WO2018195166A1

WO2018195166A1 - Methods for estimating person-specific responses to chemical or biological agents using induced pluripotent stem cells

Info

Publication number: WO2018195166A1
Application number: PCT/US2018/028117
Authority: WO
Inventors: Kevin P. Coyne; Shawn T. Coyne; Bernard Fermini
Original assignee: Coyne Scientific, Llc
Priority date: 2017-04-18
Filing date: 2018-04-18
Publication date: 2018-10-25

Abstract

A method for estimating the responses of an individual person of interest to a chemical or biological agent prior to exposing the person to that agent in vivo. Such estimations or predictions may be used to forecast the likelihood and/or magnitude of response by the person of interest. The method uses induced pluripotent stem cells (iPSCs) derived from tissues from the person of interest, as well as both stem cells and relevant medical or health information drawn from a number of individuals who have previously been exposed to the agent in vivo.

Description

METHODS FOR ESTIMATING PERSON-SPECIFIC RESPONSES TO CHEMICAL OR BIOLOGICAL AGENTS USING INDUCED PLURIPOTENT STEM CELLS

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 62/486,666 filed on April 18, 2018, which is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The invention relates generally to methods for estimating an effect of a biological or chemical agent on a person using stem cells in an assay.

BACKGROUND

Chemical and biological agents can have differing effects on different human beings, both in terms of the nature and degree of the effects, even when applied/administered/ exposed at identical dose concentrations and under identical conditions. These differences in outcomes can impose significant burdens upon society.

For example, in the field of medicine, serious adverse drug reactions (adverse drug reactions) have been estimated to cause over 100,000 hospitalizations a year, at a cost of over $30 billion, in the US alone. At the other end of the spectrum, a patient who fails to respond to an administered drug, yet would have responded to an alternative treatment, may be deprived of the beneficial impact of the foregone alternative treatment. The burden is particularly acute when the patient's malady is severe and the compounds available for treatment have high incidence of adverse drug reactions. For example, a number of anti-cancer medicines such as anthracyclines and kinase inhibitors offer strong benefits when effective, but cause serious adverse drug reactions in a significant percentage of those who take them. This health risk is likely to worsen as treatment for cancer and other ailments shifts toward combinations of several drugs, as they are predicted to do, thus exposing the patient to the combined risks of the individual components of a combination.

Currently, there is no way to accurately estimate or predict in advance of treatment whether a particular drug would fail to be effective in a particular patient or whether the treatment would produce adverse drug reactions, or both. Unfortunately, the approach is too often simply "try it and see."

The field of medicine would benefit significantly from an ability to estimate or predict for the individual patient whether a drug considered for treatment would be beneficial and/or cause the patient to suffer adverse drug reactions. Even the ability to estimate or predict only a single type of adverse drug reaction in a specific patient would be highly beneficial. Given equal likelihood of beneficial effects from two compounds, the cost-benefit ratio is higher if the clinician is able to determine in advance that one of the two compounds has significantly lower risk of adverse drug reactions.

Similarly, in the field of industrial chemicals and pesticides, individual workers in factories may have significantly different reactions to the chemicals to which they may be exposed. An ability to determine, a priori, whether a particular worker is unusually susceptible to adverse reactions to chemicals present in the factory could contribute to occupational health and safety. Recently published studies (Shinozawa, T., Nakamura, K., Shoji, M. et al. (2016), Recapitulation of clinical individual susceptibility to drug-induced QT prolongation in healthy subjects using iPSC-derived cardiomyocytes, Stem Cell Reports, 8:226-234; Burridge, P.W., Fuga Li, Y., Matsa, E. et al. (2016), Human induced pluripotent stem cell-derived

cardiomyocytes recapitulate the predilection of breast cancer patients to doxorubicin-induced cardiotoxicity, Nature Medicine, 22:547-556; and Stillitano, F., Hansen, J., Kong, C.W. et al. (2017), Modeling susceptibility to drug-induced long QT with a panel of subject-specific induced pluripotent stem cells, eLife, 6: 1-24) have demonstrated that certain functional cells (such as cardiomyocytes), derived from induced pluripotent stem cells (iPSCs) taken from a given patient, can recapitulate certain elements of reactions to a compound of the primary cells from the same patient when treated with the same compound in clinic.

