18 of the mouse CFB gene has been replaced with a genomic fragment of a human CFB gene comprising exon 1 (including the 5’ UTR) through the stop codon in exon 18. In some embodiments, the mouse genomic fragment being replaced also includes a 5’ portion (e.g., 25-35 bp, or about 31 bp) of the 3’ UTR. In some embodiments, the genomic fragment of the human CFB gene that replaces a mouse genomic fragment also includes a 5 ’ genomic sequence upstream of the 5’ UTR, wherein the 5’ genomic sequence is about 590 bp and includes one or more transcription regulatory sequences (such as promoter and / or enhancer) of the human CFB gene. As a result of the replacement, a humanized CFB gene is formed at the endogenous mouse CFB locus. In some embodiments, the humanized CFB gene comprises exon 1 through the stop codon of a human CFB gene and a 3’ portion (about 22 bp) of the 3’ UTR of the mouse CFB gene.

[0052] In some embodiments, a rodent provided herein is heterozygous for a humanized CFB gene in its genome. In some embodiments, a rodent provided herein is homozygous for a humanized CFB gene in its genome.

[0053] In some embodiments, a humanized CFB gene results in an expression of a human CFB mature protein in the serum of the rodent.

[0054] In some embodiments, rodents disclosed herein are incapable of expressing a rodent CFB protein, e.g., as a result of inactivation (e.g., deletion in full or in pail) or replacement (in full or in part) of the endogenous rodent CFB gene.Rodent Species and Strains

[0055] In some embodiments, rodents of this disclosure include, as non-limiting examples, a mouse, a rat, and a hamster. In some embodiments, a rodent is selected from the superfamily Muroidea. In some embodiments, a rodent of this disclosure is from a family selected from Calomyscidae (e.g., mouse-like hamsters), Cricetidae (e.g., hamster, New World rats and mice, voles), Muridae (true mice and rats, gerbils, spiny mice, crested rats), Nesomyidae (climbing mice, rock mice, with-tailed rats, Malagasy rats and mice), Platacanthomyidae (e.g., spiny dormice), and Spalacidae (e.g., mole rates, bamboo rats, and zokors). In some embodiments, a rodent of this disclosure is selected from a true mouse or rat (family Muridae), a gerbil, a spiny mouse, and a crested rat. In some embodiments, a mouse of this disclosure is from a member of the family Muridae.

[0056] In some embodiments, a rodent is a mouse. In some embodiments, the rodent is a mouse of a C57BL strain selected from C57BL / A, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL / 10ScSn, C57BL / 10Cr, and C57BL / 01a. In some embodiments, a rodent is a mouse of a 129 strain selected from the group consisting of a strain that is 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129Sl / Svlm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129 / SvJae, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, 129T2 (see, e.g., Festing et al., 1999, Mammalian Genome 10:836; Auerbach et al., 2000, Biotechniques 29(5): 1024- 1028, 1030, 1032; both incorporated herein by reference in their entireties). In some embodiments, a rodent is a mouse that is a mix of a 129 strain and a C57BL / 6 strain. In some embodiments, a rodent is a mouse that is a mix of aforementioned 129 strains, or a mix of aforementioned BL / 6 strains. In some embodiments, a rodent is a mouse of aBALB strain, e.g., BALB / c strain. In some embodiments, a rodent is a mouse that is a mix of aBALB strain and another aforementioned strain.

[0057] In some embodiments, a rodent is a rat. In some certain embodiments, a rat is selected from a Wistar rat, an LEA strain, a Sprague Dawley strain, a Fischer strain, F344, F6, and Dark Agouti. In some embodiments, a rat strain as described herein is a mix of two or more strains selected from the group consisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and Dark Agouti.Tissues and Cells of Genetically Modified Rodents

[0058] In some embodiments, disclosed herein is an isolated rodent cell or tissue whose genome comprises a humanized CFB gene.

[0059] In some embodiments, a tissue is selected from adipose, bladder, brain, breast, bone marrow, eye, heart, intestine, kidney, liver, lung, lymph node, muscle, pancreas, plasma, serum, skin, spleen, stomach, thymus, testis, ovum, and a combination thereof.

[0060] In some embodiments, an isolated rodent cell is a rodent embryonic stem cell. In some embodiments, an isolated rodent cell is a rodent egg, or a rodent sperm.Compositions and Methods for Making Humanized Rodents

[0061] Disclosed herein is a targeting vector (or nucleic acid construct) comprising a human CFB nucleotide sequence, desired to be integrated into a rodent locus to form a humanized CFB gene as described herein.

