Applications of PNA-laden nanoparticles for hematological disorders

  • Shipra Malik
  • Stanley Oyaghire
  • Raman BahalEmail author


Safe and efficient genome editing has been an unmitigated goal for biomedical researchers since its inception. The most prevalent strategy for gene editing is the use of engineered nucleases that induce DNA damage and take advantage of cellular DNA repair machinery. This includes meganucleases, zinc-finger nucleases, transcription activator-like effector nucleases, and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9) systems. However, the clinical viability of these nucleases is marred by their off-target cleavage activity (≥ 50% in RNA-guided endonucleases). In addition, in vivo applications of CRISPR require systemic administration of Cas9 protein, mRNA, or DNA, which presents a significant delivery challenge. The development of nucleic acid probes that can recognize specific double-stranded DNA (dsDNA) regions and activate endogenous DNA repair machinery holds great promise for gene editing applications. Triplex-forming oligonucleotides (TFOs), which were introduced more than 25 years ago, are among the most extensively studied oligomeric dsDNA-targeting agents. TFOs bind duplex DNA to create a distorted helical structure, which can stimulate DNA repair and the exchange of a nearby mutated region—otherwise leading to an undesired phenotype—for a short single-stranded donor DNA that contains the corrective nucleotide sequence. Recombination can be induced within several hundred base-pairs of the TFO binding site and has been shown to depend on triplex-induced initiation of the nucleotide excision repair pathway and engagement of the homology-dependent repair pathway. Since TFOs do not possess any direct nuclease activity, their off-target effects are minimal when compared to engineered nucleases. This review comprehensively covers the advances made in peptide nucleic acid-based TFOs for site-specific gene editing and their therapeutic applications.


PNA PLGA nanoparticles Gamma PNA Gene editing Anemia 



This work was supported by University of Connecticut startup funds. In addition, this material is based upon work supported by the State of Connecticut under the Regenerative Medicine Research Fund and Cooley’s Anemia Foundation Research Fellowship Grant application. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the State of Connecticut or Connecticut Innovations, Incorporated.


