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HIV Tat peptide enhances cellular delivery of antisense morpholino oligomers.
|Title||HIV Tat peptide enhances cellular delivery of antisense morpholino oligomers.|
|Publication Type||Journal Article|
|Year of Publication||2003|
|Authors||Moulton HM, Hase MC, Smith KM, Iversen PL|
|Journal||Antisense & nucleic acid drug development|
|Date Published||2003 Feb|
|Keywords||Gene Expression Regulation, Gene Products, tat, Hela Cells, HIV-1, Humans, Microscopy, Fluorescence, Morpholines, Morpholinos, Oligonucleotides, Antisense, Proto-Oncogene Proteins c-myc, tat Gene Products, Human Immunodeficiency Virus|
Phosphorodiamidate morpholino oligomers (PMO) are uncharged antisense molecules that bind complementary sequences of RNA, inhibiting gene expression by preventing translation or by interfering with pre-mRNA splicing. The techniques used to deliver PMO into cultured cells have been mostly mechanical methods. These delivery methods, although useful, have limitations. We investigated the ability of the HIV Tat peptide (pTat) and other cationic peptides to deliver PMO into cultured cells. Fluorescence was seen in 100% of HeLa cells treated with pTat-PMO-fluorescein conjugate. pTat-PMO conjugate targeted to c-myc mRNA downregulated c-myc reporter gene expression with an IC50 of 25 microM and achieved nearly 100% inhibition. pTat-PMO conjugate targeted to a mutant splice site of beta-globin pre-mRNA dose-dependently corrected splicing and upregulated expression of the functional reporter gene. Neither unconjugated PMO nor unconjugated pTat caused antisense activities. However, compared with mechanically mediated delivery, pTat-mediated PMO delivery required higher concentrations of PMO (>10 microM) to cause antisense activity and caused some toxicity. Most pTat-PMO conjugate was associated with cell membranes, and internalized conjugate was localized in vesicles, cytosol, and nucleus. The other three cationic peptides are much less effective than pTat. pTat significantly enhances delivery of PMO in 100% of cells assayed. pTat-mediated delivery is a much simpler procedure to perform than other delivery methods.
|Alternate Journal||Antisense Nucleic Acid Drug Dev.|