[0062] In some embodiments, a targeting vector comprises a human CFB nucleotide sequence which encodes at least a substantial portion of the mature protein sequence of a human CFB protein as described hereinabove. In some embodiments, the human CFB nucleotide sequence encodes a polypeptide comprising amino acids 26-764 of SEQ ID NO: 6, or a polypeptide comprising SEQ ID NO: 6. In some embodiments, the human CFB nucleotide sequence comprises exon 1 through the stop codon in exon 18 of a human CFB gene.

[0063] The targeting vector also includes 5’ and 3’ rodent sequences flanking the human nucleotide sequence to be integrated, also known as 5’ and 3’ homology arms, that mediate homologous recombination and integration of the human nucleotide sequence into the targetrodent locus (e.g., an endogenous rodent CFB locus), so as to form a humanized gene as described herein above. Typically, the 5’ and 3’ flanking rodent sequences in a targeting vector are identical or substantially identical (e.g., at least 98% or at least 99% identical) to the nucleotide sequences that flank the corresponding rodent nucleotide sequence at the target rodent locus that is to be replaced by the human nucleotide sequence. In some embodiments, a targeting vector comprises a humanized CFB gene as described herein above. In some embodiments, a targeting vector comprises a humanized CFB gene comprising a human CFB nucleotide sequence and a rodent CFB nucleotide sequence, as described herein above. In some embodiments, a targeting vector comprises a human CFB genomic fragment comprising a 5’ genomic sequence and exon 1 through the stop codon in exon 18 of a human CFB gene, wherein the human CFB genomic fragment is flanked by 5’ and 3’ rodent homology arms, with the 3’ rodent homology arm comprises the 3’ UTR in full or in part of a rodent CFB gene.

[0064] In some embodiments, a targeting vector comprises a selection marker gene. The selection marker gene can be inserted in an intron of the human genomic sequence to be integrated. In some embodiments, a selection marker gene is provided as a self-deleting cassette which can be deleted after a successful integration of the human nucleotide sequence.

[0065] In an embodiment, a targeting vector is generated from a bacterial artificial chromosome (BAC) clone carrying a rodent CFB genomic DNA using bacterial homologous recombination and VELOCIGENE® technology (see, e.g., U.S. 6,586,251 and Valenzuela et al. (2003) Nature Biotech. 2 l(6):652-659, incorporated herein by reference in their entireties). As a result of bacterial homologous recombination, a rodent genomic sequence is deleted from the BAC clone, and a human nucleotide sequence is inserted, resulting in a modified BAC clone carrying the human nucleotide sequence, flanked with 5’ and 3’ rodent homology arms. In some embodiments, the human nucleotide sequence can be a cDNA sequence or a human genomic DNA. The modified BAC clone, once linearized, can be introduced into rodent embryonic stem (ES) cells.

[0066] In some embodiments, the present invention provides use of a targeting vector as described herein to make a modified rodent embryonic stem (ES) cell. A targeting vector can be introduced into a rodent ES cell by, e.g., electroporation. Both mouse ES cells and rat ES cells have been described in the art. See, e.g., US 7,576,259, US 7,659,442, US 7,294,754, and US2008-0078000 A1 (all of which are incorporated herein by reference in their entireties) that describe mouse ES cells and the VELOCIMOUSE® method for making a genetically modified mouse; US 2014 / 0235933 Al (Regeneron Pharmaceuticals, Inc.), US 2014 / 0310828 Al (Regeneron Pharmaceuticals, Inc.), Tong et al. (2010) Nature 467:211-215, and Tong et al. (2011) Nat Protoc. 6(6): doi: 10.1038 / nprot.2011.338 (all of which are incorporated herein by reference in their entireties) that describe rat ES cells and methods for making a genetically modified rat, which can be used to make a modified rodent embryo, which in turn can be used to make a rodent animal.

[0067] In some embodiments, ES cells having a desirable human nucleotide sequence (e.g., a human CFB nucleotide sequence) integrated in the genome can be selected. In some embodiments, ES cells are selected based on loss of rodent allele and / or gain of human allele assays. In some embodiments, selected ES cells are then used as donor ES cells for injection into a pre-morula stage embryo (e.g., 8-cell stage embryo) by using the VELOCIMOUSE® method (see, e.g., US 7,576,259, US 7,659,442, US 7,294,754, and US 2008-0078000 Al, all of which are incorporated by reference in their entireties), or methods described in US 2014 / 0235933 Al and US 2014 / 0310828 Al, which are both incorporated by reference in their entireties. In some embodiments, an embryo comprising the donor ES cells is incubated until the blastocyst stage and then implanted into a surrogate mother to produce F0 progenies full derived from correctly targeted ES cells. Rodent pups bearing a human nucleotide sequence can be identified by genotyping of DNA isolated from tail snips using loss of rodent allele and / or gain of human allele assays.