  1. 1.
    Bak RO, Gomez-Ospina N, Porteus MH (2018) Gene editing on center stage. Trends Genet 34(8):600–611CrossRefGoogle Scholar
  2. 2.
    Doudna JA, Charpentier E (2014) Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346(6213):1258096CrossRefGoogle Scholar
  3. 3.
    Song B, Fan Y, He W, Zhu D, Niu X, Wang D, Ou Z, Luo M, Sun X (2015) Improved hematopoietic differentiation efficiency of gene-corrected beta-thalassemia induced pluripotent stem cells by CRISPR/Cas9 system. Stem Cells Dev 24(9):1053–1065CrossRefGoogle Scholar
  4. 4.
    Pattanayak V, Ramirez CL, Joung JK, Liu DR (2011) Revealing off-target cleavage specificities of zinc-finger nucleases by in vitro selection. Nat Methods 8(9):765–770CrossRefGoogle Scholar
  5. 5.
    Cradick TJ, Fine EJ, Antico CJ, Bao G (2013) CRISPR/Cas9 systems targeting β-globin and CCR5 genes have substantial off-target activity. Nucleic Acids Res 41(20):9584–9592CrossRefGoogle Scholar
  6. 6.
    Quijano E, Bahal R, Ricciardi A, Saltzman WM, Glazer PM (2017) Therapeutic peptide nucleic acids: principles, limitations, and opportunities. Yale J Biol Med 90(4):583–598PubMedPubMedCentralGoogle Scholar
  7. 7.
    Lino CA, Harper JC, Carney JP, Timlin JA (2018) Delivering CRISPR: a review of the challenges and approaches. Drug Deliv 25(1):1234–1257CrossRefGoogle Scholar
  8. 8.
    McNeer NA, Chin JY, Schleifman EB, Fields RJ, Glazer PM, Saltzman WM (2011) Nanoparticles deliver triplex-forming pnas for site-specific genomic recombination in CD34+ human hematopoietic progenitors. Mol Ther 19(1):172–180CrossRefGoogle Scholar
  9. 9.
    McNeer NA, Anandalingam K, Fields RJ, Caputo C, Kopic S, Gupta A, Quijano E, Polikoff L, Kong Y, Bahal R, Geibel JP, Glazer PM, Saltzman WM, Egan ME (2015) Correction of F508del CFTR in airway epithelium using nanoparticles delivering triplex-forming PNAs. Nat Commun 6:6952CrossRefGoogle Scholar
  10. 10.
    Egholm M, Buchardt O, Christensen L, Behrens C, Freier SM, Driver DA, Berg RH, Kim SK, Norden B, Nielsen PE (1993) PNA hybridizes to complementary oligonucleotides obeying the Watson-Crick hydrogen-bonding rules. Nature (London) 365(6446):566–568CrossRefGoogle Scholar
  11. 11.
    Nielsen PE, Egholm M, Berg RH, Buchardt O (1991) Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide. Science (Washington, D. C., 1883) 254(5037):1497–1500CrossRefGoogle Scholar
  12. 12.
    Rogers FA, Vasquez KM, Egholm M, Glazer PM (2002) Site-directed recombination via bifunctional PNA-DNA conjugates. Proc Natl Acad Sci USA 99(26):16695–16700CrossRefGoogle Scholar
  13. 13.
    Chin JY, Kuan JY, Lonkar PS, Krause DS, Seidman MM, Peterson KR, Nielsen PE, Kole R, Glazer PM (2008) Correction of a splice-site mutation in the beta-globin gene stimulated by triplex-forming peptide nucleic acids. Proc Natl Acad Sci USA 105(36):13514–13519CrossRefGoogle Scholar
  14. 14.
    McNeer NA, Schleifman EB, Cuthbert A, Brehm M, Jackson A, Cheng C, Anandalingam K, Kumar P, Shultz LD, Greiner DL, Mark Saltzman W, Glazer PM (2013) Systemic delivery of triplex-forming PNA and donor DNA by nanoparticles mediates site-specific genome editing of human hematopoietic cells in vivo. Gene Ther 20(6):658–669CrossRefGoogle Scholar
  15. 15.
    Lonkar P, Kim K-H, Kuan JY, Chin JY, Rogers FA, Knauert MP, Kole R, Nielsen PE, Glazer PM (2009) Targeted correction of a thalassemia-associated β-globin mutation induced by pseudo-complementary peptide nucleic acids. Nucleic Acids Res 37(11):3635–3644CrossRefGoogle Scholar
  16. 16.
    Bahal R, Ali McNeer N, Quijano E, Liu Y, Sulkowski P, Turchick A, Lu YC, Bhunia DC, Manna A, Greiner DL, Brehm MA, Cheng CJ, Lopez-Giraldez F, Ricciardi A, Beloor J, Krause DS, Kumar P, Gallagher PG, Braddock DT, Mark Saltzman W, Ly DH, Glazer PM (2016) In vivo correction of anaemia in beta-thalassemic mice by gammaPNA-mediated gene editing with nanoparticle delivery. Nat Commun 7:13304CrossRefGoogle Scholar
  17. 17.
    Shiraishi T, Nielsen PE (2014) Cellular delivery of peptide nucleic acids (PNAs). Methods Mol Biol 1050:193–205CrossRefGoogle Scholar
  18. 18.
    Cheng CJ, Bahal R, Babar IA, Pincus Z, Barrera F, Liu C, Svoronos A, Braddock DT, Glazer PM, Engelman DM, Saltzman WM, Slack FJ (2015) MicroRNA silencing for cancer therapy targeted to the tumour microenvironment. Nature 518(7537):107–110CrossRefGoogle Scholar
