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A novel multi-functional chimeric peptide for enhanced safe gene delivery in immunotherapy

Gene Therapy volume 32, pages 497–507 (2025)Cite this article

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Abstract

Chimeric peptides hold promising potential to be introduced as an ideal gene delivery platform based on their advantages over viral carriers, including but not limited to the safety profile and specific targeting. However, their gene transfer efficiency needs improvement. Here, we designed a new multi-functional chimeric peptide for enhanced gene delivery by adding a cyclic TAT motif to a previously designed MPG2H peptide to enable the targeting of cells with independent/dependent endocytosis cell entry mechanisms. CTATMPG2H was expressed and purified using affinity chromatography; then it was characterized through a gel retardation assay, circular dichroism (CD) spectropolarimetry, transmission electron microscopy (TEM) dynamic light scattering (DLS), and zeta potential analysis. CTATMPG2H was compared with MiRGD as a chimeric peptide control in all steps. After assessing the platform stability in various conditions, its gene transfer efficiency was evaluated in the HEK293T cell line with reporter genes. Additionally, mouse bone marrow-derived dendritic cells (BMDCs) were transfected to test CTATMPG2H potential in immunotherapy. The results illustrated a safe gene transfer profile for CTATMPG2H comparable to MiRGD and Polyethyleneimine (PEI). Flow cytometry results showed up to 48% gene transfer rate for CTATMPG2H to dendritic cells with minimal toxicity (viability rate ~80%). Moreover, the in silico investigation showed that the synergistic effects of electrostatic, hydrogen, and hydrophobic interactions enhance the stability and binding affinity of peptide-pDNA complexes, ensuring robust and specific targeting of nucleic acids. This research sets a foundation for future in vivo studies and potential clinical applications, aiming for safer and more effective gene therapy strategies.

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Fig. 1: Binding modes of peptides to circular DNA.
Fig. 2: Structural and biochemical characterization of chimeric peptides.
Fig. 3: Characterization of Nanocomplexes: Formation, Stability, and Serum Degradation Assays.
Fig. 4: Characterization of Nanocomplexes: Size, Charge, and Morphology.
Fig. 5: Gene transfer efficiency assessment on HEK293T.
Fig. 6: Gene transfer efficiency assessment on BMDCs.

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Data availability

The data supporting the findings of this study are available within the paper and its Supplementary Information. Raw Data is also provided in supplementary Information. Some parts of the datasets generated and/or analyzed during the current study are not publicly available due to the process of Patent Filling, but are available from the corresponding author on reasonable request.

References

  1. Sheridan C. Gene therapy finds its niche. Nat Biotechnol. 2011;29:121–8.

    Article  CAS  PubMed  Google Scholar 

  2. Miller AD. Human gene therapy comes of age. Nature. 1992;357:455–60.

    Article  CAS  PubMed  Google Scholar 

  3. Nayerossadat N, Ali P, Maedeh T. Viral and nonviral delivery systems for gene delivery. Adv Biomed Res. 2012;1:27.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Sabado RL, Balan S, Bhardwaj N. Dendritic cell-based immunotherapy. Cell Res. 2017;27:74–95.

    Article  CAS  PubMed  Google Scholar 

  5. Anguille S, Smits EL, Bryant C, Van Acker HH, Goossens H, Lion E, et al. Dendritic cells as pharmacological tools for cancer immunotherapys. Pharmacol Rev. 2015;67:731–53.

    Article  CAS  PubMed  Google Scholar 

  6. Alycia G, Brian R. Dendritic cells and cancer immunity. Trends Immunol. 2016;37:855–65.

    Article  Google Scholar 

  7. Mahato RI, Monera OD, Smith LC, Rolland A. Peptide-based gene delivery. Curr Opin Mol Ther. 1999;1:226–43.

    CAS  Google Scholar 

  8. Canine BF, Hatefi A. Development of recombinant cationic polymers for gene therapy research. Adv Drug Deliv Rev. 2010;62:1524–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Cheraghi R, Nazari M, Alipour M, Hosseinkhani S. Stepwise development of biomimetic chimeric peptides for gene delivery. Protein Pept Lett. 2020;27:698–710.