[0068] In some embodiments, rodents heterozygous for a humanized gene can be crossed to generate homozygous rodents.

[0069] A humanized rodent as described herein (i.e., a rodent comprising a humanized CFB gene) can be bred or crossed with another rodent. Accordingly, methods of breeding as well as progenies obtained from such breeding are also embodiments of this disclosure.

[0070] In some embodiments, a method is provided which comprises breeding a first rodent as described hereinabove, e.g., a rodent whose genome comprises a humanized CFB gene, with a second rodent, resulting in a progeny rodent whose genome comprises the humanized CFB, gene. The progeny may possess other desirable phenotypes or genetic modifications inheritedfrom the second rodent used in the breeding, e.g., a rodent comprising a humanized C3 allele such as those described in U.S. Patent 9,795,121 B2 (Rcgcncron Pharmaceuticals, Inc.) and U.S. Patent 10,765,762 B2 (Regeneron Pharmaceuticals, Inc.), the entire contents of both of which are incorporated herein by reference.

[0071] The C3 gene encodes the serum complement protein C3, which plays a central role in the activation of the classical and alternative complement activation pathways.

[0072] The human C3 gene is located on chromosome 19 at 19pl3.3-pl3.2. The human C3 gene has 41 exons and encodes a precursor polypeptide of 1663 amino acids, including a 22 amino acid signal peptide, a 645 amino acid P chain and a 992 amino acid a chain. During complement activation the a chain is cleaved, thereby generating 9 different peptides, including a 77 amino acid C3a, which is a potent pro-inflammatory anaphylatoxin.

[0073] The mouse C3 gene is located on chromosome 17 at 17 29.72 cM19. The mouse C3 gene has 41 exons and encodes a precursor polypeptide of 1663 amino acids, including a 24 amino acid signal peptide, a 642 amino acid |3 chain and a 993 amino acid a chain. During complement activation the a chain is cleaved, thereby generating 9 different peptides, including a 78 amino acid C3a, which is a potent pro-inflammatory anaphylatoxin.

[0074] The C3 gene is conserved between several species, including primates, e.g., chimpanzee, Rhesus monkey, other mammals, e.g., dog, cow, rodent, e.g., mouse, chicken, zebrafish and frog.

[0075] As described above, when CFB associates with C3b, it is cleaved by factor D to release an N-terminal fragment (Ba) and the carboxyl-terminal fragment (Bb). Bb remains bound to C3b, thereby forming the C3-convertase and ultimately the C5-convertase.

[0076] In some embodiments, a human C3 genomic DNA containing 5’ regulatory elements and all the coding exons 1 through 41 of the human C3 gene has replaced a rodent (e.g., mouse) C3 genomic DNA spanning 5’ regulatory elements and all of the coding exons 1 through 41, so as to form a humanized C3 gene at the endogenous rodent C3 locus. In some embodiments, the progeny rodent is heterozygous for the humanized CFB gene from the first rodent. In some embodiments, the progeny rodent is homozygous for the humanized CFB gene from the first rodent.

[0077] In some embodiments, a progeny rodent is provided whose genome comprises a humanized CFB gene and a humanized C3 gene.

[0078] In some embodiments, the progeny rodent is heterozygous for the humanized CFB gene from the first rodent and / or heterozygous for the humanized C3 gene from the second rodent. In some embodiments, the progeny rodent is homozygous for the humanized CFB gene from the first rodent and homozygous for the humanized C3 gene from the second rodent.

[0079] In some embodiments, disclosed herein is an in vitro method for generating a genetically modified rodent cell, comprising introducing into a rodent cell a targeting vector comprising a human CFB nucleic sequence that encodes at least a substantial portion of the mature protein sequence of a human CFB protein, flanked by rodent homology arms that mediate integration of the human CFB nucleotide sequence into an endogenous rodent CFB locus, which results in replacement of a rodent CFB genomic DNA with the human CFB nucleic acid sequence to form a humanized CFB gene as described herein, thereby generating a genetically modified rodent cell. In some embodiments, the rodent cell is mouse cell or a rat cell. In some embodiments, the rodent cell is a rodent ES cell, and the method generates a genetically modified rodent ES cell.Methods Employing the Humanized Rodents

[0080] Rodents disclosed herein provide a useful in vivo system and source of biological materials for identifying and testing compounds for their potential to treat human diseases, including complement related indications such as AMD.