  19. 19.
    Thomas SM, Sahu B, Rapireddy S, Bahal R, Wheeler SE, Procopio EM, Kim J, Joyce SC, Contrucci S, Wang Y, Chiosea SI, Lathrop KL, Watkins S, Grandis JR, Armitage BA, Ly DH (2013) Antitumor effects of EGFR antisense guanidine-based peptide nucleic acids in cancer models. ACS Chem Biol 8(2):345–352CrossRefGoogle Scholar
  20. 20.
    Egholm M, Christensen L, Dueholm KL, Buchardt O, Coull J, Nielsen PE (1995) Efficient pH-independent sequence-specific DNA binding by pseudoisocytosine-containing bis-PNA. Nucleic Acids Res 23(2):217–222CrossRefGoogle Scholar
  21. 21.
    Vasquez KM, Christensen J, Li L, Finch RA, Glazer PM (2002) Human XPA and RPA DNA repair proteins participate in specific recognition of triplex-induced helical distortions. Proc Natl Acad Sci USA 99(9):5848–5853CrossRefGoogle Scholar
  22. 22.
    Maurisse R, De Semir D, Emamekhoo H, Bedayat B, Abdolmohammadi A, Parsi H, Gruenert DC (2010) Comparative transfection of DNA into primary and transformed mammalian cells from different lineages. BMC Biotechnol 10:9CrossRefGoogle Scholar
  23. 23.
    Lohse J, Dahl O, Nielsen PE (1999) Double duplex invasion by peptide nucleic acid: a general principle for sequence-specific targeting of double-stranded DNA. Proc Natl Acad Sci USA 96(21):11804–11808CrossRefGoogle Scholar
  24. 24.
    Davis PB (2001) Cystic fibrosis. Pediatr Rev 22(8):257–264CrossRefGoogle Scholar
  25. 25.
    Davis P (2006) Cystic fibrosis since 1938. Am J Respir Crit Care Med 173(5):475–482CrossRefGoogle Scholar
  26. 26.
    Rapireddy S, Bahal R, Ly DH (2011) Strand invasion of mixed-sequence, double-helical B-DNA by γ-peptide nucleic acids containing G-Clamp nucleobases under physiological conditions. Biochemistry 50(19):3913–3918CrossRefGoogle Scholar
  27. 27.
    Bahal R, Sahu B, Rapireddy S, Lee C-M, Ly DH (2012) Sequence-unrestricted, Watson-Crick recognition of double helical B-DNA by (R)-MiniPEG-γPNAs. ChemBioChem 13(1):56–60CrossRefGoogle Scholar
  28. 28.
    He G, Rapireddy S, Bahal R, Sahu B, Ly DH (2009) Strand invasion of extended, mixed-sequence B-DNA by γPNAs. J Am Chem Soc 131(34):12088–12090CrossRefGoogle Scholar
  29. 29.
    Sahu B, Sacui I, Rapireddy S, Zanotti KJ, Bahal R, Armitage BA, Ly DH (2011) Synthesis and characterization of conformationally preorganized, (R)-diethylene glycol-containing γ-peptide nucleic acids with superior hybridization properties and water solubility. J Org Chem 76(14):5614–5627CrossRefGoogle Scholar
  30. 30.
    Dragulescu-Andrasi A, Rapireddy S, Frezza BM, Gayathri C, Gil RR, Ly DH (2006) A simple γ-backbone modification preorganizes peptide nucleic acid into a helical structure. J Am Chem Soc 128(31):10258–10267CrossRefGoogle Scholar
  31. 31.
    Sahu B, Chenna V, Lathrop KL, Thomas SM, Zon G, Livak KJ, Ly DH (2009) Synthesis of conformationally preorganized and cell-permeable guanidine-based gamma-peptide nucleic acids (gammaGPNAs). J Org Chem 74(4):1509–1516CrossRefGoogle Scholar
  32. 32.
    Singer A, Rapireddy S, Ly DH, Meller A (2012) Electronic barcoding of a viral gene at the single-molecule level. Nano Lett 12(3):1722–1728CrossRefGoogle Scholar
  33. 33.
    Bahal R, Quijano E, McNeer NA, Liu Y, Bhunia DC, Lopez-Giraldez F, Fields RJ, Saltzman WM, Ly DH, Glazer PM (2014) Single-stranded gammaPNAs for in vivo site-specific genome editing via Watson-Crick recognition. Curr Gene Ther 14(5):331–342CrossRefGoogle Scholar
  34. 34.
    Ricciardi AS, Bahal R, Farrelly JS, Quijano E, Bianchi AH, Luks VL, Putman R, Lopez-Giraldez F, Coskun S, Song E, Liu Y, Hsieh WC, Ly DH, Stitelman DH, Glazer PM, Saltzman WM (2018) In utero nanoparticle delivery for site-specific genome editing. Nat Commun 9(1):2481CrossRefGoogle Scholar
  35. 35.
    Sazani P, Gemignani F, Kang S-H, Maier MA, Manoharan M, Persmark M, Bortner D, Kole R (2002) Systemically delivered antisense oligomers upregulate gene expression in mouse tissues. Nat Biotechnol 20(12):1228–1233CrossRefGoogle Scholar
  36. 36.
    Svasti S, Suwanmanee T, Fucharoen S, Moulton HM, Nelson MH, Maeda N, Smithies O, Kole R (2009) RNA repair restores hemoglobin expression in IVS2-654 thalassemic mice. Proc Natl Acad Sci USA 106(4):1205–1210CrossRefGoogle Scholar
