FDA Mentor Names:

Minjun Chen, Ph.D.

Position and Organizational Affiliation

Minjun Chen: Senior Staff Fellow (FDA Division of Bioinformatics and Biostatistics, National Center for Toxicological Research, NCTR)

Contact Information (email/telephone)

Minjun.Chen@fda.hhs.gov / 870-543-7057

NCATS Mentor Names

Ruili Huang, Ph.D.

Position and Organizational Affiliation

Ruili Huang: Tox21 Informatics Team Lead (NCATS Division of Preclinical Innovation, DPI)

Contact Information (email/telephone)

huangru@mail.nih.gov / 301-827-0944

Research Project Summary

Despite significant efforts in recent years, the gap between basic scientific research and its clinical application—often referred to as the “valley of death” in drug development—remains substantial. Nearly 95% of drug candidates entering human trials fail to secure FDA approval [1]. A large study analyzing 608 failed drug candidate projects across four major pharmaceutical companies revealed that drug safety and toxicology were the primary causes of failure, accounting for 308 (or 51%) of the unsuccessful projects [2]. Consequently, improving the prediction of drug safety has become an urgent, unmet challenge in drug development.

The rapid evolution of artificial intelligence (AI) and machine learning (ML) techniques offers unprecedented opportunities in translational and regulatory science [3]. FDA drug labeling provides a rich resource of drug safety data, derived directly from human testing of thousands of approved drugs [4]. In addition, the FDA’s FAERS database contains millions of real-world records on adverse drug events. Together, these datasets offer vast, high-quality information on serious adverse events in humans. Moreover, the Tox21 program has screened approximately 8,500 chemicals across more than 80 high-throughput in vitro assays, generating over 120 million experimental data points [5].

This project focuses on leveraging AI technologies to analyze these large, content-rich human and in vitro toxicity datasets, with the goal of improving predictive models for assessing serious adverse drug events and supporting the FDA’s review process.

Proposed Project for TSIF Fellow

The TSIF Fellow will integrate Tox21 in vitro assay data with in silico models to develop AI tools for more accurate assessment of serious adverse drug events (defined by fatal or life-threatening events such as organ failure).

Initially, The Fellow will also leverage large language models (LLMs) to extract and interpret data from vast scientific literature and databases, enriching context of risk for serious adverse event and enhancing predictive models. Integrating these insights with in silico QSAR models, which assess chemical structure and pharmacokinetics, the Fellow will identify and flag new risks of serious adverse events from drug labels with the latest safety profiles, particularly for high-risk populations.

Then, the Fellow will apply machine learning algorithms to Tox21 data, using their expertise in deep learning and ensemble methods to identify the most predictive assays for serious adverse events. Assays focusing on specific mechanisms, such as receptor-mediated toxicity, cytotoxicity, and oxidative stress, will be prioritized to build models that provide insights into toxicity pathways. Assays will be weighted based on their biological relevance, offering a comprehensive view of complex processes like mitochondrial dysfunction.

The developed model will be validated against drugs with known serious adverse event profiles, using FDA databases and literature. The Fellow will collaborate with NCATS scientists to test compounds in the lab, refine models, and propose new assays as necessary. This role will develop advanced skills in data integration, predictive modeling, and contribute to improved risk assessment for serious adverse events, regulatory science, and the pharmaceutical industry.

Important Information: The National Center for Toxicological Research (NCTR; 3900 NCTR Rd Jefferson, AR 72079) is the only FDA Center located outside the Washington D.C. metropolitan area. In interested in this project, please discuss with the FDA mentors their relocation expectations during the TSIF fellowship.

Relevant Publications 

1. Seyhan, A.A. Lost in translation: the valley of death across preclinical and clinical divide – identification of problems and overcoming obstacles. Transl Med Commun 2019, 4(1),1-9.
2. Waring, M., Arrowsmith, J., Leach, A. et al. An analysis of the attrition of drug candidates from four major pharmaceutical companies. Nat Rev Drug Discov2015, 14, 475–486.
3. Mostafa F, Chen M. Computational models for predicting liver toxicity in the deep learning era. Frontiers in Toxicology. 2024, 19 (5):1340860. 
4. Chen M, Wu Y, Wingerd B, Liu Z, Xu J, Thakkar S, Pedersen TJ, Donnelly T, Mann N, Tong W, Wolfinger RD. Automatic text classification of drug-induced liver injury using document-term matrix and XGBoost. Frontiers in Artificial Intelligence. 2024, 7 :1401810.
5. Ann M. Richard, Ruili Huang, et al., The Tox21 10K Compound Library: Collaborative Chemistry Advancing Toxicology, Chem. Res. Toxicol. 2021, 34(2), 189–216

FDA Mentor Name

Weiming Ouyang

Position and Organizational Affiliation

Weiming Ouyang: Biologist (DPQR-III/ Office of Pharmaceutical Quality Research, OPQR; Office of Pharmaceutical Quality, OPQ; Center for Drug Evaluation and Research, CDER)

Contact Information (email/telephone)

weiming.ouyang@fda.hhs.gov / 240-402-7300

NCATS Mentor Name

Menghang Xia, Ph.D.