[0081] In some embodiments, a genetically modified rodent animal disclosed herein is used to evaluate a candidate compound that targets human CFB or another complement molecule. Candidate compounds can be, without limitation, small molecule chemical compounds, antibodies, proteins, inhibitory nucleic acids, or any combination thereof. In some embodiments, a small molecule compound is less than about 2000 Daltons in size, alternatively less than about 1500, 750, 500, 250 or 200 Daltons in size. In some embodiments, the candidate compound is an antibody. In some embodiments, the candidate compound is an antibody that specifically binds to human CFB. In some embodiments, the candidate compound is an antibody that specifically binds to another component of the complement system (e.g., C3). In some embodiments, the candidate compound is an inhibitory nucleic acid. The inhibitory nucleic acid can be, without limitation, any of an antisense oligonucleotide, a small inhibitory RNA (siRNA), or a ribozyme. In some embodiments, the inhibitory nucleic acid is an inhibitory nucleic acid targeting humanCFB. Tn some embodiments, the inhibitory nucleic acid is an inhibitory nucleic acid targeting another component of the complement system (c.g., C3).

[0082] A candidate compound such as an anti-human CFB antibody or an inhibitory nucleic acid can be administered to a rodent disclosed herein at various doses (e.g., 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / mg, 7.5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg or more). The agents may be dosed via any desired route of administration (e.g., subcutaneously, intravenously, intramuscular, intraperitoneal, etc.).

[0083] In some embodiments, genetically modified rodent animals disclosed herein are used for assessing pharmacokinetic properties of a candidate agent such as an anti-human CFB antibody inhibitory nucleic acids. The agent is administered to a genetically modified rodent animal. Blood is isolated from the animal at various time points (e.g., 0 hr, 6 hr, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or up to 30 or more days). Various assays may be performed to determine the pharmacokinetic properties which can include, but are not limited to, how an animal processes the drug into various metabolites (or detection of the presence or absence of one or more drug metabolites, including, toxic metabolites), drug halflife, circulating levels of drug after administration (e.g., serum concentration of drug), anti-drug response (e.g., anti-drug antibodies), drug absorption and distribution, route of administration, routes of excretion and / or clearance of the drug.

[0084] In some embodiments, genetically modified rodent animals disclosed herein are used to evaluate a candidate compound (e.g., a compound targeting human CFB or another component molecule) in order to determine whether the candidate compound has an effect on the rodent animals, e.g., to determine whether systemic administration of an agent targeting human CFB has an effect on the ocular CFB levels. Comparison can be made to a control rodent animal that is not administered with the agent or is administered with a control agent. Candidate compounds suitable to be evaluated in this method can be, without limitation, small molecule chemical compounds, antibodies, proteins, inhibitory nucleic acids, or any combination thereof. In some embodiments, a small molecule compound is less than about 2000 Daltons in size, alternatively less than about 1500, 750, 500, 250 or 200 Daltons in size. In some embodiments, the candidate compound is an antibody. In some embodiments, the candidate compound is an antibody that specifically binds to human CFB. In some embodiments, the candidate compound is an antibody that specifically binds to another component of the complement system (e.g., C3). In someembodiments, the candidate compound is an inhibitory nucleic acid. The inhibitory nucleic acid can be, without limitation, any of an antisense oligonucleotide, a small inhibitory RNA (siRNA), or a ribozyme. In some embodiments, the inhibitory nucleic acid is an inhibitory nucleic acid targeting human CFB. In some embodiments, the inhibitory nucleic acid is an inhibitory nucleic acid targeting another component of the complement system (e.g., C3).

[0085] The present description is further illustrated by the following examples, which should not be construed as limiting in any way. The contents of all cited references (including literature references, issued patents, and published patent applications as cited throughout this application) are hereby expressly incorporated by reference in their entireties.EXAMPLESExample 1. Humanization of an endogenous mouse CFB gene

[0086] An example of a targeting strategy for humanization of a mouse CFB gene is depicted in FIG. 1A. A targeting vector for humanization of an endogenous mouse CFB gene was constructed using bacterial artificial chromosome (BAC) clones and VELOC1GENE® technology (see, e.g., U.S. Patent No. 6,586,251 and Valenzuela et al. (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis, Nature Biotechnology 21 (6):652-659; all of the contents of which are incorporated herein by reference).