The present method builds on these recently published studies but differs from them in several important respects. Unlike the referenced studies:

1. The method provided herein involves inferences that cross-over from one or more reference person(s) to another person (i.e., the person of interest), on whom no in vivo information is available.

2. The underlying purpose of the present method is not to correlate the two analytical results of the in vitro response and clinical response of the same human being. In the case of the present method, that step is a means to an end. The purpose of the present method is to use correlations previously established between in vitro and in vivo data for a number of reference individuals exposed to the agent of interest, coupled with the in vitro score for the person of interest, in order to develop an estimation or prediction about the one analytical result that is not available - the in vivo effect on the person of interest.

3. No attempt is made to compare the underlying genetics of the reference individuals with the person of interest. Thus, the invention does not rest upon the assumption that the genetic causality is similar across individuals - only that the correlation of in vitro and in vivo reactions are consistent.

4. The method provided herein does not require the reference individuals and the person of interest to be afflicted with the same disease. The protocols and construct of the published studies indicate that their authors believe that the presence of a specific disease plays an important role in determining the reaction to the compounds that were studied. In contrast, studies using the method described herein indicate that the reactions of individual donor iPSCs to a biological or chemical agent vary widely in a way that mimics in vivo reactions, even when the donors are healthy. This provides evidence that the reactions are largely driven by the interaction of the genetics and the agent, and does not require a catalytic effect of a specific disease.

This distinction of whether or not reference donors of iPSCs must have experienced the same specific disease as the person of interest is critical to the practical application of the method provided herein. The present method requires: (1) the identification of individuals who have been exposed to the agent of interest and have experienced varying reactions, which have been documented, and (2) obtaining tissue samples from those individuals (and preferably, samples of the identical cell type). Meanwhile, the protocol of the referenced studies would further require that any reference donors must also have suffered from an identical disease as the person of interest. This additional requirement would unnecessarily increase the difficulty of locating donors capable of meeting all criteria simultaneously, perhaps rendering the protocol impractical in some cases. Further, in those cases where the additional requirement could be met, the requirement would narrow the applicability of any findings, thus unnecessarily limiting the usefulness of the effort.

SUMMARY OF THE INVENTION

The method described herein uses source tissues from a single person of interest, as well as both tissues and relevant health or medical information drawn from reference control persons who have been exposed to an agent of interest in vivo. The method further involves the reprogramming of the source tissues of these persons into iPSCs and, where appropriate, the differentiation of those cells into functional cells (such as cardiomyocytes, hepatocytes, and neurons, etc.). The resulting cells are then assayed. The in vitro results for the cells derived from the reference control individuals are used to construct a range of reactions to the assay and to determine the relationship (if any) between the in vitro results for the reference control individuals and the in vivo responses of those same individuals. The assay is conducted on the person of interest, and those in vitro results are analytically placed within the resulting range of in vitro results from the reference group in order to estimate or predict that person's in vivo reaction to the compound.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a graph showing the in vitro dose-reaction relationship for doxorubicin. Dose concentrations are shown on the X axis, and the effects on mitochondrial membrane potential are shown on the Y axis, for cardiomyocytes derived from iPSCs from four individual persons. The individuals have been hypothetically sub-grouped into two categories (one titled "Robust Group in Clinic" and the other titled "Vulnerable Group in Clinic") based on each of their

(hypothesized) cardiac reactions to doxorubicin treatment in vivo. (Data supporting this subdivision not included). Superimposed on this Figure are black and white dots, with the color of the dot corresponding to whether the individual did or did not experience adverse cardiac effects in vivo.

Figure 2 is a graph repeating Figure 1, with the addition of an in vitro dose-response relationship for cardiomyocytes derived from iPSCs from a fifth individual. That person is titled the "Person of Interest". Figure 3 is a graph showing hypothetical dose-response relationships between concentrations of doxorubicin (on the X axis), and the measured effect on mitochondrial membrane potential (on the Y axis) for cardiomyocytes derived from iPSCs from six individual persons. Superimposed on this Figure are black and white dots, corresponding to whether the individual did or did not experience adverse cardiac effects in vivo.