Position and Organizational Affiliation

Menghang Xia: Group leader (NCATS Tox21 Group; Chemical Genomics Branch, CGB; Division of Preclinical Innovation, DPI)

Contact Information (email/telephone)

mxia@mail.nih.gov/301-827-5359

Research Project Summary

T cell-mediated immune response is critical for anti-viral and anti-tumor immunity. However, under chronic viral infection and in tumor microenvironment, T cells become exhausted. Prevention of T cell exhaustion and/or restoration of the exhausted T cell function is critical for promoting the immune control of chronic viral infection and tumor growth. Our studies showed that the ubiquitin ligase COP1 is activated upon inactivation of Erk1/2, leading to degradation of c-Jun, the major component of AP-1 transcription factor that has a role in preventing T cell exhaustion. In addition to c-Jun, Foxo1 is also a substrate of COP1, which regulates T cell development, activation, tolerance and memory. Our pilot studies showed that COP1-defienct T cells express higher levels of c-Jun and Foxo1 than wild-type T cells, and mice with specific COP1 deletion in T cells exhibited a better anti-viral T cell immune response in the chronic LCMV infection model (lower virus load, more effector and memory T cells and lower percentage of exhausted T cells as compared to wild-type control mice).  These findings make COP1 an attractive target for discovery of drugs promoting T cell immunity.  This proposal includes optimization of in-house AP-1 reporter assay(s) for quantitative high throughput screening (qHTS) of drugs that can prevent Erk1/2 inactivation-induced COP1-mediated inhibition of AP1 activity, development of assay(s) to confirm the impact of selected compounds on COP1 location/function, 96-well plate flowcytometric analyses of the impact of selected compounds on the expression levels of c-Jun and Foxo1, T cell activation and cytokine production.

Proposed Project for TSIF Fellow

We have qualified AP-1 reporter gene assays. The TSIF fellow will optimize the in-house AP-1 reporter assay in a qHTS platform and then use it to screen NCATS chemical and drug library to identify compounds that can prevent Erk1/2 inactivation-induced and COP1-mediated inhibition of AP1 activity. After the primary screening, the TSIF fellow will perform data analysis and cherry-picking active compounds based on their potency and efficacy. The TSIF fellow will also develop secondary assays and perform following experiments to confirm the impact of cherry-picked  compounds from the primary screening on COP1 function based on imaging of COP1 translocation from the nuclear envelop to the nucleus, COP1 binding with its partners via the coiled-coil domain, or  COP1 binding with its substrates via the conserved consensus sequence (ESDEExxxVP[D/E]), and flowcytometric analyses of the impact of selected compounds on T cell activation and cytokine production. By working on this project, the TSIF fellow will be trained in the areas of assay development and optimization, T cell biology, drug discovery, and FDA-regulated therapeutics targeting T cell functions. The TSIF fellow will also learn knowledge about FDA regulation of therapeutics targeting T cell function, which include immune checkpoint inhibitors, anti-CD3 bispecific T cell engagers. The anticipated outcomes include delivery of qualified assay(s) for quantitative high throughput screening and confirmatory studies, and discovery of compounds that inhibit COP1 function and enhance T cell function by maintaining high expression levels of c-Jun and Foxo1 transcription factor in T cells.

Relevant Publications

1. Baessler A, and Vignali DAA. T Cell Exhaustion. Annu Rev Immunol. 2024; 42: 179-206.
2. Ouyang W, Guo P, Takeda K, Fu Q, Fang H, Frucht DM. Erk1/2 inactivation promotes a rapid redistribution of COP1 and degradation of COP1 substrates. Proc Natl Acad Sci U S A. 2020;117(8):4078-4087.
3. Lynn RC, Weber EW, Sotillo E, Gennert D, Xu P, Good Z, Anbunathan H, Lattin J, Jones R, Tieu V, Nagaraja S, Granja J, de Bourcy CFA, Majzner R, Satpathy AT, Quake SR, Monje M, Chang HY, Mackall CL. c-Jun overexpression in CAR T cells induces exhaustion resistance. Nature. 2019;576(7786):293-300.
4. Kato S, Ding J, Pisck E, Jhala US, Du K. COP1 functions as a FoxO1 ubiquitin E3 ligase to regulate FoxO1-mediated gene expression. J Biol Chem. 2008;283(51):35464-73.

FDA Mentor Name

Kyung Sung, Ph.D.

Position and Organizational Affiliation

Kyung Sung: Branch Chief (Cellular and Tissue Therapy Branch, Office of Cellular Therapy and Human Tissue, OCTHT; Center for Biologics Evaluation and Research, CBER)

Contact Information (email/telephone)

Kyung.sung@fda.hhs.gov / 240-402-7994

NCATS Mentor Name

Marc Ferrer, Ph.D.

Yi Wei Lim, Ph.D.

Position and Organizational Affiliation

Marc Ferrer: Director (NCATS 3-D Tissue Bioprinting Laboratory, Division of Preclinical Innovation, DPI)

Yi Wei Lim: Postdoctoral Fellow (NCATS 3-D Tissue Bioprinting Laboratory, Division of Preclinical Innovation, DPI)

Contact Information (email/telephone)

marc.ferrer@nih.gov / 301-480-9845

yiwei.lim@nih.gov / 414-241-4424

Research Project Summary

The skin acts as our first line of defense against external insults and harbors a highly specialized immunological niche crucial for tissue homeostasis, defense against harmful pathogens and modulating tissue repair. Dysregulation of this niche can lead to the manifestation of inflammatory skin diseases such as fibrosis. There are many etiologies of fibrotic disorders owing to their complex pathophysiological mechanisms, and it is known that macrophages are pivotal in the initiation, progression, and resolution of fibrosis.

Cell-based therapies and biological products, such as mesenchymal stromal cells (MSC) and extracellular vesicles (EVs), hold significant promise for treating skin diseases, including fibrosis manifestation such as hypertrophic scar and keloid[1]. EVs from skin cells, blood, MSCs and immune cells have demonstrated the potential to reduce fibrosis and modulate pro-fibrotic macrophage activity, offering new avenues for treatment [2, 3].

