Non-human animals used during the research and investigation of human diseases provide a useful means of better understanding the disease without the added risk of causing harm to an actual human being during the process. Many drugs, treatments and cures for human diseases have been developed by first being tested in animals (see, e.g., Greaves et al., 2004, Nat. Rev. Drug Discov. 3, 226-236; Olson et al., 2000, Regul. Toxicol. Pharmacol. 32, 56-67).
One of the drawbacks for using non-human animals in research of human diseases and preclinical trials is that a drug showing efficacy when applied to animals may not be effective in human due to the difference between target human protein and animal protein. Such disparity of drug efficacy may result in expensive pre-clinical and clinical failure. On the other hand, human-animal protein difference may lead to a miss of candidates effective in treating human diseases when animal model is used to screen or test drugs. For example, Avastin, a neutralizing antibody of human Vascular Endothelial Growth Factor (VEGF) and a drug inhibiting human cancer angiogenesis, interacts poorly with murine VEGF, while it blocks human VEGF's binding to human VEGF receptor (Ferrara et al., 2004, Nat Rev Drug Discov. 3, 391-400). This dramatic disparity is resulted from a single amino acid difference between the human epitope that Avastin binds to and the corresponding murine epitope (Fuh et al., 2006, J Biol Chem 281: 6625-31; Muller et al., 1998, Structure 6: 1153-67). It has been reported that 19 amino acid residues of VEGF participate in the interface of the complex of human VEGF and its neutralizing antibody (Muller et al., 1998, Structure 6: 1153-67). Comparison of human and mouse VEGF sequence shows that among the 19 amino acid residues only one residue (Gly88) in human VEGF differs from its counterpart in mouse (see Muller et al., 1998, Structure 6: 1153-67).
Therefore, in order to search and evaluate drug candidates in treating a human disease, there are needs to develop animal models in which a human gene is introduced to express a human protein in the animal cells and to mimic the human disease. Furthermore, in order to prevent the interaction between the drug candidates and the human protein being interfered by the endogenous animal proteins, there are needs to have endogenous animal gene corresponding to the desired human gene being damaged, such that the introduced human gene operably replaces the corresponding animal gene. Some transgenic animals in which one human gene replaces a corresponding animal gene have been made to meet the needs. For example, transgenic mice in which human VEGF gene replaces endogenous mouse VEGF gene has been made to study the role of human VEGF in normal and pathological angiogenesis (US Pat Pub No. 2010/0162415 A1).
However, transgenic mice expressing only one human gene have limitations in searching and evaluating drug candidates in treating human diseases because, to exert their effects on the human diseases, the drug candidates often need to act in a desired way on a targeted protein-protein interaction that underlies the physiological process of the human diseases. For example, a neutralizing antibody of human VEGF inhibits angiogenesis by blocking human VEGF's binding to human VEGF receptor. A neutralizing antibody candidate may not show any effect on transgenic mice expressing only human VEGF as the expressed human VEGF may not interact in the same manner with murine VEGF receptor as with human VEGF receptor. Therefore, there are needs to develop animal models expressing both human proteins involved in the targeted protein-protein interaction that underlies the physiological process of the human diseases.
This disclosure provides a transgenic animal expressing at lease two human proteins whereas a first human protein dynamically interacts with a second human protein, whereas the consequence of the interaction between the first human protein and the second human protein forms a part of a cascade of signaling pathway that direct cellular changes. In some preferred embodiments, when the second human protein interacts with a third human protein in the cascade of signaling pathway, the third human protein is also introduced into the transgenic animal. More preferably, when the third human protein interacts with a fourth human protein in the cascade of signaling pathway, the fourth human protein is also introduced into the transgenic animal, and so forth, until the whole cascade of signaling pathway is completely introduced into the transgenic animal.