[0087] Briefly, a BAC clone containing a mouse CFB gene was modified as follows. First, a DNA fragment was generated to include a mouse 5’ homology nucleotide sequence, a human CFB genomic DNA (containing a 5’ sequence of about 590 bp upstream of the 5’ UTR, exon 1 (including the 5’ UTR) through the stop codon in exon 18 (i.e., without including the 3’ UTR), and their respective introns), a self-deleting Neomycin resistance selection marker cassette (“SDC UbNeo PmCi2”) inserted within intron 10, and a 3’ mouse homology sequence. This DNA fragment includes a 5’ sequence of about 717 bp in total upstream of the start codon of human CFB which contains predicted human CFB promoter elements. This DNA fragment was used to modify the BAC clone containing the mouse CFB gene through homologous recombination in bacterial cells. As a result, a mouse CFB genomic fragment of 6123 bp in the BAC clone, which includes some of the 5’ regulatory elements and the entire 5’ UTR in exon 1 through the first 31 bp of the 3’ UTR in exon 18, was deleted and replaced by the human CFB genomic fragment. The deleted mouse genomic sequence corresponds to coordinates:chrl 7:34,856,396-34,862,518; and the inserted human genomic sequence corresponds to coordinates: hg38:chr6:31, 945, 505-31, 952, 030. The junction sequences arc also set forth in Table 4 below.Table 4

[0088] The modified BAC clone containing the human CFB genomic fragment, as described above, was used to electroporate mouse embryonic stem (ES) cells to create modified ES cells comprising a humanized CFB gene at the endogenous CFB locus. Positively targeted ES cells containing a humanized CFB gene were identified by an assay (Valenzuela et al., supra) that detected the presence of the human CFB sequences and confirmed the loss and / or retention of mouse CFB sequences. A correctly targeted ES cell clone (MAID 7610) was selected as a donor cell and injected into a pre-morula stage embryo (e.g., 8-cell stage embryo) by using the VELOCIMOUSE® method (see, e.g., Poueymirou et al., 2007, Nature Biotech. 25(1):91-99, US 7,576,259, US 7,659,442, US 7,294,754, and US 2008-0078000 Al, all of which are incorporated herein by reference in their entireties), or methods described in US 2014 / 0235933 Al and US 2014 / 0310828 Al (both of which incorporated herein by reference in their entireties). The embryo comprising the donor ES cells was incubated until blastocyst stage and then implanted into a surrogate mother to produce an F0 rodent fully derived from the donor ES cells. Mice bearing a humanized CFB allele were confirmed and identified by genotyping of DNA isolated from tails snips using a modification of allele assay (Valenzuela et al., supra) that detects the presence of the human CFB gene sequences. The humanized CFB locus before and afterdeletion of the SDC UbNeo PmCi2 cassette is depicted in FIG. IB (MAID 7610 and MAID 7611, respectively). Mice heterozygous for the humanized CFB locus were crossed to obtain homozygotes.Example 2. Double humanized C3 / CFB mice

[0089] Generation of C3 humanized mice is described in U.S. Patent 9,795,121 B2 (Regeneron Pharmaceuticals, Inc.) and U.S. Patent 10,765,762 B2 (Regeneron Pharmaceuticals, Inc.), the entire contents of both of which are incorporated herein by reference.

[0090] MAID 6149 mice - Replacement with Human C3 Promoter and Coding Exons 1 through 41: The human C3 gene containing 5’ regulatory elements and all of the coding exons 1 through 41 of the human C3 gene replaced the murine C3 gene locus spanning 5’ regulatory elements and all of the coding exons 1 through 41. Briefly, a targeted deletion of 25 kb of the mouse C3 gene was generated in mouse ES cells by replacement of coding exons 2 through 41 and including 900 bp 3’ to the polyadenylation site with a floxed neomycin cassette. The resultant mouse ES cells, 12132 ES cells, are a heterozygous C3 knockout. 12132 cells were used to generate C3 knockout mice according to procedures known in the ail. A targeting construct was generated containing mouse C3 upstream and downstream homology arms flanking a human C3 genomic sequence extending from 5’ regulatory elements upstream of coding exon 1 through coding exon 41 (including the 3’ untranslated region) and a floxed hygromycin selection cassette, and electroporated into 12132 ES cells. Correctly targeted ES cells (MAID 6148) were further electroporated with a transient Cre-expressing vector to remove the drug selection cassette. Targeted ES cell clones without drug cassette (MAID 6149) were introduced into an 8-cell stage mouse embryo. F0 mice fully derived from the donor ES cell bearing the humanized C3 gene were identified by genotyping for loss of mouse allele and gain of human allele using a modification of allele assay (see, Valenzuela et al. (2003)). MAID 6149 mice contain about 53.4 kb of human sequence including about 9 kb upstream of human C3 exon 1, the entire human C3 gene of about 42.8 kb, and about 1.5 kb of human sequence downstream of the polyA signal; and this 53.4 kb human sequence has replaced about 30.6 kb of mouse sequence including about 6.5 kb of mouse sequence upstream of mouse C3 exon 1, and the mouse C3 gene of about 24.1 kb from the beginning of exon 1 to the stop codon.

