# Developing nanoplexes for RNAi-expressing plasmids

> **NIH NIH R01** · UNIVERSITY OF MARYLAND BALTIMORE · 2022 · $339,103

## Abstract

Abstract
Histidine-lysine (HK) peptides can be tailored to transport different forms of nucleic acids such as plasmids or
siRNA into cells by altering their degree of branching and amino acid sequence. The HK carrier has shown
value in treating cancer, hypertrophic scars, and liver fibrosis in pre-clinical models. In contrast to the effective
in vitro highly-branched H2K4b peptide carrier of plasmids, the linear H2K peptide is a poor carrier in vitro, yet
surprisingly, H2K nanoplexes target tumors with very high transfection efficiency. Because H2K4b and H2K
plasmid nanoplexes differ in their accumulation and distribution within the tumor, there are clearly other
mechanisms besides EPR that govern the tumor specificity of H2K nanoplexes. Because H2K has a repeating
sequence pattern of –KHHK-, transcytosis of the nanoplex mediated by the neuropilin-1 receptor (NRP1)
through the tumor endothelium provides a rationale for enhanced tumor targeting and accumulation. Although
additional development concerning the linear peptide is required, these early findings are encouraging and are
the focus of this application. To test the overall hypothesis that characterization of HK nanoplexes containing a
shRNA plasmid targeting Kras/Raf-1 will facilitate improved antitumor activity, the following aims are planned.
Aim 1 will determine the structure-activity relationship of HK peptides as carriers of plasmids in vivo.
To transport the shRNA-inhibitory plasmids systemically to tumors, we have selected several HK polymers with
a predominant repeating amino acid sequence of –KHHK- (i.e., H2K), an effective sequence for plasmid
transport in vivo. By combining the H2K sequence with other amino acid sequence patterns, and varying the
length and number of branches, we hypothesize that derivatives of H2K will be identified that are more
effective in vivo carriers of plasmids targeting different breast tumor models. Aim 2 will delineate the
biophysical and biological properties of HK nanoplexes essential for transfection. Several biophysical
techniques will be examined, including morphology, binding, zeta potential, and stability of HK plasmid
nanoplexes to correlate their physical properties with the efficacy of plasmid delivery to tumors. By comparing
HK peptides that differ in their branching and length with in vitro and in vivo biophysical methods, our
hypothesis is that key structural features of the carrier will be identified that will affect stability and distribution
within the tumor and increase gene expression. Based on these structural and mechanistic studies, Aim 3 will
develop a targeted RNAi therapeutic with an optimized HK nanoplex toward breast tumors in mice. We
hypothesize that the specific patterns based on the linear H2K peptide and elevated levels of NRP1 in tumors
will markedly augment delivery of tumor-inhibitory plasmids (shRaf-1 and shKras) to orthotopic or metastatic
tumors. Moreover, addition of a tumor-targeting ligand to the HK nanoplex shou...

## Key facts

- **NIH application ID:** 10194491
- **Project number:** 5R01EB028534-03
- **Recipient organization:** UNIVERSITY OF MARYLAND BALTIMORE
- **Principal Investigator:** ARCHIBALD JAMES MIXSON
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $339,103
- **Award type:** 5
- **Project period:** 2019-09-15 → 2024-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10194491

## Citation

> US National Institutes of Health, RePORTER application 10194491, Developing nanoplexes for RNAi-expressing plasmids (5R01EB028534-03). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10194491. Licensed CC0.

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