DETAILED DESCRIPTION

A method is provided herein to estimate the reactions of a person of interest to a chemical or biological agent separately from that person's exposure to that agent in vivo. The estimations or predictions forecast the likelihood and/or the magnitude of response.

Definitions

The following terms, unless otherwise indicated, shall be understood to have the following meanings:

As used herein, the terms "a", "an", and "the" can refer to one or more unless specifically noted otherwise.

The use of the term "or" is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." As used herein "another" can mean at least a second or more.

Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for a test result, the method being employed to determine the value, or the variation that exists among samples. It is to be understood, although not always explicitly stated, that all numerical designations may be preceded by the term "about."

The term "comprising" or "comprises" is intended to mean that the methods include the recited elements, but not excluding others. "Consisting essentially of when used to define methods, shall mean excluding other elements that materially affect the method. For example, a method consisting essentially of the elements as defined herein would not exclude other elements that do not materially affect the basic and novel characteristic(s) of the claimed method.

"Consisting of shall mean excluding more than trace amount of other ingredients and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this invention.

"Cells" may include single cells, aggregations of similar cells, multicellular constructs, tissues (including multi-cell-type tissues), or organ constructs, and may include stem cell-derived cells, whether they be derived fully or partially from iPSCs, and may include iPSCs that have been differentiated into functional cells.

"Agent" or "agent of interest" or "compound" are used interchangeably herein and may include any chemical or biological substance that may affect any biological function of a human being, including, but not limited to, any pharmaceutical, industrial chemical, or pesticide, whether naturally occurring or manmade. In this regard, the phrase (including the word

"compound") is deemed to include any chemical element or biological molecule.

The terms "applied", "combine", and "exposed" are used interchangeably herein and are include all instances of one or more exposures, or combinations with, to include the chemical and biological agents described above, whether or not each of the incidences of exposure consists of the same concentration or dose, and/or of the same duration. The duration is of a sufficient amount of time under conditions known to those of skill in the art to allow a reaction to occur.

"Functional cells" refers to any cell types that are found in the human body or progenitor cells thereof, including cardiomyocytes, hepatocytes, and neurons.

"Person of interest" is the person for whom the in vivo reaction to the agent of interest is being estimated or predicted. It is not required by the method provided herein for the person of interest to be identified at the time the control reference panels are created and their

range/reactions established. The person of interest may be a child, adult or geriatric person. The term "child" generally includes a human approximately 17 years of age or younger, the term "adult" generally includes a human between approximately 18 years of age and 65 years of age, and the term "geriatric person" generally includes a human approximately over the age of 65.

"Bin" is defined as a specified range of reactions. Usually, but not necessarily, multiple bins are jointly specified to be mutually exclusive and collectively exhaustive, such that reactions of any one individual will lie within one bin but not any other and may form a spectrum of reactions. An illustrative binning scheme is: Bin 1 is defined as containing any observations "less than minus 1.0"; Bin 2 is defined as containing any observations "greater than or equal to minus 1.0 and less than or equal to positive 1.0"; and Bin 3 is defined as containing any observations "greater than 1.0".

"Donor" refers to any person from whom cells or tissues are obtained. "Donor" may refer to any reference person or the person of interest.

"Donor's cells" may refer to the original tissue obtained from a donor, or iPSCs derived therefrom, or cells derived from such iPSCs.

"Dose", "concentration" and "exposure" refer to the quantity of agent to which the reference person or person of interest has been exposed. In all instances, each of these phrases may be deemed to also include the duration of exposure, if duration is relevant to the existence or degree of effect/response, whether in vivo or in vitro.

"In vivo " refers to actions or events that are taken by, or act upon, a live human being. The phrase is intended to contrast with in vitro actions or events that are conducted on tissues or cells physically separated from, outside of, or derived from such human being, but that do not directly act upon the body of the living human being itself.

"Reference group", "reference control group", or "control group" refers to a sample of persons (at least one, preferably two or more, more preferably five or more) who have been exposed to the agent of interest in vivo and: (1) for whom their reactions (along one or more dimensions) to the agent in vivo have been recorded, (2) from whom tissues are collected that can be reprogrammed into iPSCs, and (3) there is an in vitro assay whose results are postulated to be correlated with the in vivo reactions. These reference groups may consist of positive and negative controls (in which groups are defined by the members of the group having strong or weak/no in vivo reactions to the agent), or may consist of multiple groups, wherein membership in each group is defined by the member's reaction in vivo falling within a specific quantified range. The reference group may be children, adults or geriatric person. The term "children" generally includes humans approximately 17 years of age or younger, the term "adults" generally includes humans between approximately 18 years of age and 65 years of age, and the term "geriatric person" generally includes humans approximately over the age of 65.