[0091] MAID 6156 mice - Replacement with Human C3 Coding Exons 2 through 41: The human C3 gene containing coding exons 2 through 41 of the human C3 gene replaced the murineC3 gene locus spanning coding exons 2 through 41 . The methods described above were basically used to replace the mouse C3 gene sequences with human C3 gene sequences. Briefly, a targeted deletion of 25 kb of the mouse C3 gene was generated in mouse ES cells by replacement of coding exons 2 through 41 and including 900 bp 3’ to the poly adenylation site with a floxed neomycin cassette. The resultant mouse ES cells, 12132 ES cells, are a heterozygous C3 knockout. A targeting construct was generated containing mouse C3 upstream and downstream homology arms flanking a human C3 sequence extending from upstream of coding exon 2 through coding exon 41 and the 3’ untranslated region and a floxed hygromycin selection cassette, and electroporated into 12132 ES cells. Correctly targeted ES cells (MAID 6155) were further electroporated with a transient Cre-expressing vector to remove the drug selection cassette. Targeted ES cell clones without drug cassette (MAID 6156) were introduced into an 8-cell stage mouse embryo. F0 mice fully derived from the donor ES cell bearing the humanized C3 gene were identified by genotyping for loss of mouse allele and gain of human allele as described above.

[0092] Homozygous MAID 6149 C3 humanized mice were crossed with homozygous MAID 7611 CFB humanized mice to obtain double humanized C3 / CFB mice that were used in the following experiments.Example 3. Reduction of Complement Factor B using CRISPR / Cas9

[0093] Purpose: Age-related macular degeneration (AMD), a leading cause of severe vision loss in people over 50, presents in two forms: dry and wet AMD. The risk of dry AMD can increase with overactivation of the alternative pathway (AP) of the complement system, a process dependent on the proteolytic cleavage of factor B (CFB). This study aimed to investigate the impact of liver knockdown CFB on ocular CFB levels.

[0094] Methods: C57BL / 6J mice or double humanized C3 / CFB mice as described above in Example 2 were used in the current study. Guide RNAs targeting mouse or human CFB were encapsulated in lipid nanoparticles along with CRISPR / Cas9 and administered intravenously to C57BL / 6J mice or double humanized C3 / CFB mice (Day 0) to knockdown CFB in the naive or humanized mice. Plasma samples were collected pre-injection (Day -5) and post-injection (at 4 weeks, 8 weeks and 10 weeks), and the eye and liver tissues were harvested at 10 weeks. The mRNA levels were quantified using quantitative polymerase chain reaction (qPCR), while mouseCfb protein levels were detected using Western blot (WB). Human CFB levels were determined using ELISA.

[0095] Results: In C57BL / 6J mice, simple Western automated Western Blot (WES) showed a 90% reduction in plasma Cfb (Figure 2A, B) and over 70% knockdown in ocular Cfb (FIG. 2C, 2D). qPCR showed more than 60-80% knockdown in liver Cfb mRNA (FIG. 3A), 60% Indel events were found by NGS sequencing (FIG. 3B). No changes in the ocular CFB mRNA were found (FIG. 3C). In double humanized C3 / CFB mice, ELISA detected a 62% reduction in liver CFB (FIG. 4A), an 83% reduction in plasma CFB (Figure 4B), and a 45% reduction in ocular CFB (FIG. 4C). Significant correlation (p=0.0069) was observed between serum & liver with a in huC3 / huCFB mice (FIG. 5A) suggested CFB found in the serum is originating from the liver. Significant correlation (p= 0.0066) was also observed in eye & serum suggested CFB circulating from the serum may contribute to the eye (FIG. 5B). Strong correlation (p= 0.047) observed between eye and liver suggests that ocular CFB may be originating from the liver (FIG. 5C).

[0096] Conclusions: The use of CRISPR / Cas9 allowed for successful knockdown of CFB in the liver and serum, which corresponded with decreased ocular CFB levels. This suggests that ocular CFB is primarily derived from the bloodstream rather than local production in the eye. These findings indicate that systemic targeting of CFB may be an appropriate approach for treating ocular complement diseases such as AMD.

Claims

WHAT IS CLAIMED IS:

1. A genetically modified rodent animal, wherein the genome of the genetically modified rodent comprises: a humanized CFB gene comprising: a rodent CFB nucleic acid sequence; and a human CFB nucleic acid sequence, wherein the humanized CFB gene encodes a polypeptide comprising a mature protein sequence substantially identical to the mature protein sequence of a human CFB protein.

