# Synthetic System for Neuron Subtype- and Context-Specific Subcellular RNA/Protein Manipulation and Bioactive Delivery

> **NIH NIH R21** · HARVARD UNIVERSITY · 2024 · $481,250

## Abstract

We propose to develop a synthetic biological system to manipulate subcellular RNAs and proteins in growth
cones or synapses of subtype- and context-specific neurons. This system has both direct, powerful near-term
experimental-investigative potential and future potential toward novel, uniquely specific forms of therapeutics/
bioactive delivery. The systems enable unique forms of subcellular functional investigation in subtype- and stage-
specific neuronal circuitry, offering generalizable modularity for manipulation of RNA and/or protein localized
to developing growth cones during circuit formation and to presynaptic compartments in maturing and mature
circuitry. Subcellular functions of proteins regulating connectivity and (dys)function of subtype-specific circuitry
are poorly understood. Based on successful preliminary studies, we propose to develop these tools for in vivo
perturbation of molecular abundances by integrating subcellular localization motifs with inducible gene
expression systems. This system will enable discovery of RNAs/proteins that function subcellularly to control
overall organization, precision, and function of distinct neural circuits, and how they are dysregulated in disease.
 We aim to integrate motifs that selectively traffick proteins to GCs and/or presynapses with an inducible
gene expression system, enabling subcellular-specific overexpression via direct motif fusion, and knockdown
via motif fusion to hfCas13d, a refined mRNA degrader. We will use data from recently developed experimental
and analytical approaches purifying subtype-specific GCs or presynapses and their parent somata or nuclei from
developing or mature mouse cortex, and quantitatively “mapping” RNAs and proteins between these subcellular
compartments. This enabled identification of molecules with subcellular localizations specific to subtypes and
stages, and/or to disease, and measuring of local translation. However, such candidates can function in multiple
subcellular domains, at multiple distinct stages/contexts. Current approaches for whole-neuron perturbation of
molecular abundances produce off-target effects, complicating investigation of subcellular-specific RNA/protein
function, thus preventing elucidation of molecular mechanisms regulating circuit formation and (dys)function.
 We aim to develop approaches for subcellular- and stage-/context-specific manipulation of molecular
abundances in developing and mature circuits. In Aim 1, we will develop a system to subcellularly manipulate
RNA/protein abundances in growth cones that regulate distinct developmental stages of circuit construction.
In Aim 2, we will develop a system to subcellularly manipulate presynaptic RNA/protein abundances later in
maturity that regulate synapse function, maintenance, and plasticity, thus are linked to memory, synaptic
weighting, and circuit function. We will employ presynaptic-localizing motifs in mature circuits. These systems
will enable elucidation of subcellular...

## Key facts

- **NIH application ID:** 11038894
- **Project number:** 1R21NS141110-01
- **Recipient organization:** HARVARD UNIVERSITY
- **Principal Investigator:** JEFFREY D MACKLIS
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $481,250
- **Award type:** 1
- **Project period:** 2024-09-20 → 2026-09-19

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/11038894

## Citation

> US National Institutes of Health, RePORTER application 11038894, Synthetic System for Neuron Subtype- and Context-Specific Subcellular RNA/Protein Manipulation and Bioactive Delivery (1R21NS141110-01). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/11038894. Licensed CC0.

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