Preliminary study of a novel transfection modality for in vivo siRNA delivery to vocal fold fibroblasts


An obstacle to clinical use of RNA-based gene suppression is instability and inefficiency of current delivery modalities. Nanoparticle delivery likely holds great promise, but the kinetics and transfection conditions must be optimized prior to in vivo utility. We investigated a RNA nanoparticle complex incorporating a lipitoid transfection reagent in comparison to a commercially available reagent.

Study Design

In vitro.


We investigated which variables influence transfection efficiency of lipitoid oligomers and a commercially available reagent across species, in vitro. These variables included duration, dose, and number of administrations, as well as serum and media conditions. The target gene was Smad3, a signaling protein in the transforming growth factor-β cascade implicated in fibroplasia in the vocal folds and other tissues.


The two reagents suppressed Smad3 mRNA for up to 96 hours; lipitoid performed favorably and comparably. Both compounds yielded 60% to 80% mRNA knockdown in rat, rabbit, and human vocal fold fibroblasts (P < 0.05 relative to control). Dose and number of administrations played a significant role in gene suppression (P < 0.05). Suppression was more dose-sensitive with lipitoid. At a constant siRNA concentration, a 50% decrease in gene expression was observed in response to a five-fold increase in lipitoid concentration. Increased number of administrations enhanced gene suppression, ∼45% decrease between one and four administrations. Neither serum nor media type altered efficiency.


Lipitoid effectively knocked down Smad3 expression across multiple transfection conditions. These preliminary data are encouraging, and lipitoid warrants further investigation with the goal of clinical utility.

Preliminary study of a novel transfection modality for in vivo siRNA delivery to vocal fold fibroblasts.

Iv Kraja, BS; Renjie Bing, MD; Nao Hiwatashi, MD, PhD; Bernard Rousseau, PhD; Danielle Nalband, BS; Kent Kirshenbaum, PhD; Ryan C. Branski, PhD. (2016)  The Laryngoscope

Multivalent Peptoid Conjugates Which Overcome Enzalutamide Resistance in Prostate Cancer Cells

Development of resistance to antiandrogens for treating advanced prostate cancer is a growing concern and extends to recently developed therapeutics, including enzalutamide. Therefore, new strategies to block androgen receptor (AR) function in prostate cancer are required. Here, we report the characterization of a multivalent conjugate presenting two bioactive ethisterone ligands arrayed as spatially defined pendant groups on a peptoid oligomer. The conjugate, named Multivalent Peptoid Conjugate 6 (MPC6), suppressed the proliferation of multiple AR-expressing prostate cancer cell lines including those that failed to respond to enzalutamide and ARN509. The structure–activity relationships of MPC6 variants were evaluated, revealing that increased spacing between ethisterone moieties and changes in peptoid topology eliminated its antiproliferative effect, suggesting that both ethisterone ligand presentation and scaffold characteristics contribute to MPC6 activity. Mechanistically, MPC6 blocked AR coactivator–peptide interaction and prevented AR intermolecular interactions. Protease sensitivity assays suggested that the MPC6-bound AR induced a receptor conformation distinct from that of dihydrotestosterone- or enzalutamide-bound AR. Pharmacologic studies revealed that MPC6 was
metabolically stable and displayed a low plasma clearance rate. Notably, MPC6 treatment reduced tumor growth and decreased Ki67 and AR expression in mouse xenograft models of enzalutamide-resistant LNCaP-abl cells. Thus, MPC6 represents a new class of compounds with the potential to combat treatment-resistant prostate cancer.

Multivalent peptoid conjugates which overcome enzalutamide resistance in prostate cancer cells

Yu Wang, Dilani C. Dehigaspitiya, Paul M. Levine, Adam A. Profit, Michael Haugbro, Keren Imberg-Kazdan, Susan K. Logan, Kent Kirshenbaum and Michael J. Garabedian
Cancer Res; 76(17); 5124 – 32.

PPII Helical Peptidomimetics Templated by Cation– p Interactions

Poly-proline type II (PPII) helical PXXP motifs are the recognition elements for a variety of protein–protein interactions that are critical for cellular signaling. Despite development of protocols for locking peptides into a-helical and b-strand conformations, there remains a lack of analogous methods for generating mimics of PPII helical structures. We describe herein a strategy to enforce PPII helical secondary structure in the 19-residue TrpPlexus miniature protein. Through sequence variation, we showed that a network of cation–pi interactions could drive the formation of PPII helical conformations for both peptide and N-substituted glycine peptoid residues. The achievement of chemically diverse PPII helical scaffolds provides a new route towards discovering peptidomimetic inhibitors of protein–protein interactions mediated by PXXP motifs.

PPII Helical Peptidomimetics Templated by Cation–π Interactions
TW Craven, R Bonneau, K Kirshenbaum
ChemBioChem 17(19): 1824-28

A Miniature Protein Stabilized by a Cation−π Interaction Network

The design of folded miniature proteins is predicated on establishing noncovalent interactions that direct the self-assembly of discrete thermostable tertiary structures. In this work, we describe how a network of cation−π interactions present in proteins containing “WSXWS motifs” can be emulated to stabilize the core of a miniature protein. This 19-residue protein sequence recapitulates a set of interdigitated arginine and tryptophan residues that stabilize a distinctive β-strand:loop:PPII-helix topology. Validation of the compact fold determined by NMR was carried out by mutagenesis of the cation−π network and by comparison to the corresponding disulfide-bridged structure. These results support the involvement of a coordinated set of cation−π interactions that stabilize the tertiary structure.

Timothy W. Craven, Min-Kyu Cho, Nathaniel J. Traaseth, Richard Bonneau, and Kent Kirshenbaum. (2016), A Miniature Protein Stabilized by a Cation−π Interaction Network. J. Am. Chem. Soc. 138(5), 1543-1550 Link