2. The genetically modified rodent animal of claim 1, wherein the polypeptide comprises a signal peptide substantially identical to the signal peptide of the human CFB protein or the signal peptide of a rodent CFB protein.

3. The genetically modified rodent animal of claim 1 or 2, wherein the polypeptide is substantially identical in sequence to the human CFB protein.

4. The genetically modified rodent animal of any one of claims 1-3, wherein the human CFB nucleic acid sequence comprises a nucleotide sequence of a human CFB gene beginning at the start codon and ending at the stop codon of the human CFB gene.

5. The genetically modified rodent animal of claim 4, wherein the nucleotide sequence of the human CFB gene further comprises the 5’ untranslated region (5’ UTR) of the human CFB gene.

6. The genetically modified rodent animal of claim 5, wherein the nucleotide sequence of the human CFB gene further comprises a 5’ nucleotide sequence upstream of the stall of the 5’ UTR of the human CFB gene.

7. The genetically modified rodent animal of claim 6, wherein the nucleotide sequence of the human CFB gene comprises a 5’ regulatory sequence of the human CFB gene.

8. The genetically modified rodent animal of any one of claims 1-7, wherein the rodent CFB nucleic acid sequence comprises a 5’ upstream sequence of a rodent CFB gene up to the 5’ start of exon 1 of the rodent CFB gene.

9. The genetically modified rodent animal of any one of claims 1-8, wherein the rodent CFB nucleic acid sequence comprises a 5’ regulatory sequence of a rodent CFB gene.

10. The genetically modified rodent animal of any one of claims 1-9, wherein the rodent CFB nucleic acid sequence comprises the 3’ UTR or a portion thereof of a rodent CFB gene.

11. The genetically modified rodent animal of any one of claims 7-10, wherein the rodent CFB gene is an endogenous rodent CFB gene.

12. The genetically modified rodent animal of any one of claims 1-11, wherein the humanized CFB gene is located at an endogenous rodent CFB locus.

13. The genetically modified rodent animal of claim 11, wherein the humanized CFB gene is a result of replacement of a rodent CFB genomic DNA at an endogenous rodent CFB locus with the human CFB nucleic acid.

14. The genetically modified rodent animal of claim 13, wherein the humanized CFB gene is a result of replacement of a rodent genomic DNA comprising exon 1 through the first 31 bp of the 3’ UTR of an endogenous rodent CFB gene with the human CFB nucleic acid, and wherein the human CFB nucleic acid comprises exon 1 through the stop codon of a human CFB gene.

15. The genetically modified rodent animal of claim 14, wherein the human CFB nucleic acid comprises a 5’ nucleotide sequence upstream of the start of the 5’ UTR of the human CFB gene.

16. The genetically modified rodent animal of any one of claims 1-15, wherein the rodent is heterozygous for the humanized CFB gene.

17. The genetically modified rodent animal of any one of claims 1-15, wherein the rodent is homozygous for the humanized CFB gene.

18. The genetically modified rodent animal of any one of claims 1-16, wherein the rodent 7expresses the polypeptide encoded by the humanized CFB gene in the serum.

19. The genetically modified rodent animal of any one of claims 1-18, wherein the genome of the rodent further comprises a humanized C3 gene encoding a human C3 protein.

20. The genetically modified rodent animal of claim 19, wherein the humanized C3 gene comprises exon 1 through exon 41 of the human C3 gene.

21. The genetically modified rodent animal of claim 20, wherein the humanized C3 gene further comprises the promoter of the human C3 gene.

22. The genetically modified rodent animal of any one of claims 19-21, wherein the humanized C3 gene is located at an endogenous rodent C3 locus.

23. The genetically modified rodent animal of claim 22, wherein the humanized C3 gene is a result of a replacement of an endogenous rodent C3 genomic DNA comprising exon 1 through exon 1 with a human C3 nucleic acid comprising exon 1 through exon 41 of the human C3 gene.

24. The genetically modified rodent animal of any one of claims 19-23, wherein the rodent animal is heterozygous for the humanized C3 gene.

25. The genetically modified rodent animal of any one of claims 19-23, wherein the rodent animal is homozygous for the humanized C3 gene.

26. The genetically modified rodent animal of any one of claims 1-25, wherein the rodent animal is a mouse or a rat.

27. An isolated rodent tissue or cell, wherein the genome of the isolated rodent tissue or cell comprises a humanized CFB gene comprising a rodent CFB nucleic acid sequence and a human CFB nucleic acid sequence, wherein the humanized CFB gene encodes a polypeptide comprising a mature protein sequence substantially identical to the mature protein sequence of a human CFB protein.