  37. 37.
    Chen X, Goncalves MA (2016) Engineered viruses as genome editing devices. Mol Ther 24(3):447–457CrossRefGoogle Scholar
  38. 38.
    Liu C, Wang J, Huang S, Yu L, Wang Y, Chen H, Wang D (2018) Self-assembled nanoparticles for cellular delivery of peptide nucleic acid using amphiphilic N,N,N-trimethyl-O-alkyl chitosan derivatives. J Mater Sci Mater Med 29(8):114CrossRefGoogle Scholar
  39. 39.
    Baek A, Baek YM, Kim HM, Jun BH, Kim DE (2018) Polyethylene glycol-engrafted graphene oxide as biocompatible materials for peptide nucleic acid delivery into cells. Bioconjug Chem 29(2):528–537CrossRefGoogle Scholar
  40. 40.
    Bertucci A, Prasetyanto EA, Septiadi D, Manicardi A, Brognara E, Gambari R, Corradini R, De Cola L (2015) Combined delivery of temozolomide and anti-miR221 PNA using mesoporous silica nanoparticles induces apoptosis in resistant glioma cells. Small 11(42):5687–5695CrossRefGoogle Scholar
  41. 41.
    Ma X, Devi G, Qu Q, Toh DF, Chen G, Zhao Y (2014) Intracellular delivery of antisense peptide nucleic acid by fluorescent mesoporous silica nanoparticles. Bioconjug Chem 25(8):1412–1420CrossRefGoogle Scholar
  42. 42.
    Arayachukiat S, Seemork J, Pan-In P, Amornwachirabodee K, Sangphech N, Sansureerungsikul T, Sathornsantikun K, Vilaivan C, Shigyou K, Pienpinijtham P, Vilaivan T, Palaga T, Banlunara W, Hamada T, Wanichwecharungruang S (2015) Bringing macromolecules into cells and evading endosomes by oxidized carbon nanoparticles. Nano Lett 15(5):3370–3376CrossRefGoogle Scholar
  43. 43.
    Beavers KR, Mares JW, Swartz CM, Zhao Y, Weiss SM, Duvall CL (2014) In situ synthesis of peptide nucleic acids in porous silicon for drug delivery and biosensing. Bioconjug Chem 25(7):1192–1197CrossRefGoogle Scholar
  44. 44.
    Beavers KR, Werfel TA, Shen T, Kavanaugh TE, Kilchrist KV, Mares JW, Fain JS, Wiese CB, Vickers KC, Weiss SM, Duvall CL (2016) Porous silicon and polymer nanocomposites for delivery of peptide nucleic acids as anti-MicroRNA therapies. Adv Mater 28(36):7984–7992CrossRefGoogle Scholar
  45. 45.
    Bertucci A, Lulf H, Septiadi D, Manicardi A, Corradini R, De Cola L (2014) Intracellular delivery of peptide nucleic acid and organic molecules using zeolite-L nanocrystals. Adv Healthc Mater 3(11):1812–1817CrossRefGoogle Scholar
  46. 46.
    Cheng CJ, Saltzman WM (2012) Polymer nanoparticle-mediated delivery of microRNA inhibition and alternative splicing. Mol Pharm 9(5):1481–1488CrossRefGoogle Scholar
  47. 47.
    Bahal R, McNeer NA, Ly DH, Saltzman WM, Glazer PM (2013) Nanoparticle for delivery of antisense gammaPNA oligomers targeting CCR5. Artif DNA PNA XNA 4(2):49–57CrossRefGoogle Scholar
  48. 48.
    Gupta A, Quijano E, Liu Y, Bahal R, Scanlon SE, Song E, Hsieh WC, Braddock DE, Ly DH, Saltzman WM, Glazer PM (2017) Anti-tumor activity of miniPEG-gamma-modified PNAs to inhibit MicroRNA-210 for cancer therapy. Mol Ther Nucleic Acids 9:111–119CrossRefGoogle Scholar
  49. 49.
    Fields RJ, Quijano E, McNeer NA, Caputo C, Bahal R, Anandalingam K, Egan ME, Glazer PM, Saltzman WM (2015) Modified poly(lactic-co-glycolic acid) nanoparticles for enhanced cellular uptake and gene editing in the lung. Adv Healthc Mater 4(3):361–366CrossRefGoogle Scholar
  50. 50.
    Babar IA, Cheng CJ, Booth CJ, Liang X, Weidhaas JB, Saltzman WM, Slack FJ (2012) Nanoparticle-based therapy in an in vivo microRNA-155 (miR-155)-dependent mouse model of lymphoma. Proc Natl Acad Sci USA 109(26):E1695–E1704CrossRefGoogle Scholar
  51. 51.
    Fang H, Zhang K, Shen G, Wooley KL, Taylor JS (2009) Cationic shell-cross-linked knedel-like (cSCK) nanoparticles for highly efficient PNA delivery. Mol Pharm 6(2):615–626CrossRefGoogle Scholar
  52. 52.
    Langer K, Coester C, Weber C, von Briesen H, Kreuter J (2000) Preparation of avidin-labeled protein nanoparticles as carriers for biotinylated peptide nucleic acid. Eur J Pharm Biopharm 49(3):303–307CrossRefGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2018

Authors and Affiliations

  1. 1.Department of Pharmaceutical SciencesUniversity of ConnecticutStorrsUSA
  2. 2.Department of Therapeutic RadiologyYale UniversityNew HavenUSA

Personalised recommendations