Current testing of these therapies often relies on traditional 2D monolayer in vitro models or in vivo animal models, which do not fully replicate human tissue complexity. To address this gap, there is a growing need to develop more advanced experimental tools to assess mechanisms of action, better characterize biologics, and provide stronger support for nonclinical testing.

This project aims to assess the feasibility of using a 3D immunocompetent skin fibrosis model to test the functional activity of both cell-based therapies and biological products, focusing on their anti-fibrotic effects and their interactions with immune cells in a more physiologically relevant environment.

Proposed Project for TSIF Fellow

Given the current trends in product development, we will begin by testing both mesenchymal stromal cells (MSC) and MSC-derived extracellular vesicles (EVs). This approach reflects the growing interest in these products, though future studies will explore additional cell therapies and biological products to assess their broader potential in treating skin fibrosis.

The Sung lab (FDA) has expertise in developing new approaches for the manufacture and characterization of both MSC and EVs, while the Ferrer lab (NCATS) has developed an immunocompetent 3D skin model by incorporating primary human macrophages and have successfully model skin fibrosis using fibrotic inducer, TGFβ. The fellow will work across both labs, developing the 3D model in the Ferrer Lab and manufacturing and characterizing MSCs and MSC-derived EVs in the Sung Lab. We anticipate the fellow to derive EVs from both stimulated and unstimulated MSCs to examine their impact on fibrotic markers, macrophage function, and plasticity within the 3D skin fibrosis model.

Preconditioning strategies, such as cytokine stimulation or chemical agents, will be explored to assess the immunomodulatory and anti-fibrotic capabilities of both MSCs and MSC-derived EVs. The study will also evaluate their effect on fibrosis by assessing established markers such as collagen and myofibroblast staining, alongside a panel of fibrosis-associated factors secreted by the 3D tissue constructs. The effectiveness of both MSCs and MSC-derived EVs will be compared to TGFβ treated tissues, which will serve as a positive control.

Relevant Publications

1. Yu, H., et al., Exosomes: The emerging mechanisms and potential clinical applications in dermatology. Int J Biol Sci, 2024. 20(5): p. 1778-1795.
2. Yu, Y., et al., The Therapeutic Effects of Exosomes Derived from Human Umbilical Cord Mesenchymal Stem Cells on Scleroderma. Tissue Eng Regen Med, 2022. 19(1): p. 141-150.
3. Xie, L., et al., Bone marrow mesenchymal stem cell-derived exosomes alleviate skin fibrosis in systemic sclerosis by inhibiting the IL-33/ST2 axis via the delivery of microRNA-214. Mol Immunol, 2023. 157: p. 146-157.

FDA Mentor Name

Chava Kimchi-Sarfaty, Ph.D.

Upendra Katneni, Ph.D.

Position and Organizational Affiliation

Chava Kimchi-Sarfaty: Associate Director for Research (FDA Office of Therapeutic Products, OTP; Center for Biologics Evaluation and Research, CBER)

Upendra Katneni: Staff Fellow (FDA Office of Therapeutic Products, OTP; Center for Biologics Evaluation and Research, CBER)

Contact Information (email/telephone)

Chava.kimchi-sarfaty@fda.hhs.gov / 240-402-8203

upendra.katneni@fda.hhs.gov

NCATS Mentor Names

Catherine Chen, Ph.D.

Position and Organizational Affiliation

Catherine Chen: Associate Group Leader (Biology group, Therapeutics Development Branch; Division of Preclinical Innovation, DPI)

Contact Information (email/telephone)

catherine.chen@nih.gov/301-827-5365

Research Project Summary

Recombinant protein therapeutics and gene therapies have emerged as key players for treating a variety of clinical conditions. During the drug design of these therapeutics, codon optimization (CO), a technique wherein synonymous variants are systematically substituted into the gene coding sequence, is commonly leveraged to enhance protein yield. However, our prior studies have highlighted numerous potential functional effects of CO that can negatively impact the therapeutic, such as changes to protein structure, activity, post-translational modifications, and immunogenicity [1-2]. Currently, effects of CO on therapeutic protein characteristics are not clearly understood. In this proposal, we investigate novel CO approaches for a truncated-ADAMTS13 protein with functional ADAMTS13 activity (commonly referred to as MDTCS) and Niemann-Pick disease type C1 (NPC1well characterized by NCATS studies. Loss of ADAMTS13 activity in plasma results in a thrombotic disorder referred to as Thrombotic Thrombocytopenic Purpura (TTP). Loss of function mutations in NPC1 causes the great majority of Niemann-Pick disease type C1. Both serve as examples for rare diseases. The overarching goal of this project is to evaluate novel designs of codon-optimization together with cellular factors that will enable enhanced expression of MDTCS and NPC1 proteins while preserving their native structure and function. 

Proposed Project for TSIF Fellow

In this project, the prospective TSIF fellow will be leading studies at both the FDA and NCATS research centers to address the following aims:

1. Assess the effects of novel strategies of targeted CO on the physicochemical properties of recombinant proteins in the context various delivery systems. The evaluated strategies will include CO based on evolutionary conservation of coding sequences, islands of slow translation and cellular tRNA availability. This part will be done at FDA.
2. Evaluate the effects of cellular tRNAome on the translation and co-translational folding of CO recombinant proteins. This part will be done at FDA.
3. Evaluate the effect of expression levels on recombinant protein quality characteristics and cellular metabolism. Purified recombinant MDTCS expressed from targeted integration of single or multiple copies of transgenes using CRISPR/Cas9-mediated DNA integration will be evaluated. This part will be done at NCATS.
4. Evaluate the effects of NPC1 CO on expression levels, ER stress markers, and in vitro disease phenotypes in NPC1 patient iPSC-derived neuronal and hepatocytes. This part will be done at both FDA and NCATS.