The term "researcher" includes, and may be used interchangeably with, the terms clinician, physician, healthcare provider, scientist and laboratory technician and refers to an individual who conducts in vivo and/or in vitro experiments or assays. Method Overview

The method provided herein is directed towards situations in which a person of interest has been, or in the future may be exposed to one or more chemical or biological agents that have previously caused reactions in some, but not all, other persons who have been exposed to the same agent(s). The method covers reactions that cannot be quantified beyond their presence or absence (e.g. death, effects that are reported qualitatively - such as nausea or headaches). The method recognizes that in vivo data for any individual may only be collectible at one dose concentration, regimen, and duration.

Although the reference control group consists of individuals who have been exposed to the agent of interest in vivo, it is not a requirement that any individuals of the reference control group have experienced any malady or disease being experienced by the person of interest. Further, it is not a requirement of the method that the in vivo reactions of reference persons have been demonstrated to exhibit a dose response relationship with the agent of interest. Indeed, owing to the potentially limited access to acceptable tissue samples from persons having been exposed to the agent in vivo, data across exposures may not be available on reference individuals.

Given that gender, ancestry, race or ethnic group, and age have been shown to affect drug and other chemical agent responses, the researcher may choose to limit membership in the reference group (or groups) to individuals who match the person of interest on one or more of these dimensions. However, this is not a requirement of the method, as the researcher may not have access to a large enough pool of potential control persons to render this additional restriction feasible.

In a relatively simple form of the method provided herein, the method provides for the identification of persons who have been exposed to the agent of interest in vivo, and who have had either no such reaction or only a limited reaction, and the procurement of source tissues, or cells, from such individuals. This portion of the reference control group forms a "negative" control group. The method also provides for the identification of persons who have been treated with the agent of interest in vivo and who have had a higher degree of reaction and the procurement of source tissues, or cells, from such individuals This portion of the reference control group forms a "positive" control group. Third, the methods provide for the procurement of similar source tissues, or cells, from the person of interest for whom the reaction to the agent of interest is to be estimated.

If data is available, the researcher or clinician may test to determine whether there is a statistical relationship between the dose, duration and quantified effect of reaction {in vivo or in vitro) within each of the groups, or indeed, across the control groups. If so, then the researcher or clinician can further attempt to establish a relationship between the in vitro assay and the in vivo result in any way, including onset thresholds, severity of effects, dose escalation strategies, etc. Any such activities are included within the scope of this invention.

At various points in the process, any or all of the cells procured or created during the process may be cryopreserved and later thawed.

For purposes of simplicity, much of description of the method provided herein and many of the examples provided below focus on the identification of the potential for adverse drug reactions associated with pharmaceutical compounds, and the potential severity thereof.

However, the application of the method for the purposes of identifying and/or sizing beneficial therapeutic effects of pharmaceutical compounds is explicitly considered within the scope of the present invention, as are estimations or predictions associated with exposures to industrial chemicals, pesticides, and any other chemical or biological substance or agent. Further, while the description herein of the method is limited to discussion of a single compound, the use of the method to compare multiple compounds lies within the scope of this invention.

Method Steps

The steps of the method are described as follows. Throughout the steps, the

reprogramming, differentiation, cell culturing and assaying steps are conducted using protocols that are sufficiently consistent across all persons involved in the study such that any significant differences in in vitro reactions are deemed to be due to biological differences among the underlying individuals, and not due to variations in protocols or processes among the samples.

The steps, which may be undertaken in any appropriate order, are:

1. The researcher/clinician selects an agent of interest.

2. The researcher identifies at least one, preferably two or more, and more preferably five or more persons who have been exposed to the agent in vivo and who have collectively experienced significantly different reactions (from each other) when exposed to concentrations (for relevant durations) that are relevantly close to the concentration of interest being contemplated for the person of interest. These individuals are designated as reference persons.

