# Developing cell-penetrating miniproteins as a new class of therapeutics

> **NIH NIH R21** · NORTHWESTERN UNIVERSITY · 2021 · $231,323

## Abstract

Project Summary
Current protein therapeutics have a major limitation: they generally cannot cross the cellular membrane or
interact with cytosolic targets. The ability to design protein therapeutics that enter the cell cytosol would enable
new therapeutic strategies across many disease areas, including cancer, autoimmunity, and neurological
disease. Therapeutic “miniproteins” (30-60 residues in length) have the potential to address this challenge, and
several miniproteins capable of efficiently reaching the cell cytosol have recently been identified. However, we
lack a general understanding of the “design rules” for cell-penetrating miniproteins, limiting the development of
this class of molecules. Furthermore, current approaches to measure cytosolic delivery require measuring each
protein individually, which is slow and labor intensive. This makes it impossible to test large numbers of
miniproteins to develop a robust, quantitative understanding of the determinants of cytosolic delivery.
In this exploratory project, we will develop a new approach to measure delivery for each different protein in a
large mixed pool, using targeted mass spectrometry to individually identify each miniprotein sequence. In our
approach, a soluble mixed pool containing thousands of designed miniprotein sequences is incubated with cells,
and miniproteins that enter those cells are captured by a cytosolic target. Miniproteins captured by the target are
then purified out of the cellular contents and identified and quantified using targeted proteomics. The amount of
each protein in the captured sample (relative to the starting sample) will provide a quantitative measure of
delivery efficiency. This approach is unprecedented, and we will test and optimize this approach using different
positive and negative control miniproteins, different library sizes, and different cell lines.
With this method in hand, we will use approaches we previously pioneered to computationally design thousands
of candidate cell-penetrating miniproteins with intentionally diverse sequence and structural properties. We will
then quantify cytosolic delivery for these new proteins using our new high-throughput approach, creating
unprecedented large-scale data on delivery efficiency. We will then use these data to build machine learning
models that predict miniprotein delivery based on sequence and structural properties. Finally, we will repeatedly
iterate, designing new miniprotein libraries based on our predictive models of delivery, testing these designs
using our high-throughput experimental approach, and further updating our models. This iterative design-test-
learn approach will build a robust, predictive understanding of the determinants of delivery. Ultimately, the ability
to design cell-penetrating miniproteins will unlock a wide range of new therapeutic targets inside the cell.

## Key facts

- **NIH application ID:** 10289040
- **Project number:** 1R21GM143560-01
- **Recipient organization:** NORTHWESTERN UNIVERSITY
- **Principal Investigator:** Gabriel Jacob Rocklin
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $231,323
- **Award type:** 1
- **Project period:** 2021-08-01 → 2023-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10289040

## Citation

> US National Institutes of Health, RePORTER application 10289040, Developing cell-penetrating miniproteins as a new class of therapeutics (1R21GM143560-01). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10289040. Licensed CC0.

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