On the cusp of an iPSC-informed ALS therapy: an interview with Gabriel R. Linares

In this interview, Gabriel R. Linares (Keck School of Medicine of the University of Southern California; USA) exclusively discusses some of his innovative research: automation, stem cells and potential amyotrophic lateral sclerosis (ALS) therapies.

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Oct 21, 2019
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Please can you introduce yourself, your institution and your research interest? 

I am a postdoctoral scholar research associate at the Keck School of Medicine at the University of Southern California (CA, USA). I conduct research with Justin Ichida, whose laboratory is part of the University’s Department of Stem Cell Biology and Regenerative Medicine. A major research focus of ours concerns discerning the mechanisms involved in the pathogenesis of amyotrophic lateral sclerosis (ALS) – a fatal neurodegenerative disorder – using patient-derived motor neurons. We employ induced pluripotent stem cell (iPSC) technology to create in vitro models of ALS patients.

There are two types of ALS: sporadic and familial. The majority of ALS cases are sporadic; only about 10% of ALS cases are familial and associated with known genetic mutations. Despite intense research efforts, only four drugs have been approved by the US FDA for the treatment of ALS. Moreover, these drugs benefit a very limited number of patients and demonstrate modest effects in slowing their disease progression. To address this unmet medical need, we performed an unbiased phenotypic screen, using FDA-approved compounds, to identify potential therapeutics that can slow or stop the degeneration of ALS motor neurons. Validated hits are being tested in a diverse panel of ALS patients to identify drugs that are broadly efficacious in both familial and sporadic cases. 

What prompted you to study iPSCs as a potential therapeutic treatment for neurodegenerative diseases? 

To date, the majority of drug discovery applications have relied on the use of immortalized cell lines, artificial overexpression systems and animal models of human diseases. While these models have provided insights into some aspects of disease mechanisms, it is possible that they may not accurately and faithfully recapitulate the precise disease pathogenesis observed in humans. This may explain why many promising drugs have failed when trialed in humans due to a lack of efficacy or safety.

“The ability to identify promising drug candidates, and test them on human motor neurons in vitro, prior to clinical testing, will help bridge the gaps in the drug discovery pipeline.”

One way to potentially circumvent these challenges is to use biologically relevant cells – such as iPSCs – that are derived directly from a patient and thus yield a representative human cellular model. Our laboratory previously reported that abnormal cellular and molecular ALS phenotypes could be recapitulated in vitro using induced motor neurons produced by transcription factor-mediated conversion [1]. Based on these findings, we sought to utilize this patient-derived disease model as a discovery platform to screen and evaluate therapeutic candidates for ALS. The ability to identify promising drug candidates, and test them on human motor neurons in vitro, prior to clinical testing, will help bridge the gaps in the drug discovery pipeline. Ultimately, this strategy will greatly improve preclinical drug discovery and increase the likelihood of discovering better treatments for ALS.

What are some of the advantages of employing automated cell screening protocols in stem cell research? 

Automated cell screening protocols, such as high-throughput screening, allow one to test a chemical library containing thousands of drugs and evaluate their effects on modulating a specific molecular target or phenotypic characteristic associated with a disease. In terms of drug discovery efforts, it is ideal to pair this technology with a biologically relevant cell-based assay. The translational gap that exists between preclinical and clinical studies is currently being addressed by the use of human-derived stem cells in high-throughput screens for drug discovery. The hope is that using such physiologically relevant cells will increase the efficiency of the screening process and potentially lead to lower attrition rates in a cost-effective manner.

“…it is imperative to have a highly sensitive and reproducible assay in place before conducting a robust experiment.”

An advantage of using cells for a high-throughput screen is that they can be easily converted into a scalable assay that can be miniaturized and used to probe the effects of thousands of drugs contained within a library of interest. The miniaturization of the assay benefits the researcher by saving costs on precious samples and reagents, especially for subsequent compound/target validation experiments and counter screens. Importantly, the success of the screen for a given therapeutic area is dependent upon a strong disease phenotype, such as survival or a change in the expression and/or activity of an enzyme or gene. Therefore, it is imperative to have a highly sensitive and reproducible assay in place before conducting a robust experiment.

How do you apply automated processes in other areas of your research? 

The primary endpoint of our high-throughput screens is motor neuron survival. Therefore, high-content imaging is another automated process that we frequently employ. Our collaborators at DRVision Technologies LLC (WA, USA) are also working on developing an automated cell recipe to evaluate motor neuron survival by both cross-sectional and longitudinal analyses. In this regard, they are applying machine learning methods to count the motor neurons and follow their rate of neurodegeneration over time. The implementation of this automated image analysis will allow one to rapidly detect neurodegeneration, thereby enabling the easy quantification of large, screen-generated datasets.

