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Genetic variations (e.g., polymorphic alleles) within and among human patient populations underlie, to a large extent, differences in individual disposition to diseases, disease manifestation, disease severity, and response to treatment (e.g., to drug treatment). The prevalent animal and cellular models for human disease and drug discovery provide a poor representation of the genotypic/phenotypic spectrum extant in the patient populations to be treated. For example, strains of mice and rats commonly used in drug discovery are highly inbred, and thus only represent a very narrow range of possible genotype/phenotype combinations in mice or rats, let alone humans. Likewise, the relatively small number of human cell lines used for drug screening may reflect the genotypic/phenotypic scope of the individuals from which they were derived, but not that of a genetically diverse population. Further, most human cell lines are quite limited in their capacity to generate or phenocopy specific differentiated cell types (e.g., neurons, cardiomyocytes, and hepatocytes) affected by a particular health condition. Also, the cell lines are not representative of cell populations in a subject, since cell lines have been altered to indefinitely replicate. Importantly, in many cases animal models or genetically modified cell models of disease simply fail to adequately recapitulate the cellular disease phenotypes as they actually occur in a human patient's cells. Thus, typical preclinical drug discovery strategies miss many genotype/phenotypes that are present in the human population and will have a direct impact on the therapeutic efficacy and toxicity of a candidate drug compound. A practical consequence of these facts is that more often than not lead compounds fail in human clinical trials despite successful preclinical testing in animal models and transformed cell line models, as mentioned above. Ideally, drug screening and drug target discovery would be performed in biological models that recapitulate the genetic and phenotypic diversity present in a human patient population and the appropriate disease state at the cellular level, well before the clinical trial stage. These drug discovery paradigms are illustrated schematically in FIG. 1. In the traditional drug discovery model (left), candidate therapeutic agents are selected for clinical trials in patients based on their action on specific drug targets and their efficacy/lack of toxicity in animal models. In an alternative drug discovery model (right) the disease-relevant cells derived from patient iPSC lines, as described herein, are the starting point for identification of lead compounds based on their ability to ameliorate a disease-relevant cellular phenotype in patient derived cells.
Accordingly, the present disclosure describes human induced pluripotent stem cell lines from selected individuals (e.g., patients), genetically diverse panels of such cell lines, differentiated cells derived from such cell lines, and methods for their use in disease modeling, drug discovery, diagnostics, and individualized therapy.
“Candidate drug compound,” as used herein, refers to any test compound to be assayed for its ability to affect a functional endpoint. Some examples of such functional endpoints are ligand binding to a receptor, receptor antagonism, receptor agonism, protein-protein interactions, enzymatic activities, transcriptional responses, etc.
“Correcting” a phenotype, as used herein, refers to altering a phenotype such that it more closely approximates a normal phenotype.
“iPSC donor,” as used herein, refers to a subject, e.g. a human patient from which one or more induced stem cell lines have been generated. Generally, the genome of an iPSC line corresponds to that of its iPSC donor.
“Phenomic analysis,” as used herein, refers to the analysis of phenotypes (e.g., resting calcium level, gene expression profiles, apoptotic index, electrophysiological properties, sensitivity to free radicals, compound uptake and extrusion, kinase activity, second messenger pathway responses) exhibited by a particular type of cell (e.g., cardiomyocytes).
“Phenome,” as used herein refers to the set of phenotypes that is subject and cell-type specific. For example, the phenome of hepatocytes and cardiomyocytes from the same individual will be quite distinct even though they share the same genome.
An “endogenous allele,” as used herein, refers to a naturally occurring allele that is native to the genome of a cell, i.e., an allele that is not introduced by recombinant methodologies.
An “iPSC-derived cell,” as used herein, refers to a cell that is generated from an iPSC either by proliferation of the iPSC to generate more iPSCs, or by differentiation of the iPSC into a different cell type. iPSC-derived cells include cells not differentiated directly from an iPSC, but from an intermediary cell type, e.g., a glial progenitor cell, a neural stem cell, or a cardiac progenitor cell.
A “normal” phenotype, as used herein, refers to a phenotype (e.g., apoptotic rate, resting calcium level, kinase activity, gene expression level) that falls within a range of phenotypes found in healthy individuals or that are not associated with (e.g., predictive of) a health condition.