Through studies that investigate these aims, the fellow will be able to learn and apply sensitive and novel techniques including structural analysis techniques (e.g., circular dichroism spectra analysis and post-translational modification analysis), biochemical/functional assays (e.g., immunoblotting, enzyme activity assays and Biacore SPR Octet analysis), and ER stress markers. The fellow will lead projects through unique collaborations with the FDA biotechnology CORE and bioinformatics teams. The fellow’s contributions will help to reveal novel insights into the effects of CO on recombinant protein expression, which will inform upon future FDA regulations on the use of codon optimization for therapeutic products. Work on these studies will be presented at national conferences to inform the broad research community on the implications for the use of these redesign approaches for generating translational therapeutics.

Relevant Publications

1. Katneni UK, Alexaki A, Hunt RC, Hamasaki-Katagiri N, Hettiarachchi GK, Kames JM, McGill JR, Holcomb DD, Athey JC, Lin B: Structural, functional, and immunogenicity implications of F9 gene recoding. Blood Advances 2022.
2. Alexaki A, Hettiarachchi GK, Athey JC, Katneni UK, Simhadri V, Hamasaki-Katagiri N, Nanavaty P, Lin B, Takeda K, Freedberg D: Effects of codon optimization on coagulation factor IX translation and structure: Implications for protein and gene therapies. Scientific reports 2019, 9:1-15.

FDA Mentor Name

Kang Chen

Position and Organizational Affiliation

Kang Chen: Research Chemist (FDA Center for Drug Evaluation and Research, CDER)

Contact Information (email/telephone)

Kang.Chen@fda.hhs.gov / 240-402-5550

NCATS Mentor Names

Christopher LeClair, Ph.D.

Dingyin Tao, Ph.D.

Position and Organizational Affiliation

Christopher LeClair: Director (NCATS Analytical Chemistry Core, Division of Preclinical Innovation, DPI) 

Dingyin Tao: Lead, (NCATS Mass Spectrometry Team, Analytical Chemistry Core, Division of Preclinical Innovation, DPI)

Contact Information (email/telephone)

leclairc@mail.nih.gov / 301-480-9941

dingyin.tao@nih.gov / 301-827-7176

Research Project Summary

As of March 2020, drug applications for certain biological products previously regulated under section 505 of the FD&C Act are now assessed and licensed under the 351(a/k) BLA pathway of the PHS Act. This regulatory transition enabled the possibility of new biosimilar applications for these products, which if approved, could reduce direct patient cost. Included in these regulated biological products are growth hormone drugs composed of the individual proteins or protein mixtures of human chorionic gonadotrophin (hCG), follicle stimulating hormone (FSH), luteinizing hormone (LH), human growth hormone (hGH) and human thyroid stimulating hormone (hTSH), which are naturally sourced from human urine or recombinantly expressed in various host cells. Biosimilar applicants need to provide appropriate data to demonstrate the protein higher order structure (HOS), oligomerization, glycosylation pattern, and bioassay results are of sufficient similarity between the biosimilar product and the innovator product. This necessitates developing modern analytical methods for successful analysis of complex protein mixtures specifically related to human growth hormones. The current proposal will evaluate similar growth hormone drug products with varied sources of production and formulation. Chemical, structural, and biological activity results will be analyzed holistically to assess correlations among them and identify characteristics of process features. Both the agency and biosimilar drug developers will benefit from these modern analytical approaches that would foster approval of future biosimilar hormone mixtures.

Proposed Project for TSIF Fellow

Under the guidance of mentors at the FDA and NCATS, the fellow will develop analytical methods for the analysis of human growth hormones, which are an important category of biological products. This will be achieved using various techniques and technology that include but is not limited to (i) high-performance liquid chromatography mass spectrometry (HPLC-MS), (ii) nuclear magnetic resonance (NMR), (iii) dynamics light scattering (DLS), (iv) fast protein liquid chromatography (FPLC), and (v) in vitro cell based bioassay. The fellow will conduct analysis and gather data on chemical modifications (e.g., protein glycosylation, higher order structure, and oligomerization) and their effect in the bioassay of hormone drugs. The implementation of applicable biological product analysis in the drug regulatory field will offer greater assurance of drug quality and minimize potential adverse effects introduced through the use of biosimilar versions or changes to the manufacturing process. This research will provide CMC reviewers the necessary information, especially glycan and structural variation reflected in MS and NMR spectral data, to effectively evaluate physiochemical differences. The application of state-of-the-art analytical techniques at the FDA and NCATS for drug product quality characterization will be a unique regulatory research and development experience not available from other programs. Research results will be disseminated through professional meetings, peer-reviewed publications, and potential regulatory guidance.