    Article  CAS  PubMed  Google Scholar 

  10. Rusiecka, I, Gągało, I & Kocić, I Cell-penetrating peptides improve pharmacokinetics and pharmacodynamics of anticancer drugs. Tissue Barriers 2022;10:1965418.

  11. Zhang X, Lei T, Du H. Prospect of cell penetrating peptides in stem cell tracking. Stem Cell Res Ther. 2021;12:457.

  12. Sadeghian I, Heidari R, Sadeghian S, Raee MJ, Negahdaripour M. Potential of cell-penetrating peptides (CPPs) in delivery of antiviral therapeutics and vaccines. Eur J Pharm Sci. 2022;169:106094.

  13. Zhang Z, Baxter AE, Ren D, Qin K, Chen Z, Collins SM et al. Efficient engineering of human and mouse primary cells using peptide-assisted genome editing. Nat Biotechnol. 2023. https://doi.org/10.1038/s41587-023-01756-1.

  14. Morris MC, Vidal P, Chaloin L, Heitz F, Divita G. A new peptide vector for efficient delivery of oligonucleotides into mammalian cells. Nucleic Acids Res. 1997;25:2730–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Deshayes S, Morris M, Heitz F, Divita G. Delivery of proteins and nucleic acids using a non-covalent peptide-based strategy. Adv Drug Deliv Rev. 2008;60:537–47.

    Article  CAS  PubMed  Google Scholar 

  16. Simeoni F, Morris MC, Heitz F, Divita G. Insight into the mechanism of the peptide-based gene delivery system MPG: Implications for delivery of siRNA into mammalian cells. Nucleic Acids Res. 2003;31:2717–24.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Sadeghian F, Hosseinkhani S, Alizadeh A, Hatefi A. Design, engineering and preparation of a multi-domain fusion vector for gene delivery. Int J Pharm. 2012;427:393–9.

    Article  CAS  PubMed  Google Scholar 

  18. Majidi A, Nikkhah M, Sadeghian F, Hosseinkhani S. Development of novel recombinant biomimetic chimeric MPG-based peptide as nanocarriers for gene delivery: Imitation of a real cargo. Eur J Pharm Biopharm. 2016;107:191–204.

    Article  CAS  PubMed  Google Scholar 

  19. Cheraghi R, Nazari M, Alipour M, Majidi A, Hosseinkhani S. Development of a Targeted anti-HER2 scFv Chimeric Peptide for Gene Delivery into HER2-Positive Breast Cancer Cells. Int J Pharm. 2016;515:632–43.

    Article  CAS  PubMed  Google Scholar 

  20. Alipour M, Majidi A, Molaabasi F, Sheikhnejad R, Hosseinkhani S. In vivo tumor gene delivery using novel peptideticles: pH-responsive and ligand targeted core–shell nanoassembly. Int J Cancer. 2018;143:2017–28.

    Article  CAS  PubMed  Google Scholar 

  21. Alipour M, Hosseinkhani S, Sheikhnejad R, Cheraghi R. Nano-biomimetic carriers are implicated in mechanistic evaluation of intracellular gene delivery. Sci Rep. 2017;7:41507.

  22. Zohrabi T, Hosseinkhani S. Ternary nanocomplexes of metallic nanoclusters and recombinant peptides for fluorescence imaging and enhanced gene delivery. Mol Biotechnol. 2020;62:495–507.

    Article  CAS  PubMed  Google Scholar 

  23. Golestanipour A, Nikkhah M, Aalami A, Hosseinkhani S. Gene delivery to tobacco root cells with single-walled carbon nanotubes and cell-penetrating fusogenic peptides. Mol Biotechnol. 2018;60:863–78.

    Article  CAS  PubMed  Google Scholar 

  24. Ghafary SM, Nikkhah M, Hatamie S, Hosseinkhani S. Simultaneous gene delivery and tracking through preparation of photo-luminescent nanoparticles based on graphene quantum dots and chimeric peptides. Sci Rep. 2017;7:9552.