28. The isolated rodent tissue or cell of claim 27, wherein the polypeptide is substantially identical in sequence to the human CFB protein.

29. The isolated rodent tissue or cell of claim 27 or 28, wherein the genome further comprises a humanized C3 gene encoding a human C3 protein.

30. The isolated rodent tissue or cell of any one of claims 27-29, wherein the isolated rodent cell is a rodent embryonic stem cell, or a rodent germ cell.

31. The isolated rodent tissue or cell according to any one of claims 27-30, wherein the isolated rodent tissue or cell is a mouse tissue or cell, or a rat tissue or cell.

32. A rodent embryo, comprising a rodent embryonic stem cell according to claim 30.

33. A method of making a genetically modified rodent animal, comprising: modifying a rodent genome to comprise a humanized CFB gene, wherein the humanized CFB gene comprises a rodent CFB nucleic acid sequence and a human CFB nucleic acid sequence, and encodes a polypeptide comprising a mature protein sequence substantially identical with the mature protein sequence of a human CFB protein; and making the rodent comprising the modified rodent genome.

34. The method of claim 33, wherein said modifying comprises introducing a nucleic acid molecule comprising the human CFB nucleic acid sequence into the genome of a rodent embryonic stem (ES) cell,obtaining a rodent ES cell in which the human CFB nucleic acid sequence has been integrated into an endogenous CFB locus to replace a rodent CFB genomic DNA thereby forming the humanized CFB gene, and generating a rodent animal from the obtained rodent ES cell.

35. The method of claim 33 or 34, wherein the polypeptide is substantially identical in sequence to the human CFB protein.

36. The method according to any one of claims 33-35, wherein the human CFB nucleic acid sequence comprises exon 1 through the stop codon in exon 18 of a human CFB gene.

37. The method of claim 36, wherein the human CFB nucleic acid comprises a 5’ nucleotide sequence upstream of the start of the 5’ UTR of the human CFB gene.

38. The method according to any one of claims 33-37, wherein the rodent CFB nucleic acid sequence comprises a 5’ regulatory sequence of a rodent CFB gene.

39. The method according to any one of claims 33-38, wherein the rodent is heterozygous for the humanized CFB gene.

40. The method according to any one of claims 33-38, wherein the rodent is homozygous for the humanized CFB gene.

41. The method according to any one of claims 33-40, further comprising modifying the rodent genome to comprise a humanized C3 gene that encodes a human C3 protein.

42. The method according to any one of claims 33-41, wherein the rodent is a mouse or a rat.

43. A targeting nucleic acid construct, comprisinga human CFB nucleic acid sequence to be integrated into a rodent CFB gene at an endogenous rodent CFB locus, flanked by a 5' nucleotide sequence and a 3' nucleotide sequence that are homologous to nucleotide sequences at the rodent CFB locus, wherein integration of the human CFB nucleic acid sequence into the rodent CFB gene results in a replacement of a rodent CFB genomic DNA with the human CFB nucleic acid sequence thereby forming a humanized CFB gene, and wherein the humanized CFB gene encodes a polypeptide comprising the mature protein sequence of a human CFB protein.

44. The targeting nucleic acid of claim 43, wherein the human CFB nucleic acid sequence comprises exon 1 through the stop codon in exon 18 of a human CFB gene.

45. The targeting nucleic acid of claim 43 or 44, wherein the rodent is a mouse or a rat.

46. An in vitro method for generating a genetically modified rodent cell, comprising introducing the targeting nucleic acid construct according to any of claims 43-45 into a rodent cell, whereby the human CFB nucleic acid sequence is integrated into the endogenous rodent CFB gene locus resulting in a replacement of a rodent CFB genomic DNA with the human CFB nucleic acid sequence to form a humanized CFB gene, thereby generating the genetically modified rodent cell.

47. The method of claim 46, wherein the rodent cell is a rodent ES cell.

48. A method of assessing pharmacokinetic properties of a candidate compound, comprising: administering the candidate compound to a rodent animal according to any of claims 1-26; performing one or more assays to determine the pharmacokinetic properties of the candidate compound in the rodent animal.

49. A method for screening candidate compounds targeting a component of the human complement system, comprising:administering a candidate compound to a rodent animal according to any of claims 1-26; and performing one or more assays to determine whether the candidate compound has an effect on the complement system of the rodent animal.

50. The method of claim 48 or 49, wherein the candidate compound is a compound tailing human CFB .

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