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 literature.
  • Synthetic promoter-YB_TATA core promoter with FOXO1, STAT and NF-κB response elements for inducible, context-specific activation.

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 gating. Alternative activation domains (HSV VP16, human p65) for stronger, more stable expression.

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 conditions. 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 system. 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 insertions.

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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 targeting.

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 cells.

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). 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 diet.
  • ob/ob (leptin-deficient) and db/db (leptin receptor-mutant) mice.

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.