  25. Moasses Ghafary S, Rahimjazi E, Hamzehil H, Modarres Mousavi M, Nikkhah M, Hosseinkhani S. et al. Design and preparation of a theranostic peptideticle for targeted cancer therapy: Peptide-based codelivery of doxorubicin/curcumin and graphene quantum dots. Nanomed Nanotechnol Biol Med. 2022;42.

  26. Alipour M, Sheikhnejad R, Fouani MH, Bardania H, Hosseinkhani S. DNAi-peptide nanohybrid smart particles target BCL-2 oncogene and induce apoptosis in breast cancer cells. Biomed Pharmacother. 2023;166.

  27. Wang H, Feng Z, Xu B. Supramolecular assemblies of peptides or nucleopeptides for gene delivery. Theranostics. 2019;9:3213–22.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Lehto T, Ezzat K, Wood MJA, Andaloussi EL. S. Peptides for nucleic acid delivery. Adv Drug Deliv Rev. 2016;106:172–82.

    Article  CAS  PubMed  Google Scholar 

  29. Thapa RK, Sullivan MO. Gene delivery by peptide-assisted transport. Curr Opin Biomed Eng. 2018;7:71–82.

    Article  PubMed  PubMed Central  Google Scholar 

  30. Hoyer J, Neundorf I. Peptide vectors for the nonviral delivery of nucleic acids. Acc Chem Res. 2012;45:1048–56.

    Article  CAS  PubMed  Google Scholar 

  31. Wang X, Tai Z, Tian J, Zhang W, Yao C, Zhang L, et al. Reducible chimeric polypeptide consisting of octa-D-arginine and tetra-L-histidine peptides as an efficient gene delivery vector. Int J Nanomed. 2015;10:4669–90.

    CAS  Google Scholar 

  32. Salomone F, Cardarelli F, Signore G, Boccardi C, Beltram F. In vitro efficient transfection by CM18-Tat11 hybrid peptide: a new tool for gene-delivery applications. PLoS ONE 2013;8:e70108.

  33. Teo SLY, Rennick JJ, Yuen D, Al-Wassiti H, Johnston APR, Pouton CW. Unravelling cytosolic delivery of cell penetrating peptides with a quantitative endosomal escape assay. Nat Commun. 2021;12:3721.

  34. Sahni A, Qian Z, Pei D. Cell-penetrating peptides escape the endosome by inducing vesicle budding and collapse. ACS Chem Biol. 2020;15:2485–92.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Zhao Y, Jiang H, Yu J, Wang L, Du J. Engineered histidine-rich peptides enhance endosomal escape for antibody-targeted intracellular delivery of functional proteins. Angew Chemie Int Ed Engl. 2023;62:e202304692.

  36. Khamehchian S, Nikkhah M, Madani R, Hosseinkhani S. Enhanced and selective permeability of gold nanoparticles functionalized with cell penetrating peptide derived from maurocalcine animal toxin. J Biomed Mater Res Part A. 2016;104:2693–2700.

    Article  CAS  Google Scholar 

  37. Patel SG, Sayers EJ, He L, Narayan R, Williams TL, Mills EM, et al. Cell-penetrating peptide sequence and modification dependent uptake and subcellular distribution of green florescent protein in different cell lines. Sci Rep 2019;9:6298.

  38. Sajid MI, Moazzam M, Stueber R, Park SE, Cho Y, Malik NUA, et al. Applications of amphipathic and cationic cyclic cell-penetrating peptides: significant therapeutic delivery tool. Peptides 2021;141:170542.

  39. Tsuchiya K, Gimenez-Dejoz J, Numata K. Molecular dynamics simulation of complexation between plasmid DNA and cationic peptides. Polym J. 2023;55:1109–14.

    Article  CAS  Google Scholar 

  40. Reichelt P, Schwarz C, Donzeau M. Single step protocol to purify recombinant proteins with low endotoxin contents. Protein Expr Purif. 2006;46:483–8.

    Article  CAS  PubMed  Google Scholar 

  41. Cheraghi R, Alipour M, Nazari M, Hosseinkhani S. Optimization of conditions for gene delivery system based on PEI. Mol Pharm. 2017;4:8–16.

