X29 - Novel HER2 Targeting Tandem Peptide for delivery of CD44 siRNA into HER2+ Breast Cancer in vitro and in vivo

Friday, October 10, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

Tristan D. Dover

Graduate Research Student
Clemson University
Anderson, South Carolina, United States

Co-Author(s)

MS

Morgan Stevens

Student
Clemson University, United States
Audreanna Miserendino

PhD Student
UNC Eshelman School of Pharmacy
Fort Mill, South Carolina, United States
KA

Kharimat Lora Alatise

Graduate Research Assistant
Clemson University, United States
AV

Anusha Vemula

Graduate Student
Department of Bioengineering, Clemson University; Department of Biological Sciences, Clemson University
Clemson, South Carolina, United States
James W. Kalogeros

Research Associate
VitaKey Inc.
Cary, North Carolina, United States
ST

Sevrina Tekle

Lab Manager
Spelman College, United States
BB

Brian Booth

Associate Professor
Clemson University
Clemson, South Carolina, United States

Last Author(s)

Angela Alexander-Bryant

Associate Professor
Clemson University, United States

Primary Investigator(s)

Angela Alexander-Bryant

Associate Professor
Clemson University, United States

Introduction: : Human epidermal growth factor receptor 2 (HER2) is overexpressed in 25%-30% of breast cancers and is associated with worse prognoses, higher mortality rates, and shorter remission times.1 Although current therapies for treating HER2+ breast cancer have improved outcomes, significant challenges remain, including toxicity due to off-target effects and lack of efficacy, resulting in the development of resistance to treatment. RNA interference (RNAi), using small interfering RNA (siRNA) to silence overexpressed oncogenes in tumor cells, offers an advanced treatment approach.2 Despite its potential, siRNA delivery faces many biological barriers, including degradation by nucleases, insufficient cellular internalization, and endosomal entrapment. Many vectors have been explored to overcome these obstacles. Peptide-based nanocarriers have emerged as promising tools for nucleic acid delivery due to their high tunability, ability to protect cargo, and capacity to facilitate targeted delivery of genetic therapies.3 Our lab has developed a fusogenic peptide, termed DIV3W, and demonstrated its ability to protect siRNA from degradation in physiological environments, facilitate efficient siRNA internalization, and endosomal escape of bioactive siRNA.4 To target HER2+ breast cancer, we combined the DIV3W peptide with the P25 HER2-targeting peptide to create a tandem peptide capable of targeting HER2+ breast cancer and delivering siRNA. We aim to examine the ability of P25-DIV3W tandem peptide to target HER2+ breast cancer cells in vitro and in vivo and deliver siRNA targeting CD44, an oncogene upregulated in HER2+ breast cancer.

Materials and

Methods: : Two different HER2+ cell lines were used in this study: SKBR3 and BT474. To confirm specific targeting, a receptor blocking experiment was conducted by incubating HER2+ breast cancer cells with or without P25 HER2-targeting peptide alone for 1 hour, followed by treatment with the P25-DIV3W peptide complexed with Cy5-siRNA (P25-DIV3W:Cy5-siRNA). Fluorescent imaging was performed to visualize siRNA internalization. PCR was used to quantify knockdown of the CD44 gene at the mRNA level, while Western blotting was used to examine knockdown at the protein level. For PCR and western blots, P25-DIV3W amine to phosphate (N:P) ratios of 20:1 through 60:1 of peptide complexed with siRNA were incubated with HER2+ cell lines to determine which ratio would facilitate knockdown most efficiently. A non-targeting control siRNA and a CD44-specific siRNA were used to examine knockdown. examined to determine whether the tandem peptide efficiently delivers siRNA to HER2+ tumors. SKBR3 cells were injected into the mammary fat pad to allow to tumor formation for 7 days. Non-targeting Cy5.5-siRNA, DIV3W/Cy5.5-siRNA, P25/Cy5.5-siRNA, and P25-DIV3W/Cy5.5-siRNA were injected intratumorally, and IVIS spectral imaging of each mouse was conducted to determine siRNA biodistribution at 1, 3, 6, 12, 24, 48, and 72 hours after treatment.

Results, Conclusions, and Discussions:: When blocking the HER2 receptor with the P25 targeting peptide, we found that the tandem peptide complexes were prevented from internalizing into cancer cells, as evidenced by the lack of siRNA signal compared to unblocked cells treated with the complexes (Figure 1A). This suggests that the P25-DIV3W peptide requires the HER2 receptor for internalization. PCR results indicated that the highest knockdown of the CD44 gene mediated by the tandem peptide complexes occurred at N:P ratios between 30:1 and 60:1 (Figure 1B). Similarly, western blotting revealed the greatest reduction of CD44 protein at the 30:1 and 40:1 ratios. After in vivo administration of treatments, biodistribution data obtained via IVIS imaging showed that the accumulation of siRNA was greatest when delivered with the P25-DIV3W tandem peptide compared to the P25 targeting peptide or DIV3W fusogenic peptide alone. The mice treated with the tandem peptide retained the highest siRNA signal across a 72-hour period. This suggests that the tandem peptide facilitates uptake of siRNA into tumor cells in vivo. Overall, this data indicates that the P25-DIV3W tandem peptide can deliver siRNA for gene knockdown in vitro, specifically internalize into HER2+ breast cancer cells in vitro, and internalize into breast tumor tissue in vivo.

Acknowledgements and/or References (Optional): : This research was supported in part by the Dabo Swinney All-In Foundation as well as Materials Assembly and Design Excellence in South Carolina (MADE in SC) under the National Science Foundation EPSCoR Program under NSF Award #OIA-1655740. Data presented in this material was partially collected using SC BioCRAFT facilities supported by the National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health under award number P30 GM131959.