Relevant Publications

1.  Chen, K.; Long, D.; Lute, S.; Levy, M.; Brorson, K.; Keire D. Simple NMR methods for evaluating higher order structures of monoclonal antibody therapeutics with quinary structure. J. Pharm. Biomed. Anal. 2016, 128, 398-407.
2. Patil, S.; Keire, D.; Chen, K. Comparison of NMR and Dynamic Light Scattering for measuring diffusion coefficients of formulated insulin: implications for particle size distribution measurements in drug products. AAPS Journal 2017, 19, 1760-1766.
3. Peng, J.; Patil, S.; Keire, D.; Chen K. Chemical Structure and Composition of Major Glycans Covalently Linked to Therapeutic Monoclonal Antibodies by Middle-Down Nuclear Magnetic Resonance. Anal. Chem. 2018, 90, 11016-11024.
4. Xie, T. et al. The ELISA Detectability and Potency of Pegfilgrastim Decrease in Physiological Conditions: Key Roles for Aggregation and Individual Variability, Scientific Report, 2020, 10, 2476.
5. Zhuo, Y.; Keire, D.; Chen, K. Minor N-glycan Mapping of monoclonal Antibody Therapeutics using Middle-down NMR Spectroscopy, Mol. Pharm. 2021, 18, 441.
6. Biel, T. et al, An etanercept O-glycovariant with heightened potency, Molecular Therapy - Methods & Clinical Development 2022, 124, 135.
7. Shipman, J.; Sommers, C.; Keire, D.; Chen, K.; Zhu, H. Comprehensive N-Glycan Mapping using Parallel Reaction Monitoring LC-MS/MS, Pharm. Res. 2023, 40, 1399.
8. Wang, K.; Chen, K. Direct Assessment of Oligomerization of Chemically Modified Peptides and Proteins in Formulations using DLS and DOSY-NMR. Pharm. Res. 2023, 40, 1329. 
9. Qiao, X.; Tao, D.; Qu, Y.; Sun, L.; Gao, L.; Zhang, X.; Liang, Z.; Zhang, L.; Zhang, Y. Large-scale N-glycoproteome map of rat brain tissue: simultaneous characterization of insoluble and soluble protein fractions. Proteomics 2011, 11, 4274-4278.
10. Yan, W.; Zhong, Y.; Hu, X.; Xu, T.; Zhang, Y.; Kales, S.; Qu, Y.; Talley, D. C.; Baljinnyam, B.; LeClair, C. A.; Simeonov, A.; Polster, B. M.; Huang, R.; Ye, Y.; Rai, G.; Henderson, M. J.; Tao, D.; Fang, S. Auranofin targets UBA1 and enhances UBA1 activity by facilitating ubiquitin trans-thioesterification to E2 ubiquitin-conjugating enzymes. Nat. Commun. 2023, 14, 4798.
11. Burns, A. P.; Zhang, Y. Q.; Xu, T.; Wei, Z.; Yao, Q.; Fang, Y.; Cebotaru, V.; Xia, M.; Hall, M. D.; Huang, R.; Simeonov, A.; LeClair, C. A.; Tao, D. A Universal and High-Throughput Proteomics Sample Preparation Platform. Anal. Chem. 2021, 93, 8423-8431.

FDA Mentor Names

Ronit Mazor, Ph.D.

Zhaohui Ye, Ph.D.

Sojin Bing, Ph.D.

Position and Organizational Affiliation

Ronit Mazor: Principal Investigator (Gene Transfer and Immunogenicity Branch, GTIB; Office of Generic Drugs, OGD, Office of Therapeutic Products, OTP; Center for Biologics Evaluation and Research, CBER)

Zhaohui Ye: Branch Chief (Gene Transfer and Immunogenicity Branch, GTIB; Office of Generic Drugs, OGD, Office of Therapeutic Products, OTP; Center for Biologics Evaluation and Research, CBER)

Sojin Bing: Staff Fellow (Gene Transfer and Immunogenicity Branch, GTIB; Office of Generic Drugs, OGD, Office of Therapeutic Products, OTP; Center for Biologics Evaluation and Research, CBER)

Contact Information (email/telephone)

Ronit.Mazor@fda.hhs.gov / 301-796-5415

zhaohui.ye@fda.hhs.gov / 240-402-7471

sojin.bing@fda.hhs.gov / 240-402-7339

NCATS Mentor Names

Min Jae Song, Ph.D.

Position and Organizational Affiliation

Min Jae Song: Team Lead (NCATS 3D Tissue Bioprinting Lab, Division of Preclinical Innovation, DPI)

Contact Information (email/telephone)

minjae.song@nih.gov/301-402-8076

NEI Collaborators Names

Ruchi Sharma, Ph.D.

Kapil Bharti, Ph.D.

Position and Organizational Affiliation

Ruchi Sharma: Staff Scientist (Ocular Stem Cells and Translational Research, OSCTR; National Eye Institute, NEI)

Kapil Bharti: Senior Investigator, Scientific Director (National Eye Institute, NEI)

Contact Information (email/telephone)

fnu.ruchi2@nih.gov/301-451-3081

kapil.bharti@nih.gov/301-451-9372

Research Project Summary

NCATS in collaboration with NEI, has developed a bioprinted outer blood retina barrier (BRB) tissue model recapitulating the basic tissue architecture and barrier functions by containing essential cellular components in the context of three-dimensional (3D) scaffold [1]. The model has further demonstrated relevant disease phenotypes such as outer BRB degeneration and choroidal neovascularization (CNV) by biochemical stimulation. The use of human iPSCs in the model scales up patient-specific tissue models for extensive applications to therapeutic development. The pluripotency of the iPSCs further allows for gene-phenotype studies using the gene modified iPSCs [1]. Differentiation protocols of iPSCs into RPE, endothelial cells, pericytes and fibroblasts have been well established at NEI, demonstrating in vivo phenotypes and functions [2,3].                  