3. The researcher determines what in vitro tests exist that are seen as correlating with the in vivo effects that are to be estimated or predicted for the person of interest.

4. The researcher collects tissue samples, exposure information, and relevant health and/or medical information on the reactions to the agent from the reference persons. In one embodiment, the method involves separating the reference persons into groups that fit within distinct bins of degree of reaction that are of interest (e.g., no adverse reaction, developed pro-arrhythmia). The tissue collected preferably contains the same type of cells from each of the persons, more preferably from peripheral blood in all cases. However, the tissue samples need not be fresh (i.e., may be cryopreserved), nor necessarily be from the same type of tissue. Further, the reference persons need not be still alive.

5. The researcher conducts the in vitro assays on relevant functional cells that have been derived from the tissue samples collected in the previous step, and determines the in vitro test scores for each reference person, and groups those results by bin.

6. The researcher conducts the identical assay on functional cells derived from tissues taken from the person of interest. The person of interest need not be still alive, as long as the source tissues collected from that person are still alive, or viable, and capable of being programmed into stem cells.

7. The researcher compares the in vitro response of the patient of interest (from the previous step) to those of the bins of reference patients (determined in step 5 above) to estimate (via mathematical means or interpolation) the potential reaction of the patient of interest to the compound.

The present method is not restrictive as to the statistical, mathematical, graphical or other methods to be used to compare the various data points and/or generate the estimation or prediction of the likelihood or degree of reaction of the person of interest. Further, when developing an estimation or prediction, the researcher is not restricted to examining only in vitro data corresponding to the actual exposure/dose concentration experienced in vivo by reference persons or only to in vivo or in vitro data that corresponds to the exposure/dose concentration for the person of interest. The following simplified illustrations are merely two (of many possible) illustrations of how the data may be used to develop an estimation or prediction.

In a first illustration, four reference persons have been treated in clinic with doxorubicin at a dose that produced a blood plasma concentration of 0.3 micromolar. Two of the reference persons suffered adverse cardiac outcomes, and two did not. The first two of these are referred to herein as the "Vulnerable Group in Clinic", and are designated with black dots in Figures 1 through 3 (wherein the dots will be placed at points corresponding to a value of 0.3 micromolar on the X axis of the figures) and thereafter. The second two are referred to as the "Robust Group in Clinic", and are designated with white dots in the figures. Because their dose in clinic also produced a blood plasma concentration of 0.3 micromolar, the white dots are also placed at points corresponding to a value of 0.3 micromolar on the X axis of the figures.

The researcher produces iPSC-derived cardiomyocytes from the four persons, and conducts an assay to measure changes in the mitochondrial membrane potential (MMP) across a spectrum of concentrations, plotting the results (including results interpolated between observations) in Figure 1. (Note that the white and black dots are placed on the graph at points on the Y axis corresponding the intersections of their X axis placement and their value on the in vitro assay at that dose.) The researcher notes that there is a distinct separation of the in vitro reactions of the members of the Vulnerable Group in Clinic from the Robust Group in Clinic.

The researcher conducts the in vitro assay on the person of interest, and superimposes that person's in vitro reaction onto the preceding graph (Figure 2). The person of interest's mitochondrial membrane potential (MMP) values lie within the range of values of the Robust Group at the 0.3 micromolar concentration, but fall rapidly thereafter to a level that is below the range exhibited by the Vulnerable Group. This suggests to the researcher that if the person of interest is treated with doxorubicin, the risk to that person would increase dramatically if the dose concentration exceeds 0.3 micromolar.

In a second illustration, the researcher is faced with an apparent anomaly. Of six reference patients, the three who suffered adverse cardiac outcomes were ones who received a lower dose than the three who received higher doses. However, when the researcher follows the graphing directions described for Figure 1, the likely cause of the apparent anomaly is revealed (Figure 3). The differences in the in vitro dose-response relationships among the six individuals clearly establishes that the three individuals who experienced adverse cardiac outcomes show demonstrably more sensitivity (as judged through the in vitro assay) than do the three who did not experience such outcomes.

If the person of interest were to also be tested with the same in vitro assay (not shown in Figure 3), the researcher could ascertain that person's dose-response relationship, and compare it to the dose response relationships of the six reference persons to estimate or predict the person of interest's likely reactions in vivo.

