Using Humanization we can take advantage of the ancient biology between humans and other organisms to create stand-ins – patient avatars – for drug screening studies. In this blog, we will focus on models of inborn errors of metabolism (IEM), as these genetic conditions can lead to hypersensitivity to the metabolite. Since stressor condition hypersensitivity can be used to detect favorable drug effects, IEM model systems are ideal tools for phenotypic screens to find molecules that alleviate the metabolic stress occurring from the deficiency. We discuss the model organisms used in hypersensitivity screens, and why they are advantageous to drug discovery. Ultimately, showcasing this approach’s potential to be widely generalized to many genetic disorders.
A wide variety of genetic conditions can lead to chemical hypersensitivity. While this phenomenon can be life-threatening, it can also be beneficial to the discovery of therapeutics as it enables researchers to use a hypersensitivity assay in drug screening studies. In this approach, Humanized model systems (such as nematodes, zebrafish, or iPSC that have the patient’s genetic defect installed) act as patient avatars, or stand-ins for the patient. These models are especially valuable for identifying therapeutics, and expediting the process to clinical trials as they have short lifespans, no ethical considerations, and are highly translatable.
Chemical Hypersensitivity Due to Metabolic Block
One group of genetic conditions that can have this phenomenon occur are inborn errors of metabolism (IEM), as clinical variants in their genes lead to the build up of toxic metabolites . An example of an IEM that would lead to chemical hypersensitivity is a defective ENZ 2 gene. If this variation inhibited the enzymatic conversion of metabolite 2 into metabolite 3, it would cause a metabolic pathway blockade (Figure 1). As a result, patients with this condition could experience hypersensitivity with exposure to metabolite 1: exposure to metabolite 1 leads to toxic metabolite 2 build up, which leads to activation of reactive oxygen species, and ultimately, paralysis and death.
Figure 1. Hypothetical metabolic pathway. When the second enzyme (ENZ 2) gene is defective, metabolite 2 can build up to toxic levels that lead to paralysis and death.
Model Systems using the Fluidics Paradigm
A variety of model systems are possible for use in testing for metabolite hypersensitivity. Starting with the simple model organisms, a human gene associated with disease can be installed as a gene replacement. In the case of metabolic disorders, the high degree of sequence conservation in these ancient genes often enable the human gene to rescue the function of the removed ortholog (the animal's version of the disease gene). When we add in iPSC and then install clinical variants in the human gene locus, we get three types of model systems: C. elegans nematode, zebrafish, and iPSC to model clinical variants (Figure 2).
Figure 2. Humanized C. elegans nematodes, and zebrafish create wild-type Avatars (wt-Avatars). When clinical variants are installed, three types of Avatars with the same variation seen in the patient are created (var-Avatar): animal models zebrafish and C. elegans nematode, and iPSCs.
These models are advantageous for drug discovery because they fit the fluidics paradigm — the zebrafish and nematode can live their entire lifecycle in liquid, and differentiated iPSC can be hooked up in biocircuits with microfluidics. Since the organisms exist in fluid, assessment of oral bioavailability is simply done by adding drugs to the liquid growth media. In this liquid environment, the first step is to create the wt-Avatars. In the nematode, the evolutionary distance often renders a gene to gene comparison with low homology (sequence identity under 75%). As a result, only a portion of the pathogenic alleles can be modeled as amino acid substitution in the nematode's native locus. To work around this we use Whole Gene Humanization - CRISPR gene editing to remove the coding sequence at the native locus and replace it with the human gene coding sequence. When the human sequence restores normal function, we know we are looking at a high degree of conservation of biology.
In zebrafish, the orthologous gene is often at a sequence identity that is equal to or greater than 75% of the human sequence. This often renders the zebrafish's gene sufficient for modeling the patient condition as a single amino acid substitution, yet occasionally whole-gene humanization will need to be deployed. For iPSC, we source from a healthy patient (reference line - "wt Avatar") and make the single amino acid variation to model the patient variant condition (var-Avatar). Bottomline: modeling a patient condition requires the creation of a wild-type humanized animal or the use of an unmodified line (wt-Avatar) and then the insertion of the missense variant that models the genetic variant of the patient (var-Avatar). In regards to IEM deficiencies, this system enables detection of phenotypic abnormalities that are often accentuated by metabolite exposure.
Detection of Hypersensitivity Phenotypes
To use these model systems for drug discovery, we can look to hypersensitivity in stress response as a screenable phenotype. In our inborn errors of metabolism model, we can take advantage of the stress response from metabolite build up. As previously mentioned, the loss of enzyme ("ENZ 2") leads to hypersensitivity to upstream metabolite 1 in the var-Avatar animal model (Figure 3, red line).
Figure 3. LD50 curves are generated for var-Avatar (red line ) and wt-Avatar (green line) upon exposure to different concentrations of metabolite. An intermediate metabolite concentration is used to discriminate for hypersensitivity in var-Avatar lines (dotted blue line). A drug with possible therapeutic use leads to restoration of normal metabolite sensitivity (black line).
Identifying Drug Candidates
A drug library can be screened with the hypersensitivity assay to find candidate therapeutics. This is done by administering a candidate drug to the var-Avatar animal model and then exposing the animal to the metabolite — the drug is identified as possibly therapeutic when it leads to restoration of normal metabolite sensitivity. To learn more about the details of our Three Phase Platform for Drug Repurposing, download our white paper and get the detailed steps involved in creating simplified drug discovery.