Choroideremia (CHM) is a rare inherited retinal degeneration that affects the outer retinal layers at the back of the eye, leading to degeneration of Retinal Pigment Epithelium (RPE) and Choroid, eventually leading to irreversible vision loss. CHM is caused by a mutation in a gene located in the X-chromosome, which encodes Rab escort protein 1 (REP1), causes degenerations of the photoreceptor, RPE, and choroid in the outer BRB where the clinical manifestations include pigmentary clumping in the RPE layer, CNV, and macular edema. Currently, there is no approved treatment for Choroideremia, and the adenovirus associated vector (AAV)-based gene therapy strategies are one of the potential solutions to the unmet need by repairing the CHM gene. Despite the promising results of AAV2-REP1 in phase 1/2 trials, it failed to meet its primary endpoint of best-corrected visual acuity in phase 3 STAR trial [4]. This discrepancy reemphasizes the critical issues in the predictability of preclinical studies and the need for high level models to investigate the factors contributing to this discrepancy [5]. The 3D bioprinted outer BRB model using choroideremia patient-derived iPSC provides extensive opportunities in assessing various gene therapy strategies, and the goal of the proposal is to help complement the current preclinical studies of gene therapy by improving predictability.

The proposed work, combined with NCATS’ longstanding research interest in rare diseases, NEI’s and FDA commitment to developing treatments for rare inherited retinal degenerations such as choroideremia using cell and gene-based therapies, aims to provide a benchmark for validation and standardization of new approach methods for FDA regulatory decision-making regarding the effectiveness and safety of gene therapy products.

Proposed Project for TSIF Fellow

The selected fellow will develop choroideremia in-vitro models and lead the assessment of the gene therapy products on it as below.

3D Bioprinting choroideremia models at NCATS: The selected fellow will participate in bioprinting outer blood retina barrier (BRB) tissues in a transwell® format using patient-derived and CHM gene-modified iPSC-derived cells at NCATS. The fellow will lead the validation of the disease models based on the disease phenotypes by confocal microscopy-based imaging, barrier functions by transepithelial electrical resistance, phagocytic ability of RPE cells, degenerative or senescent changes in the choriocapillaris, and other cellular endophenotypes developed in consultations with Dr. Kapil Bharti and Dr. Ruchi Sharma at NEI. 

Assessment of gene therapy products at NCATS and FDA: The Fellow will work on the assessment of gene therapy products (fluorescence labeled AAVs from NEI and FDA; AAV-REP1 from NEI or from commercial sources) on the outer BRB model, including efficacy for reversing a choroideremia phenotype and toxicity. The assessment will include quantitative evaluations of AAV transduction efficacy, disease phenotype, barrier functions, cytokine secretions, and qualitative histology evaluations, in both healthy and disease BRBs.

The Fellow will present experiment results at the monthly meeting and share the data with NCATS and FDA mentors. The Fellow will present this work in conferences and work on publication in a scientific journal.

Relevant Publications

1. Song, M.J., Quinn, R., Nguyen, E. et al.Bioprinted 3D outer retina barrier uncovers RPE-dependent choroidal phenotype in advanced macular degeneration. Nat Methods 20, 149–161 (2023). https://doi.org/10.1038/s41592-022-01701-1.
2. Sharma R, George A, Nimmagadda M, Ortolan D, Karla BS, Qureshy Z, Bose D, Dejene R, Liang G, Wan Q, Chang J, Jha BS, Memon O, Miyagishima KJ, Rising A, Lal M, Hanson E, King R, Campos MM, Ferrer M, Amaral J, McGaughey D, Bharti K. Epithelial phenotype restoring drugs suppress macular degeneration phenotypes in an iPSC model. Nat Commun. 2021 Dec 15;12(1):7293. doi: 10.1038/s41467-021-27488-x. PMID: 34911940; PMCID: PMC8674335.
3. Park TS, Hirday R, Ali A, Megersa R, Villasmil R, Nguyen E, Bharti K. Protocol to generate endothelial cells, pericytes, and fibroblasts in one differentiation round from human-induced pluripotent stem cells. STAR Protoc. 2023 May 6;4(2):102292. doi: 10.1016/j.xpro.2023.102292. Epub ahead of print. PMID: 37149860; PMCID: PMC10189549.
4. MacLaren RE, Fischer MD, Gow JA, Lam BL, Sankila EK, Girach A, Panda S, Yoon D, Zhao G, Pennesi ME. Subretinal timrepigene emparvovec in adult men with choroideremia: a randomized phase 3 trial. Nat Med. 2023 Oct;29(10):2464-2472. doi: 10.1038/s41591-023-02520-3. Epub 2023 Oct 9. PMID: 37814062; PMCID: PMC10579095.
5. Zhai Y, Xu M, Radziwon A, Dimopoulos IS, Crichton P, Mah R, MacLaren RE, Somani R, Tennant MT, MacDonald IM. AAV2-Mediated Gene Therapy for Choroideremia: 5-Year Results and Alternate Anti-sense Oligonucleotide Therapy. Am J Ophthalmol. 2023 Apr;248:145-156. doi: 10.1016/j.ajo.2022.12.022. Epub 2022 Dec 26. PMID: 36581191.

FDA Mentor Names

Ross Marklein, Ph.D.

Jin Han, Ph.D.

Position and Organizational Affiliation

Ross Marklein: Senior Staff Fellow (Office of Therapeutic Products, OTP; Cellular and Tissue Therapy Branch, CTTB; Center for Biologics Evaluation and Research, CBER)

Jin Han: Staff Fellow (Office of Therapeutic Products, OTP; Cellular and Tissue Therapy Branch, CTTB; Center for Biologics Evaluation and Research, CBER)

Contact Information (email/telephone)

ross.marklein@fda.hhs.gov / 301-796-8712

jin.han@fda.hhs.gov / 240-402-1849

NCATS Mentor Name

Carlos Tristan, Ph.D.

