Alu retroelements propagate via retrotransposition by hijacking long interspersed nuclear element-1 (L1) reverse transcriptase (RT) and endonuclease activities. Reverse transcription of Alu RNA into complementary DNA (cDNA) is presumed to occur exclusively in the nucleus at the genomic integration site. Whether Alu cDNA is synthesized independently of genomic integration is unknown. Alu RNA promotes retinal pigmented epithelium (RPE) death in geographic atrophy, an untreatable type of age-related macular degeneration. We report that Alu RNA-induced RPE degeneration is mediated via cytoplasmic L1–reverse-transcribed Alu cDNA independently of retrotransposition. Alu RNA did not induce cDNA production or RPE degeneration in L1-inhibited animals or human cells. Alu reverse transcription can be initiated in the cytoplasm via self-priming of Alu RNA. In four health insurance databases, use of nucleoside RT inhibitors was associated with reduced risk of developing atrophic macular degeneration (pooled adjusted hazard ratio, 0.616; 95% confidence interval, 0.493–0.770), thus identifying inhibitors of this Alu replication cycle shunt as potential therapies for a major cause of blindness.

Cyclic peptides containing tryptophan (W) and arginine (R) residues, [WR] 5 , [WR] 6 , [WR] 7 , [WR] 8 , and [WR] 9 , were synthesized through Fmoc solid-phase chemistry to compare their molecular transporter efficiency. The in vitro cytotoxicity of the peptides was evaluated using human leukemia carcinoma cell line (CCRF-CEM) and normal kidney cell line (LLC-PK1). [WR] 6 , [WR] 7 , [WR] 8 , and [WR] 9 were not significantly cytotoxic to LLC-PK1cells at a concentration of 10 μM after 3 h incubation. Among all the peptides, [WR] 9 was found to be a more efficient transporter than [WR] 5 , [WR] 6 , [WR] 7 , and [WR] 8 in CCRF-CEM cells for delivery of a cell-impermeable fluorescence-labeled negatively charged phosphopeptide (F′-GpYEEI). [WR] 9 (10 μM) improved the cellular uptake of F′-GpYEEI (2 μM) by 20-fold. The cellular uptake of a fluorescent conjugate of [WR] 9 , F′-[W 9 R 8 K], was increased in a concentration- and time-dependent pattern in CCRF-CEM cells. The uptake of F′-[W 9 R 8 K] was slightly reduced in CCRF-CEM cells in the presence of different endocytic inhibitors, such as nystatin, 5-( N -ethyl- N -isopropyl)amiloride, chlorpromazine, chloroquine, and methyl β-cyclodextrin. Furthermore, the uptake of F′-[W 9 R 8 K] was shown to be temperature-dependent and slightly adenosine 5′-triphosphate-dependent. The intracellular/cellular localization (in the nucleus and cytoplasm) of F′-[W 9 R 8 K] was confirmed by fluorescent microscopy in CCRF-CEM cells. These studies suggest that large cyclic peptides containing arginine and tryptophan can be used as a molecular transporter of specific compounds.

A number of amphiphilic cyclic peptides—[FR]4, [WR]5, and [WK]5—containing hydrophobic and positively-charged amino acids were synthesized by Fmoc/tBu solid-phase peptide methods and evaluated for their efficiency in intracellular delivery of siRNA to triple-negative breast cancer cell lines, MDA-MB-231 and MDA-MB-468, in the presence and absence of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). Among the peptides, [WR]5, which contains alternate tryptophan (W) and arginine (R) residues, was found to be the most efficient in the delivery of siRNA by improving the delivery by more than 3-fold when compared to other synthesized cyclic peptides that were not efficient. The data also showed that co-formulation of [WR]5 with lipid DOPE significantly enhanced the efficiency of siRNA delivery by up to ~2-fold compared to peptide alone. Based on the data indicating the efficiency of [WR]5 in siRNA delivery, peptides containing arginine residues on the ring and tryptophan residues on the side chain, [R6K]W6 and [R5K]W5, were also evaluated, and demonstrated improved delivery of siRNA. The presence of DOPE again enhanced the siRNA delivery in most cases. [WR]5, [R5K]W5, and [R6K]W6 did not show any significant toxicity in MDA-MB-231, MDA-MB-468, and AU565 WT cells at N/P ratios of 20:1 or less, in the presence and absence of DOPE. Silencing of kinesin spindle protein (KSP) and Janus kinase 2 (JAK2) was evaluated in MDA-MB-231 cells in the presence of the peptides. The addition of DOPE significantly enhanced the silencing efficiency for all selected peptides. In conclusion, peptides containing tryptophan and arginine residues were found to enhance siRNA delivery and to generate silencing of targeted proteins in the presence of DOPE.