Assay Consistency

In an alternative embodiment of the present method, the researcher's purpose is to determine whether the reactions measured by a particular in vitro assay are consistent from agent to agent (e.g., that the assay produces values for individuals who are not affected by an agent in vivo consistently fall below a specific value regardless of the agent involved, whereas the assay produces values for individuals who are affected by an agent in vivo that consistently fall above that score). Should the researcher find that this is true, then the researcher may later be able to utilize the assay to estimate or predict the response of the person of interest to an agent for whom the researcher does not have a reference control group.

Therefore, the researcher repeats Steps 1 through 5 of the method described above on two or more agents, and compares results of these analyses to determine whether the results are consistent across the agents.

As above, this embodiment of the present method is not restrictive as to the statistical, mathematical, graphical or other methods to be used to compare the various data points and/or conclude whether and how the assay meets the test of consistency. Further, the present invention is not restrictive as to any biological or chemical similarity among the agents involved in conducting this embodiment.

Disclosed herein are methods which can be used with an array of materials useful for carrying out any one or more disclosed method. Where a method is disclosed and a number of modifications to the method are discussed, each and every combination and permutation of the method, and the modifications that are possible, are specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of this disclosure. Thus, if there are a variety of additional steps that can be performed, it is understood that each additional step can be performed with any specific method step or combination of method steps of the disclosed methods, and in any order or permutation, unless otherwise indicated, and that each such combination or subset of combinations is specifically contemplated.

EXAMPLES

The examples below are intended to further illustrate certain aspects of the method described herein, and are not intended to limit the scope of the claims.

Example 1

A clinician sees a patient who has cancer and expects that he may become a candidate for treatment with a chemotherapy agent of interest, an anthracycline, in the near future

(approximately six months). The clinician knows from the warning label that between 20% and 35% of anthracycline patients experience significant levels of cardiac adverse drug reactions. Instead of prescribing a costly and intrusive regimen of post-dosing monitoring of the patient to detect whether the patient will experience an adverse drug reaction, the clinician initiates a records search for others who have taken the drug for any reason and could be candidates for one of the required control groups, positive (Vulnerable) or negative (Robust). Simultaneously, the clinician arranges for the patient of interest to provide source tissues, or cells, for conversion into the appropriate pluripotent stem cells, and further differentiation into functional cells (in this case, into cardiomyocytes). The clinician is able to assemble a positive and a negative control group consisting of 10 qualified representatives each and procures source cells from each. The clinician then assays the cardiomyocytes derived from the iPSCs from each member of the control groups and the person of interest. The clinician plots the response curves for each member of the positive and negative control groups, as well as the patient of interest, and determines that results for the patient of interest results falls within the distribution of the plots of the negative control group, and well outside of the distribution of the positive control group. Therefore, the clinician decides that it will be safe for the drug to be prescribed for the patient of interest. Example 2

A pharmaceutical company recognizes that one of its most effective drugs for high risk cases produces unsafe levels of adverse drug reactions in a significant percentage of patients. The company identifies previous patients who did and did not experience such adverse drug reactions, and uses their information and cells to create reference control groups. The company creates the required iPSC-derived cardiomyocytes, performs the assays, and establishes the statistical validity of the construct.

Subsequently, when a clinician believes that a patient may become a candidate for treatment with the drug, the clinician obtains the appropriate tissue containing the same type of cells that were used to construct the reference group. The resulting assay provides the patient of interest's in vitro test values that are then mathematically compared to the control group's predetermined test values.

The "diagnostic" of the patient's suitability for receiving the drug is available in a timely fashion and the cost of determining the individual patient's adverse drug reaction susceptibility is limited to the one wet-lab workload.

Example 3

A clinician determines that a patient may at some point become a candidate for any one of a number of compounds for which the procedures in Example 2 have been performed in the past. The clinician desires to minimize the time between the choice of compound, and the beginning of treatment with the chosen compound. Therefore, the clinician collects tissue samples from the patient well before the choice must be made, and has a third party service provider reprogram the tissues into iPSCs and differentiate the patient's iPSCs into functional cells (in this case, cardiomyocytes) and cryopreserve the resulting cells. As the time for the choice of compound approaches (e.g., four weeks from the intended administration), the clinician has the third party vendor prepare the cells for testing, and conduct the necessary assay using each of the compounds under consideration. The clinician then compares the patient's results with the results for the control groups (provided by the pharmaceutical company, as defined in Example 2 above), and determines the safest choice of compound.

