A group of investigators this week published data demonstrating that a peptide-based delivery technology under development at Traversa Therapeutics can carry siRNAs into a variety of primary and transformed cells — including T cells, human umbilical vein endothelial cells, and human embryonic stem cells — with no toxicity or immune-response activation.
The team, which was led by Steven Dowdy, a Traversa co-founder and researcher at the University of California, San Diego, wrote that the findings indicate that the technology could be useful for basic research, target screening, and therapeutic applications.
Traversa has for some time been looking to ink partnerships applying the technology to both RNAi drugs and reagents (see RNAi News, 4/2/2009). In an e-mail this week, Traversa President and CEO Hans Petersen said that he hopes the newly published data, which appears in the online edition of Nature Biotechnology, will facilitate such arrangements.
The technology, called PTD-DRBD, comprises protein transduction domains linked with a double-stranded RNA-binding domain.
According to Traversa, which holds the exclusive rights to the technology, siRNAs coated with PTD-DRBD molecules bind to cell-surface proteoglycans, which stimulates macropinocytosis. The drug then enters the cell inside a macropinosome, at which point the pH inside the vesicle drops and the siRNA is released from the PTD-DRBD molecules into the cytoplasm.
Looking to demonstrate the ability of the technology to deliver siRNAs, Dowdy and colleagues generated a human H1299 lung adenocarcinoma reporter cell line using destabilized green and red fluorescent proteins, which allowed them to directly determine the magnitude of a single-cell RNAi response, the investigators wrote in Nature Biotechnology.
"PTD-DRBD delivery of GFP-specific siRNAs resulted in a substantial GFP knockdown with little to no alteration of the internal [destabilized red fluorescent protein] control," they wrote. "Similar RNAi responses were induced with three additional GFP siRNAs delivered by PTD-DRBDs."
Additionally, none of the controls used — non-specific control siRNAs and PTD delivery peptide alone — induced RNAi, they added.
Notably, PTD-DRBD-mediated GFP siRNA delivery also resulted in a "substantially stronger" RNAi response versus lipofectamine-delivered siRNAs, and there was little or no alteration of viability detected in cells treated with the PTD-DRBD-delivered siRNAs.
"We next targeted endogenous glyceraldehyde 3-phosphate dehydrogenase mRNA by PTD-DRBD-mediated RNAi," the researchers wrote. Treating H1299 cells with one of two sequence-independent GAPDH siRNAs delivered using the PTD-DRBD technology resulted in an RNAi response, but the negative controls did not.
The team noted that PTD-DRBD-delivered siRNAs triggered a "near-maximal RNAi response" in the cells six hours after treatment, which was "significantly earlier" than control lipofectamine delivery of the same siRNAs. This, they wrote, suggests that siRNAs delivered with Traversa's technology "rapidly enter the cytoplasm and are loaded into the RNA-induced silencing complex."
Further analysis of the treated cells revealed a "dramatic reduction" in the target GAPDH mRNA with a "limited number of both up- and down-regulated genes," according to the paper. However, none of the genes affected are present in an innate immunity response pathway or congregate into a specific genetic pathway, it adds.
The investigators then turned to an especially difficult-to-transfect cell type, tumorigenic Jurkat T cells, to further examine the PTD-DRBD technology.
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PTD-DRBD delivery of GFP-specific siRNAs into Jurkat T cells resulted in "a strong GFP RNAi response" in the entire cellular population, the team wrote. In comparison, siRNAs delivered using the lipofection reagents Lipofection-2000 or RNAiMax induced limited RNAi responses.
"Moreover, PTD-DRBD-delivered GAPDH siRNA resulted in a strong GAPDH RNAi response in Jurkat T cells as measured by qRT-PCR, whereas the two lipofection reagents performed poorly," they added.
Next, primary murine T cells were treated with CD4-specific siRNAs delivered using the PTD-DRBD technology. According to the paper, the entire cell population underwent an RNAi response after 24 hours, while control siRNAs had no effect.
"Similarly, PTD-DRBD-mediated delivery of CD8-specific siRNAs into primary T cells resulted in a CD8-specific RNAi response with no change in CD4 levels," the paper states. No RNAi responses were detected in primary T cells using lipofection reagents.
The PTD-DRBD technology was then tested in human umbilical vein endothelial cells and human embryonic stem cells.
"Treatment of primary [human umbilical vein endothelial cells] with one of two sequence-independent GAPDH siRNAs delivered by PTD-DRBD resulted in a GAPDH RNAi response" that was first detected by qRT-PCR after six hours and peaked at 12 hours, the team wrote.
"In contrast, none of the PTD-DRBD negative controls induced a GAPDH RNAi response," and there was little or no alteration of cell viability detected in those cells treated with siRNAs delivered using the delivery technology.
The researchers then evaluated the ability of the PTD-DRBD technology to deliver siRNAs into H9 human embryonic stem cells stably expressing GFP, and found that the oligos induced "a marked GFP RNAi response" throughout the cell colony.
In order to prove the potential of Traversa's technology for in vivo applications, Dowdy and his team assessed its immunogenicity in primary human peripheral blood mononuclear cells and its ability to deliver siRNAs into a reporter mouse model.
The investigators treated transgenic mice expressing luciferase in the nasal and tracheal passages with intranasal doses of either phosphate-buffered saline, PTD-DRBD combined with luciferase-specific siRNAs, or PTD-DRBD with a control siRNA.
While no change in luciferase expression was detected in the control animals, "an extensive reduction" was detected throughout the nasal and tracheal passages of mice receiving the luciferase-specific siRNAs.
Noting that delivery has become the "rate-limiting barrier to efficient cell culture and preclinical and clinical usage of siRNA therapeutics," Dowdy and his colleagues wrote that current siRNA delivery approaches "generally do not target the entire population or even a high percentage of cells, especially primary cells, and often result in some degree of cytotoxicity and alterations in cell biology.
"In contrast, the PTD-DRBD siRNA delivery approach described here fulfills many of the criteria for an efficient siRNA delivery system for primary cells," they added.
They cautioned, however, that additional studies are required to fully ascertain the delivery technology's in vivo utility.
Such studies are already underway through technology-evaluation deals Traversa has signed with a number of undisclosed companies, including ones interested in using the PTD-DRBD approach for ocular diseases, metabolic disorders, and cancer.
In April, Traversa's Petersen told RNAi News that the company hopes that at least two of these preliminary arrangements will have matured into broader research agreements this year.