Position and Organizational Affiliation

Carlos Tristan: Director (NCATS Stem Cell Translation Laboratory; Division of Preclinical Innovation DPI)

Contact Information (email/telephone)

carlos.tristan@nih.gov/301-827-0682

Research Project Summary

Mesenchymal stromal cell (MSC) derived extracellular vesicles (MSC-EVs) are secreted membrane-bound nanoparticles currently being explored as therapies for neurodegenerative diseases due to their ability to modulate neuroinflammation. However, clinical translation has been unsuccessful due in part to challenges such as MSC-EV heterogeneity and unknown mechanisms of action. MSC-EV heterogeneity can occur due to differences in donor and tissue source of the MSCs, as well as non-standardized use of undefined (heterogeneous) media components such as fetal bovine serum and platelet lysate for large-scale MSC expansion. Induced pluripotent stem cell (iPSC) derived MSCs (iMSCs) have been proposed as a promising source of MSCs (and MSC-EVs) as iPSCs have extensive proliferation potential and different batches of MSCs could be manufactured from the same, consistent isogenic cell source. However, this differentiation process is not defined or well understood. In terms of mechanism of action, there is evidence that MSC-EVs can modulate neuroinflammation and promote recovery of brain function, however; it is unclear how MSC-EVs exert their therapeutic effect and whether neurovascular cell-types such as pericytes can mediate this effect. Therefore, the overall goal of the TSIF project is to develop a defined protocol for manufacturing iMSCs that produce iMSC-derived EVs (iMSC-EVs) with ability to modulate pericyte function in the context of neuroinflammation.

Proposed Project for TSIF Fellow

For Aim 1 (FDA-focused), the fellow will develop a defined media to differentiate iPSCs into immunomodulatory iMSCs. The fellow will establish a protocol to differentiate multiple batches of iMSCs from a single iPSC line then compare the effects of different media on iMSC differentiation and subsequent production of iMSC-EVs. Finally, the fellow will perform transcriptomics on iMSCs at various stages of differentiation process to identify pathways regulating iMSC differentiation.

For Aim 2 (NCATS-focused), we will investigate how pericytes respond to iMSC-EV treatment. The fellow will develop an assay to profile the effect of iMSC-EVs on pericyte function and transcriptome. The fellow will then explore whether pericytes treated with iMSC-EVs can indirectly regulate the following iPSC-derived neuron outcomes relevant to neuroinflammation: neuronal survival (viability assay), maturation (RNA-Seq, neurite outgrowth) and function (multi-electrode arrays) using an indirect co-culture assay (iMSC-EV treated pericyte conditioned medium transferred to neuron cultures). For iMSC-EV batches that induce the greatest neuronal response (mediated by pericytes), we will profile the pericyte secretome and neuronal transcriptome to identify key secreted factors and signaling pathways associated with the observed response.

The proposed project will leverage the expertise of both NCATS and FDA labs to provide comprehensive training in the following areas: regulatory science, cell/EV manufacturing, iPSC technology, high throughput screening, image analysis, transcriptomics, and machine learning.

Relevant Publications

1. Larey AM, Spoerer TM, Daga KR, Morfin MG, Hynds HM, Carpenter J, et al. High throughput screening of mesenchymal stromal cell morphological response to inflammatory signals for bioreactor-based manufacturing of extracellular vesicles that modulate microglia. Bioact Mater. 2024;37:153-71.
2. Daga KR, Larey AM, Morfin MG, Chen K, Bitarafan S, Carpenter JM, et al. Microglia Morphological Response to Mesenchymal Stromal Cell Extracellular Vesicles Demonstrates EV Therapeutic Potential for Modulating Neuroinflammation. J Biol Eng. 2024.
3. Tristan CA, Ormanoglu P, Slamecka J, Malley C, Chu PH, Jovanovic VM, et al. Robotic high-throughput biomanufacturing and functional differentiation of human pluripotent stem cells. Stem Cell Reports. 2021;16(12):3076-92.

FDA Mentor Names

Sangeeta Khare, M.S., Ph.D.

Kuppan Gokulan, Ph.D.

Position and Organizational Affiliation

Sangeeta Khare: Principal Investigator/Research Microbiologist (FDA Division of Microbiology, National Center for Toxicological Research, NCTR)

Kuppan Gokulan: Principal Investigator/Research Microbiologist (FDA Division of Microbiology, National Center for Toxicological Research, NCTR)

Contact Information (email/telephone)

sangeeta.khare@fda.hhs.gov / 870-543-7519

Kuppan.gokulan@fda.hhs.gov / 870-543-7467

NCATS Mentor Names

Xin Xu, Ph.D.

Elias Carvalho Padilha, Ph.D.

Menghang Xia, Ph.D.

Position and Organizational Affiliation

Xin Xu: Sr. Scientist, Director (NCATS Drug Metabolism and Pharmacokinetics (DMPK) Core; Division of Preclinical Innovation, DPI)

Elias Carvalho Padilha: Staff Scientist (NCATS Drug Metabolism and Pharmacokinetics (DMPK) Core; Division of Preclinical Innovation, DPI)

Menghang Xia: Group leader (NCATS Tox21 Group; Chemical Genomics Branch, CGB; Division of Preclinical Innovation, DPI)

Contact Information (email/telephone)

xin.xu3@nih.gov / 301-480-9844
elias.padilha@nih.gov / 301-827-1813
mxia@mail.nih.gov / 301-827-5359