The methods of the appended claims are not limited in scope by the specific methods described herein, which are intended as illustrations of a few aspects of the claims and any methods that are functionally equivalent are within the scope of this disclosure. Various modifications of the methods in addition to those shown and described herein are intended to fall within the scope of the appended claims. Further, while only certain representative methods, and aspects of these methods are specifically described, other methods and combinations of various features of the methods are intended to fall within the scope of the appended claims, even if not specifically recited. Thus, a combination of steps, elements, components, or constituents may be explicitly mentioned herein; however, all other combinations of steps, elements, components, and constituents are included, even though not explicitly stated. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entireties for all purposes.

Claims

CLAIMS What is claimed is:

1. A method of estimating an in vivo reaction of a person of interest to a chemical or biological agent of interest comprising:

a. determining whether reactions to a particular in vitro assay by stem cell-derived cells whose source tissue was taken from a first reference group of individuals designated as negative controls, who have previously been exposed to the agent of interest and exhibited a lesser reaction in vivo to that agent, are different than reactions to the same in vitro assay by stem cell-derived cells taken from a second reference group of individuals designated as positive controls, who have previously been exposed to the same agent, wherein the positive controls are individuals that exhibited a larger reaction in vivo to that agent;

b. conducting the same in vitro assay on stem cell-derived cells whose source tissue was taken from the person of interest and comparing the reaction of the stem cell-derived cells from the person of interest to the reactions of the stem cell-derived cells from the reference groups in step a.

2. The method of Claim 1 wherein the stem cell-derived cell is a cardiomyocyte.

3. The method of Claim 1 wherein the stem cell-derived cell is a hepatocyte.

4. The method of Claim 1 wherein the stem cell-derived cell is a neuron.

5. The method of Claim 1 wherein the reference persons were not treated for the same malady for which the person of interest is afflicted, or for which the agent is being considered for use as a treatment.

6. The method of Claim 1 wherein cells are cryopreserved at any step for any of the reference persons or person of interest.

7. The method of Claim 1 wherein reference persons are not classified into negative and positive controls, but into multiple bins, wherein the reactions form a spectrum of reactions from most negative to most positive.

8. The method of Claim 1 comprising two or more compounds and further comprising comparing the reaction of the stem cell-derived cells whose source tissue was taken from the person of interest to the reactions of the stem cell-derived cells whose source tissue was taken from the reference group in step a for each of the two or more compounds.

9. The method of Claim 1 wherein the in vitro reactions of the reference persons can be mathematically, statistically, or graphically related to the in vivo reactions of the reference persons, such that the resulting relationship can be used in combination with the in vitro value of the person of interest to estimate the in vivo reaction of the person of interest.

10. The method of Claim 1 wherein the person of interest is a child.

11. The method of Claim 1 wherein some or all of the members of the reference group are children.

12. The method of Claim 1 wherein the person of interest is a geriatric person.

13. The method of Claim 1 wherein some or all of the members of the reference group are geriatric persons.

14. The method of Claim 1 wherein the members of the reference group are chosen to be of the same ancestry or gender as the person of interest.

15. A method of estimating an in vivo reaction of a person of interest to a chemical or biological agent of interest comprising:

a. determining whether reactions to a particular in vitro assay by stem cell-derived cells whose source tissue was taken from a reference group of individuals designated as negative controls, who have previously been exposed to the agent of interest and exhibited a lesser reaction in vivo to that agent, are different than the reactions to the same in vitro assay by stem cell-derived cells whose source tissue was taken from a second reference group of individuals designated as positive controls, who have previously been exposed to the same agent, wherein the positive controls are individuals that exhibited a larger reaction in vivo to that agent;

b. repeating step a. for at least one additional agent; c. comparing the results for consistency, and determining the threshold value for the assay; and

d. conducting the same in vitro assay using the agent of interest on the person of interest and comparing in vitro test reactions of the person of interest to the threshold value developed in step c.