What prompted you to study the effects of mood stabilizing pharmacological agents on the activities of stem cells? 

Stem cell-based therapy has recently emerged as a potential therapeutic avenue for treating neurodegenerative disorders. However, donor cell loss after transplantation presents a major obstacle for the clinical application of stem cells. One potential strategy to boost donor cell survival is to precondition stem cells, prior to transplantation, with compounds to enhance their survival and therapeutic efficacy.

“Stem cell-based therapy has recently emerged as a potential therapeutic avenue for treating neurodegenerative disorders.”

Following the completion of my PhD at Loma Linda University (CA, USA), I did a brief postdoc at the National Institute of Mental Health (part of the National Institutes of Health) (MD, USA) with De-Maw Chuang. His group had shown that certain mood stabilizers – lithium and valproic acid (VPA) – exert robust beneficial effects in diverse, preclinical models of neurological and neuropsychiatric diseases. Given that lithium and VPA induce multifaceted, pro-survival signaling pathways, we hypothesized that preconditioning bone marrow-derived mesenchymal stem cells (MSCs) with these mood stabilizers would enhance their therapeutic efficacy and facilitate functional recovery in a transgenic mouse model of Huntington’s disease. We found that lithium and VPA preconditioning prior to intranasal stem cell delivery significantly increased MSC survival in the brain post-transplantation, and enhanced the biological activity and therapeutic properties of the MSCs. This resulted in functional improvements and reduced neuropathological features in Huntington’s disease mice [2] and suggests that preconditioning with lithium and VPA may serve as an effective strategy for improving the therapeutic efficacy of stem cell-based therapies.  

How translatable is your research to the clinic and patient population?

For generations, the paradigm of clinical medicine has embodied a ‘one-size-fits-all’ approach; physicians look at the average patient response to a given treatment and use this information to prescribe medicine to the masses. This standard of care assumes that all patients that exhibit similar symptoms will respond identically when treated for a particular abnormality. However, it turns out that we are not all identical – a person’s physiology, lifestyle and environment contribute to individual variability. A more useful medical model is a precision medicine-based approach in which individual variability is taken into account for treatment and prevention strategies. In this context, our research using ALS patient-specific motor neurons to determine how diseased cells respond to drugs is a paradigm shift towards translational research and precision medicine.

“…our research using ALS patient-specific motor neurons to determine how diseased cells respond to drugs is a paradigm shift towards translational research and precision medicine.”

The results from the drug screen we conducted demonstrate highly varied drug responses among familial and sporadic ALS patients. This indicates that the implementation of tailored treatments would be helpful in the management of patients. The ability to stratify patient responders versus non-responders for a given treatment will help improve medical decision making for clinicians. We envision that the list of drugs generated from our screen will be able to be used as a unique blueprint for each patient and lead to tailored treatments – sparing patients from treatments that are not likely to work.                 

What is the future direction of your research? 

At the completion of this project it is our expectation that we will have determined novel molecular targets and key downstream signaling pathways implicated in the pathogenesis of ALS. In doing so, we hope to also determine whether any common therapeutic targets overlap across familial and sporadic forms of ALS. One intriguing question that arose during our phenotypic screen was: what governs whether an ALS patient is a responder versus a non-responder for a given treatment? We plan to investigate if there are specific genetic signatures that may allow one to predict the ability of a patient to respond effectively to a given treatment.

“We envision that the list of drugs generated from our screen will be able to be used as a unique blueprint for each patient and lead to tailored treatments – sparing patients from treatments that are not likely to work.”                 

In our present experiments, we utilize a 2D cell culture system as an in vitro model of ALS. It would be great to evaluate the effects of drugs which have been found beneficial in our 2D system, in a human, 3D brain organoid model of ALS. Organoid models are particularly exciting because they recapitulate the developmental processes of the developing brain and contain different cell types that can potentially model the complex network of interactions that exist in the brain. However, it remains to be determined whether this type of model would recapitulate the ALS disease processes observed in aged individuals.    

References:

[1] Shi Y, Lin S, Staats KA et al. Haploinsufficiency leads to neurodegeneration in C9ORF72 ALS/FTD human induced motor neurons. Nat Med. 24 (3):313-325, 2018.

 [2] Linares GR, Chiu CT, Scheuing L et al  Preconditioning  mesenchymal stem cells with the mood stabilizers lithium and valproic acid enhances therapeutic efficacy in a mouse model of Huntington’s Disease. Exp. Neurol. 281:81-92, 2016.  

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