III. Induced Stem Cell Lines for Drug Screening and Drug Target Discovery
The present disclosure provides human induced pluripotent stem cell (iPSC) lines, panels of stem cell lines, and methods for their use in drug discovery, diagnostic, and therapeutic methods as described in detail below. The induced pluripotent stem cell lines disclosed herein are characterized by long term self renewal, a normal karyotype, and the developmental potential to differentiate into a wide variety of cell types (e.g., neurons, cardiomyocytes, and hepatocytes). Induced pluripotent stem cell lines can be differentiated into cell lineages of all three germ layers, i.e., ectoderm, mesoderm, and endoderm.
An important nexus exists between a subject (e.g., a patient) and iPSC lines generated from that subject. First, all of the genotypes of iPSC lines and those of the corresponding subject are identical. Thus, genotype-phenotype correlations, uncovered in one are informative for the other, and vice versa. Second, differentiated cells (e.g., neurons) derived ex vivo from an iPSC line will exhibit a complete set of cellular phenotypes (referred to herein as a “phenome”) that are very similar, if not identical, to those of differentiated cells in vivo in the corresponding subject. This point is particularly relevant for developing therapeutics targeted to cells that cannot be routinely obtained from patients (e.g., neurons, cardiomyocytes, hepatocytes, or pancreatic cells). For example, in the case of a patient suffering from a neurodegenerative disease (e.g., parkinson's disease), dopaminergic neurons, which are typically affected by this condition, can be obtained non-invasively by differentiating an iPSC line from the subject, and can then be screened in multiple assays. Thus, iPSC lines provide a renewable source of differentiated cells (e.g., inaccessible differentiated cells) in which pathological cellular phenotypes that are associated with a disease, cell type, and individual may be examined and screened against test compounds. An exemplary, non-limiting embodiment of this approach to disease modeling and drug discovery is schematically illustrated in FIG. 2. iPSC lines and iPSC-derived cells (e.g., motor neurons) are also useful for predicting the efficacy and/or adverse side effects of a candidate drug compound in specific individuals or groups of individuals, as schematically illustrated in FIG. 3. For example, test compounds can be tested for toxicity in hepatocytes differentiated from a genetically diverse panel of induced pluripotent stem cells. Toxicity testing in iPSC-derived hepatocytes can reveal both the overall likelihood of toxicity of a test compound in a target patient population, and the likelihood of toxicity in specific patients within that population.
In effect, iPSC lines and iPSC-derived cells (e.g., pancreatic cells) can serve as “cellular avatars,” that reveal cellular phenotypes that are disease, cell-type, and subject-specific to the extent the phenotypes are determined or predisposed by the genome. Collectively, panels of patient induced stem cell lines will represent a wide range of genotype/phenotype combinations in a patient population. Thus, they are useful for developing therapeutics that are effective and safe across a wide range of the relevant target population, or for determining which individuals can be treated effectively and safely with a given therapeutic agent.
B. Screening and Selection of Subject Samples
Some of the methods described herein utilize induced stem cell lines or panels of induced stem cell lines derived from subjects that meet one or more pre-determined criteria. In some cases subjects and cellular samples from such subjects may be selected for the generation of induced stem cell lines and panels of induced stem cell lines based on one or more of such pre-determined criteria. These include, but are not limited to, the presence or absence of a health condition in a subject, one or more positive diagnostic criteria for a health condition, a family medical history indicating a predisposition or recurrence of a health condition, the presence or absence of a genotype associated with a health condition, or the presence of at least one polymorphic allele that is not already represented in a panel of induced stem cell lines.
In some cases, a panel of induced stem cell lines is generated specifically from individuals diagnosed with a health condition, and from subjects that are free of the health condition. Such health conditions include, without limitation, neurodegenerative disorders; neurological disorders such as cognitive impairment, and mood disorders; auditory disease such as deafness; osteoporosis; cardiovascular diseases; diabetes; metabolic disorders; respiratory diseases; drug sensitivity conditions; eye diseases such as macular degeneration; immunological disorders; hematological diseases; kidney diseases; proliferative disorders; genetic disorders, traumatic injury, stroke, organ failure, or loss of limb.