Future Steps

Preclinical Studies: Laboratory Testing

We are designing minimal promoters for precise transcriptional control in AgRP neurons. The natural AgRP promoter, enriched for FOXO1, STAT3 and glucocorticoid receptor binding, provides a strong foundation.

Minimal Promoter Engineering for Neuron-Specific Control

Our approach includes:

  • Minimal AgRP promoter-truncated sequence reported in the literature1.
  • Synthetic promoter-YB_TATA core promoter with FOXO1, STAT and NF-κB response elements for inducible, context-specific activation2.

HDAC6 knockdown

We are developing miR-E shRNA constructs in lentiviral vectors to silence HDAC6 by >90%, validated through α-tubulin hyperacetylation. Transcriptomic analysis confirms minimal off-target effects, while tetracycline-inducible promoters allow temporal control. This enables precise and reversible epigenetic modulation for therapeutic testing.

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Split transcription systems: Beyond GAL4/VP16

To overcome limitations of conventional GAL4/VP16 systems (context sensitivity, cytotoxicity at high expression levels) we are investigating next-generation split transcription systems:

Split-intein GAL4 platforms for spatial and temporal gating3,4. Alternative activation domains (HSV VP16, human p65) for stronger, more stable expression5.

This ensures robust, low-background activity suitable for layered neuromodulatory circuits.

Orthogonal circuits and CRISPR-based kill switches

We plan to explore orthogonal circuits to insulate synthetic pathways from host signaling. As a safety layer, we are considering CRISPR-based kill switches that could trigger apoptosis or transgene silencing under defined conditions6,7. Though still conceptual, these systems offer a promising route to long-term biocontainment and fail-safe control in therapeutic settings.

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Lenti-Virus Development

From 2nd to 3rd Generation LVs

We plan to implement third-generation self-inactivating (SIN) lentiviral vectors, which use a four-plasmid system8. This modular approach effectively prevents the formation of replication-competent lentiviruses (RCLs).

Targeted integration into the rDNA locus

To address random integration concerns, we are exploring directed integration into the ribosomal DNA (rDNA) locus, a naturally transcriptionally active region tolerant of insertions9.

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AgRP Neuron-Specific Targeting

Precision in the Hypothalamus

AgRP neurons in the hypothalamus are key regulators of appetite and energy balance. About 30% of these neurons express leptin receptors (LepRb), providing a well-defined molecular entry point for selective targeting10.

We propose pseudotyping lentiviral vectors with engineered rabies virus glycoprotein (RVG) to achieve up to 95% targeting specificity.

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Translational Safety Assessment

Using hInGeTox Platform

To strengthen translational safety, we plan to integrate advanced genotoxicity screening tools such as hInGeTox - a platform utilizing human iPSC-derived hepatocyte-like cells11.

This platform supports both short-term (30-day) and extended long-term (up to 700-day) clonal tracking, simulating the equivalent of a full murine lifespan in vitro.

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Proof-of-Concept Design

Human Neuronal Models for Testing

We will validate our strategy using human iPSC-derived hypothalamic arcuate organoids (ARCOs)12. These 3D models differentiate into mature AgRP neurons expressing functional leptin receptors.

Single-cell RNA-seq confirms strong transcriptomic similarity to native human arcuate tissue, enabling accurate therapeutic assessment.

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Pre-Clinical Studies: Animal Testing

Non-Human Testing for Efficacy and Safety

Therapeutic efficacy will be validated across both diet-induced and genetic obesity models:

  • C57BL/6N mice on 60% fat Western-style diet13,14.
  • ob/ob (leptin-deficient) and db/db (leptin receptor-mutant) mice15,16.

Long-term safety studies (>12 months) will assess therapeutic durability, immune response and resistance development.

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Phase 0: Early Human Studies

Microdosing and Pharmacology

Prior to full-scale trials, exploratory Phase 0 studies provide critical preliminary human data while minimizing participant risk. This involves a small cohort (10-15 participants) receiving subtherapeutic doses.

Key Endpoints

  • Vector biodistribution and transduction efficiency.
  • Early pharmacodynamic readouts.
  • Safety and tolerability assessment.
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AI/ML-Driven Optimization

Computational Framework

Our clinical translation framework incorporates AI and machine learning pipelines to:

  • Predict therapeutic responsiveness.
  • Optimize dosing schedules.
  • Guide patient stratification.

By integrating multi-omic datasets, these models identify optimal treatment regimens while minimizing trial-and-error during early clinical testing.

References

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  1. Brown, A. M., Mayfield, D. K., Volaufova, J., & Argyropoulos, G. (2001). The gene structure and minimal promoter of the human agouti related protein. Gene
  2. Graves, D. T., & Milovanova, T. N. (2019). Mucosal Immunity and the FOXO1 Transcription Factors. Frontiers in Immunology
  3. Luan, H., Diao, F., Scott, R. L., & White, B. H. (2020). The Drosophila Split Gal4 System for Neural Circuit Mapping. Frontiers in Neural Circuits
  4. Ewen-Campen, B., Luan, H., Xu, J., et al. (2023). split-intein Gal4 provides intersectional genetic labeling that is fully repressible by Gal80. bioRxiv
  5. Bae, … (2024). Split Proteins and Reassembly Modules for Biological Applications. ChemBioChem
  6. Rottinghaus, A. G., Ferreiro, A., Fishbein, S. R. S., Dantas, G., & Moon, T. S. (2022). Genetically stable CRISPR-based kill switches for engineered microbes. Nature Communications
  7. Asin-Garcia, E., Martín-Pascual, M., de Buck, C., Allewijn, M., Müller, A., & Martins dos Santos, V. A. P. (2024). GenoMine: a CRISPR-Cas9-based kill switch for biocontainment of Pseudomonas putida. Frontiers in Bioengineering and Biotechnology
  8. Fang, E., He, G., Chang, Y., He, Q., Chen, P., & Hu, K. (2025). Application Advances of Lentiviral Vectors: From Gene Therapy to Vaccine Development. Molecular Biotechnology
  9. Schenkwein, D., Afzal, S., Nousiainen, A., Schmidt, M., & Ylä-Herttuala, S. (2020). Efficient Nuclease-Directed Integration of Lentivirus Vectors into the Human Ribosomal DNA Locus. Molecular Therapy
  10. Bell, B. B., Harlan, S. M., Morgan, D. A., Guo, D. F., & Rahmouni, K. (2018). Differential contribution of POMC and AgRP neurons to the regulation of regional autonomic nerve activity by leptin. Molecular Metabolism
  11. Suleman, S., Alhaque, S., Guo, A., et al. (2025). hInGeTox: a human-based in vitro platform to evaluate lentivirus/host interactions that contribute to genotoxicity. Gene Therapy
  12. Generation of hypothalamic arcuate organoids from human induced pluripotent stem cells. (2021). Cell Stem Cell
  13. Hintze, K. J., Benninghoff, A. D., Cho, C. E., & Ward, R. E. (2018). Modeling the Western Diet for Preclinical Investigations. Advances in Nutrition
  14. Lang, P., Hasselwander, S., Li, H., & Xia, N. (2019). Effects of different diets used in diet-induced obesity models on insulin resistance and vascular dysfunction in C57BL/6 mice. Scientific Reports
  15. Drel, V. R., Mashtalir, N., Ilnytska, O., et al. (2006). The leptin-deficient (ob/ob) mouse: a new animal model of peripheral neuropathy of type 2 diabetes and obesity. Diabetes
  16. Db/Db Mouse – an overview. ScienceDirect Topics