Research Project Summary

Transepithelial electrical resistance (TEER) is an in vitro method to measure the barrier function of cellular monolayers that has been widely utilized in biological research in areas such as drug screening, infectious diseases, cancer metastasis, and more ^1-5. The NCATS Drug Metabolism and Pharmacokinetics (DMPK) Core in partnership with Applied Biophysics developed the TEER96 device (https://www.biophysics.com/teer96.php) to increase the throughput of TEER measurements. The device also allows for the continuous read of TEER values through time in a sterile and temperature-controlled environment.  Given the gained functionality, a unique opportunity is presented to utilize this technology to live-monitor the impact of chemicals to cell monolayers cultured in transwell systems. The DMPK group conducted a screening of orally available toxins in Caco-2 intestinal cell monolayers while monitoring TEER over 72 hours. In the preliminary study, several compounds showed concentration dependent monolayer disrupting properties measurable by TEER, however, the mechanisms involved in this barrier disruption remain elusive. This project aims to expand the TEER screening of oral toxins to the intestinal monolayer model and elucidate barrier disruption mechanisms with the goal of developing a new gut toxicity screening paradigm.

The project will leverage the NCATS expertise with the TEER96, high-throughput screening, and automation and, at the NCTR, the project will benefit from Dr. Khare’s expertise in gut biology and toxicology to unveil the mechanistic features of the gut barrier disruption by orally available environmental toxins. The data generated from this project will provide valuable information for FDA drug regulation and public health.

Proposed Project for TSIF Fellow

The Fellow selected for this collaboration’s goal will be to develop a new gut toxicity screening paradigm. The Fellow will lead the development of high-throughput assay for the intestinal barrier monitoring using TEER and further investigate the molecular mechanism of the food grade toxins involved in the disruption of the intestinal epithelial layer. The Fellow will work 50% time at the NCATS and 50% time at the NCTR/FDA. The proposed work will be as follow:

NCATS: Test the potential intestinal toxicity of compounds from the Tox21 10k library that may be orally ingested from food or water. These test compounds include pesticides, antibiotics, fragrances, food coloring compounds that are usually present in food or water.

NCATS will perform TEER96 screening in human epithelial (Caco-2) cells and share the data of TEER results with NCTR Collaborator. Samples (cell extracts) will be sent to NCTR for gene expression analysis and (supernatants) cytotoxicity measurement. After gene expression analysis at NCTR.

NCTR: Complete cell extraction and perform gene expression on samples related to cell monolayer disruption, cell death, and other relevant mechanisms which can influence the observed TEER profile. Cytotoxicity measurements will be measured from supernatants. Data will be shared with NCATS.

The Fellow will present experiment results at the monthly meeting and share the data with NCATS and FDA mentors. The Fellow will participate in data interpretation and writing scientific report(s) with TEER profiles and gene expression information to be considered for publication in a scientific journal.

Important Information: The National Center for Toxicological Research (NCTR; 3900 NCTR Rd Jefferson, AR 72079) is the only FDA Center located outside the Washington D.C. metropolitan area. In interested in this project, please discuss with the FDA mentors their relocation expectations during the TSIF fellowship. 

Relevant Publications

1. Gokulan K, Kolluru P, Cerniglia CE, Khare S. Dose-Dependent Effects of Aloin on the Intestinal Bacterial Community Structure, Short Chain Fatty Acids Metabolism and Intestinal Epithelial Cell Permeability. Frontiers in Microbiology 2019;10.
2. Gokulan K, Kumar A, Lahiani MH, Sutherland VL, Cerniglia CE, Khare S. Differential Toxicological Outcome of Corn Oil Exposure in Rats and Mice as Assessed by Microbial Composition, Epithelial Permeability, and Ileal Mucosa-Associated Immune Status. Toxicological Sciences 2021;180:89-102.
3. Hao HH, Gokulan K, Pineiro SA, et al. Effects of Acute and Chronic Exposure to Residual Level Erythromycin on Human Intestinal Epithelium Cell Permeability and Cytotoxicity. Microorganisms 2019;7. 
4. Orr SE, Gokulan K, Boudreau M, Cerniglia CE, Khare S. Alteration in the mRNA expression of genes associated with gastrointestinal permeability and ileal TNF- secretion due to the exposure of silver nanoparticles in Sprague-Dawley rats. Journal of Nanobiotechnology 2019;17.
5. Williams KM, Gokulan K, Cerniglia CE, Khare S. Size and dose dependent effects of silver nanoparticle exposure on intestinal permeability in an in vitro model of the human gut epithelium. Journal of Nanobiotechnology 2016;14.
6. Siramshetty V, Williams J, Nguyen T, et al. Validating ADME QSAR Models Using Marketed Drugs. Slas Discovery 2021;26:1326-1336.
7. Williams J, Siramshetty V, Nguyen DT, et al. Using in vitro ADME data for lead compound selection: An emphasis on PAMPA pH 5 permeability and oral bioavailability. Bioorganic & Medicinal Chemistry 2022;56.
8. Gokulan K, Mathur A, Kumar A, Vanlandingham MM, Khare S. Route of Arsenic Exposure Differentially Impacts the Expression of Genes Involved in Gut-Mucosa-Associated Immune Responses and Gastrointestinal Permeability. International Journal of Molecular Sciences 2023;24.
9. Parajuli P, Gokulan K, Khare S. Preclinical In Vitro Model to Assess the Changes in Permeability and Cytotoxicity of Polarized Intestinal Epithelial Cells during Exposure Mimicking Oral or Intravenous Routes: An Example of Arsenite Exposure. International Journal of Molecular Sciences 2022;23. 
10. Skrzydlewski P, Twaruzek M, Grajewski J. Cytotoxicity of Mycotoxins and Their Combinations on Different Cell Lines: A Review. Toxins 2022;14.