    CAS  Google Scholar 

  42. Yu B, Pettitt BM, Iwahara J. Dynamics of ionic interactions at protein-nucleic acid interfaces. Acc Chem Res. 2020;53:1802–10.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Solovyev AY, Tarnovskaya SI, Chernova IA, Shataeva LK, Skorik YA. The interaction of amino acids, peptides, and proteins with DNA. Int J Biol Macromol. 2015;78:39–45.

    Article  CAS  PubMed  Google Scholar 

  44. Faltejsková K, Jakubec D, Vondrášek J. Hydrophobic amino acids as universal elements of protein-induced DNA structure deformation. Int J Mol Sci 2020;21:3986.

  45. Kane JF. Effects of rare codon clusters on high-level expression of heterologous proteins in Escherichia coli. Curr Opin Biotechnol. 1995;6:494–500.

    Article  CAS  PubMed  Google Scholar 

  46. Morris MC, Deshayes S, Heitz F, Divita G. Cell-penetrating peptides: from molecular mechanisms to therapeutics. Biol Cell. 2008;100:201–17.

    Article  CAS  PubMed  Google Scholar 

  47. Eiríksdóttir E, Konate K, Langel Ü, Divita G, Deshayes S. Secondary structure of cell-penetrating peptides controls membrane interaction and insertion. Biochim Biophys Acta - Biomembr. 2010;1798:1119–28.

    Article  Google Scholar 

  48. Veldhoen S, Laufer SD, Trampe A, Restle T. Cellular delivery of small interfering RNA by a non-covalently attached cell-penetrating peptide: Quantitative analysis of uptake and biological effect. Nucleic Acids Res. 2006;34:6561–73.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Yoo JW, Doshi N, Mitragotri S. Adaptive micro and nanoparticles: Temporal control over carrier properties to facilitate drug delivery. Adv Drug Deliv Rev. 2011;63:1247–56.

    Article  CAS  PubMed  Google Scholar 

  50. Pappalardo JS, Langellotti CA, Giacomo SD, Olivera V, Quattrocchi V, Zamorano P, et al. In vitro transfection of bone marrow-derived dendritic cells with TATp-liposomes. Int J Nanomed. 2014;9:963–73.

    Article  Google Scholar 

  51. Haines AMR, Irvine AS, Mountain A, Charlesworth J, Farrow NA, Husain RD, et al. CL22 - A novel cationic peptide for efficient transfection of mammalian cells. Gene Ther. 2001;8:99–110.

    Article  CAS  PubMed  Google Scholar 

  52. Tan PH, Beutelspacher SC, Wang Y, McClure MO, Ritter MA, Lombardi G, et al. Immunolipoplexes: An efficient, nonviral alternative for transfection of human dendritic cells with potential for clinical vaccination. Mol Ther. 2005;11:790–800.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported by the National Institute for Medical Research Development (NIMAD) (grant number 400369).

Funding

This work was supported by the National Institute for Medical Research Development (NIMAD) (grant number 400369).

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Authors and Affiliations

  1. Department of Nanobiotechnology, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran

    Mahdiyar Dehshiri

  2. Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran

    Shokouh Rezaei & Saman Hosseinkhani

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  1. Mahdiyar Dehshiri
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  2. Shokouh Rezaei
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Contributions

M.D conducted all the wet lab experiments, prepared the manuscript and figures. S.R conducted in silico investigations and prepared related figures. S.H supervised the project. All authors reviewed the final manuscript.

Corresponding author

Correspondence to Saman Hosseinkhani.

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The authors declare no competing interests.

Ethics approval and consent to participate

All protocols and procedures in the animal study and all experiments described were approved by the Research Ethics Committee of the Faculty of Biological Science – Tarbiat Modares University (Approval ID: IR.MODARES.REC.1400.313) and National Institute for Medical Research Development (Approval ID: IR.NIMAD.REC.1400.177) which was in accordance to the ethical principles and the national norms and standards for conducting medical research in Iran.

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Dehshiri, M., Rezaei, S. & Hosseinkhani, S. A novel multi-functional chimeric peptide for enhanced safe gene delivery in immunotherapy. Gene Ther 32, 497–507 (2025). https://doi.org/10.1038/s41434-025-00538-5

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