Advancements of 599 peptide carrier design through stereochemical and/or amino acid modifications


Technology: Researchers at MUSC have developed a cell-penetrating peptide (CPP) specifically designed to overcome challenges associated with small interfering RNA (siRNA)-drug delivery for cancer treatment. Investigators previously generated the 599 peptide that silenced the CIP2A oncogene and suppressed tumor growth in vivo (Figure 1).


Figure 1. Demonstration of 599 peptide-mediated delivery of an siRNA designed to target the CIP2A oncogene (siCIP2A), in which the complex was administered intratumorally in a xenograft floor-of-mouth tumor mouse model. Inhibition of tumor growth was observed 8 days post initial treatment compared to control treatments.


The group further modified the 599 peptide carrier by incorporating either different stereochemical patterns of L/D-amino acids or a specific D-amino acid substitution within the 599 peptide design, with one particular 599 peptide variant, RD3AD, that contained the specific D-amino acid substitution, capable of improving the efficiency of siRNA delivery into cancer cells without any associated toxicity. In particular, data showed that RD3AD significantly improved siCIP2A delivery into cancer cells ~2-fold, at the peptide:siRNA molar ratio of 50:1, in comparison to the 599 parent peptide, and that the delivery efficiency was also ~4-fold greater compared to the commercially available product Lipofectamine 3000 (Figure 2A). Interestingly, RD3AD also mediated a more ordered binding of siRNAs to specific cellular projections, identified as filopodia, (Figure 2B) that coincidentally also resulted in the most enhanced gene silencing among the peptide variants, at approximately 80%, in comparison to 599, which only displayed ~55% gene silencing efficiency (Figure 2C). Thus, collectively, these data imply that the RD3AD peptide design modification could be responsible for directing a more efficient mode of siRNA drug cell entry and that this peptide carrier could have a high therapeutic potential as a delivery vehicle for RNA interference (RNAi)-based therapies.






Figure 2. siRNA delivery, cellular localization, and gene silencing efficiency of 599 and its peptide variants. (A) Quantitative siCIP2A delivery into CAL 27 cancer cells via 599 and its peptide variants at 30:1 and 50:1 peptide:siRNA molar ratios. A commercially available lipid-based transfection reagent (LTR) Lipofectamine 3000 (LF3000) was used as a positive control. (B) Confocal fluorescence microscopy analysis of CAL 27 cancer cells treated with DY547-siCIP2A (red) in complex with 599 or the RD3AD peptide variant at 50:1 peptide:siRNA molar ratios. Filopodia and nuclei are stained green and blue, respectively. Arrows in the RD3AD panel show siRNA-containing foci localized along filopodia. (C) Real-time PCR analysis of CIP2A mRNA levels in CAL 27 cancer cells 48 hours post-treatment with a control non-targeting siRNA (siNT) or siCIP2A in complex with either 599 or its peptide variants at 50:1 peptide:siRNA molar ratios.


Overview: The discovery that exogenous siRNAs could induce sequence-specific inhibition of gene expression has resulted in the investigation of the use of RNAi-based therapeutic approaches to treat many diseases, including cancer. Peptide carriers have been explored to overcome the barriers of nucleic acid transport and CPPs have proven to be promising vehicles in mediating their efficient delivery into cells/tissues. Moreover, the market for CPPs is immense because they can be used for drug delivery, gene therapy, and disease diagnosis, and the peptide therapeutics market has the potential to reach $50.6 billion by 2026.


Applications:  siRNA delivery, nucleic acid drug delivery, gene therapy


Advantages:  The RD3AD peptide carrier has higher siRNA delivery efficiency into cancer cells, resulting in enhanced gene silencing, most likely caused by its ability to mediate associations with filopodia. Intriguingly, filopodia are highly dynamic, elongated, and thin cellular processes that have been reported to facilitate the highly efficient cell entry of viruses, bacteria, activated receptors, lipo/polyplexes, and exosomes via rapid, but directed, retrograde trafficking towards the cell body, where at the filopodial base, endocytic hot spots potentially allow for easier cell entry.


Key Words: siRNA, peptide, CPP, carrier, filopodia, drug delivery, gene therapy, RNAi, stereochemistry, L/D-amino acids, cancer



Holjencin CE, Feinberg CR, Hedrick T, Halsey G, Williams RD, Patel PV, Biles E, Cummings JC, Wagner C, Vyavahare N, Jakymiw A. “Advancing peptide siRNA-carrier designs through L/D-amino acid stereochemical modifications to enhance gene silencing.(2021) Molecular Therapy - Nucleic Acids, 24:462-476.


Background Publication(s):

    1. Alexander-Bryant AA, Dumitriu A, Attaway CC, Yu H, Jakymiw A. “Fusogenic-oligoarginine peptide-mediated silencing of the CIP2A oncogene suppresses oral cancer tumor growth in vivo.” (2015) Journal of Controlled Release 218:72-81. PMID: 26386438, PMCID: PMC4646222
    2. Cantini L, Attaway CC, Butler B, Andino LM, Sokolosky ML, Jakymiw A. “Fusogenic-oligoarginine peptide-mediated delivery of siRNAs targeting the CIP2A oncogene into oral cancer cells.” (2013) PLoS ONE 8(9):e73348. PMID: 24019920, PMCID: PMC3760901


Inventors: Andrew Jakymiw, Charles Holjencin


Patent Status: Provisional Filed on 10/02/2020


MUSC-FRD Technology ID:     P20111



Patent Information:
For Information, Contact:
Docket BioPharma
Zucker Institute of Innovation Commercialization powered by MUSC
Andrew Jakymiw
Charles Holjencin
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