Effective delivery of antisense peptide nucleic acid oligomers into cells by anthrax protective antigen.
Peptide nucleic acid (PNA) is highly stable and binds to complementary RNA and DNA with high affinity, but it resists cellular uptake, thereby limiting its bioavailability. We investigated whether protectiveantigen (PA, a non-toxic component of anthrax toxin) could transport antisense PNA oligomers into reporter cells that contain luciferase transgenes with mutant beta-globin IVS2 intronic inserts, which permit aberrant pre-mRNA splicing and impair luciferase expression. PNA oligomers antisense to mutant splice sites in these IVS2 inserts induced luciferase expression when effectively delivered into the cells. PNA 18-mers with C-terminal poly-lysine tails [PNA(Lys)(8)] demonstrated modest sequence-specific antisense activity by themselves at micromolar concentrations in luc-IVS2 reporter cell cultures. However, this activity was greatly amplified by PA. Antisense PNA(Lys)(8) with but not without PA also corrected the IVS2-654 beta-globin splice defect in cultured erythroid precursor cells from a patient with beta-thalassemia [genotype, IVS2-654(beta(0)/beta(E))], providing further evidence that anthrax PA can effectively transport antisense PNA oligomers into cells.
Wright
Daniel G
DG
Molecular Medicine Section, Department of Medicine, Boston University School of Medicine, Boston, MA, USA. dw341u@nih.gov
Zhang
Ying
Y
Murphy
John R
JR
D000942:Antigens, Bacterial / Q000378:metabolism*; D001427:Bacterial Toxins / Q000378:metabolism*; D001692:Biological Transport; D005914:Globins / Q000235:genetics; D006801:Humans; D007438:Introns; D008156:Luciferases / Q000235:genetics; D020319:Oligodeoxyribonucleotides, Antisense / Q000235:genetics / Q000378:metabolism*; D020135:Peptide Nucleic Acids / Q000235:genetics / Q000378:metabolism*; D022821:RNA Splice Sites; D012326:RNA Splicing; D014162:Transfection / Q000379:methods*; D017086:beta-Thalassemia / Q000235:genetics
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D020319:Oligodeoxyribonucleotides, Antisense; D020135:Peptide Nucleic Acids; D022821:RNA Splice Sites; C030325:anthrax toxin; D005914:Globins; D008156:Luciferases
Science. 1991 Dec 6;254(5037):1497-500
1962210
J Biol Chem. 1993 Jun 5;268(16):12077-82
8505330
Proc Natl Acad Sci U S A. 2000 Aug 15;97(17):9591-6
10944225
Anal Biochem. 1995 Jul 1;228(2):336-42
8572316
Adv Drug Deliv Rev. 2003 Feb 10;55(2):267-80
12564980
Infect Immun. 2004 Nov;72(11):6313-7
15501759
J Biol Chem. 2007 Mar 30;282(13):9834-9845
17251181
Nucleic Acids Res. 2001 Oct 1;29(19):3965-74
11574678
Adv Drug Deliv Rev. 2000 Oct 31;44(1):23-34
11035195
Biochemistry. 2002 Apr 9;41(14):4503-10
11926811
Biotechniques. 1989 Nov-Dec;7(10):1116-22
2698191
Protein Eng. 1987 Dec;1(6):493-8
3334101
Biochemistry. 1998 May 5;37(18):6235-9
9572837
Nat Biotechnol. 2002 Dec;20(12):1228-33
12426578
Oligonucleotides. 2006 Spring;16(1):43-57
16584294
J Clin Invest. 2003 Aug;112(4):481-6
12925686
FASEB J. 2000 Jun;14(9):1041-60
10834926
Curr Opin Hematol. 2005 Jan;12(1):40-4
15604890
Infect Immun. 1991 Oct;59(10):3381-6
1909998
Antisense Nucleic Acid Drug Dev. 1996 Fall;6(3):169-75
8915501
Biochim Biophys Acta. 1999 Dec 10;1489(1):45-52
10806996
Lancet. 2001 Aug 11;358(9280):489-97
11513935
Nat Biotechnol. 2000 Mar;18(3):300-3
10700145
Biochim Biophys Acta. 1999 Dec 10;1489(1):31-44
10806995
Biochemistry. 1998 Nov 10;37(45):15737-46
9843379
Expert Opin Biol Ther. 2007 Jul;7(7):1021-34
17665991
J Biol Chem. 1998 Apr 10;273(15):8835-41
9535863
Biochim Biophys Acta. 1999 Dec 10;1489(1):141-58
10807004
Biochemistry. 2001 Jan 9;40(1):53-64
11141056
Curr Opin Biotechnol. 2002 Oct;13(5):468-72
12459339
J Cell Biol. 2003 Mar 31;160(7):1139-50
12668662
Proc Natl Acad Sci U S A. 1996 Aug 6;93(16):8437-42
8710889
Semin Oncol. 2006 Feb;33(1 Suppl 3):S11-6
16516670
Antisense Nucleic Acid Drug Dev. 2000 Apr;10(2):117-21
10805163