By Kevin E. Noonan --
A research paper in the June 2009 edition of Nature Biotechnology (Eguchi et al., 2009, "Efficient siRNA delivery into primary cells by a peptide transduction domain–dsRNA binding domain fusion protein," Nature Biotechnology 27: 567-71) describes a new system for introducing short interfering RNAs (siRNAs) into mammalian cells that may help this technology become practicable for therapeutic uses. The promise of RNA interference, mediated by siRNAs, has revolutionized the prospects for modulating gene expression as a way to achieve therapeutic aims in disease treatment. First identified in worms (Fire et al., 1998, "Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans," Nature 391: 806–11), plants (Hamilton et al., 1999, "A species of small antisense RNA in posttranscriptional gene silencing in plants," Science 286: 950–52) and then in mammalian cells (Elbashir et al., 2001, "Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells," Nature 411: 494–98), siRNAs form the basis for 81 U.S. patents and 2,125 published patent applications. Their benefits reside principally in their ability to specifically reduce or even enhance (Li, 2008, "Small RNA-Mediated Gene Activation," In: RNA and the Regulation of Gene Expression: A Hidden Layer of Complexity, Caister Academic Press) gene expression in mammalian cells.
However, a serious impediment to further development of siRNA, particularly for therapeutic uses, is that it is difficult to effectively introduce these molecules into mammalian cells. siRNAs are double-stranded RNA molecules, typically 21 nucleotides in length and having a 2 nucleotide overhang at the 3' end of each strand. These molecules are thus large (> 14,000 kDa) and highly negatively charged, and cannot cross the cellular plasma membrane. Methods for introducing siRNAs have included encapsulation in liposome, complexing with cationic lipids, cholesterol, and condensing polymers, or conjugation with antibodies. While these methods have proven effective with adherent tumor cells in vitro, they have been less effective in the types of cells that are attractive cellular targets in vivo (such as lymphocytes). One approach, conjugation with cell-penetrating peptides (PTD) like TAT protein from human immunodeficiency virus or poly(Arg), resulted in cytotoxicity associated with limited siRNA delivery into target cells (Meade et al., 2008, "Enhancing the cellular uptake of siRNA duplexes following noncovalent packaging with protein transduction domain peptides," Adv. Drug Deliv. Rev. 60: 530-36), despite the effectiveness of such PTD carriers in other contexts (Gump et al., 2007, TAT transduction: The molecular mechanism and therapeutic prospects," Trends Molec. Med. 13: 443-48).
The Nature Biotechnology paper is from a group at the Howard Hughes Institute at the University of California, San Diego School of Medicine headed by Steven F. Dowdy. The paper describes complexes of siRNAs with a fusion protein comprising a TAT-derived PTD conjugated to a double-stranded (ds) RNA-binding domain (DRBD) that binds to siRNA with an avidity of KD ~ 10-9. A canonical example of such a sequence is the following (provided on-line in supplementary materials):
MGRKKRRQRRRGHSGRKKRRQRRRGHIYPYDVPVPDYAGDPGRKKRRQRRRGDP
AGDLSAGFFMEELNTYRQKQGVVLKYQELPNSGPPHDRRFTFQVIIDGREFPEG
EGRSKKEAKNAAAKLAVEILNEKKAAALEHHHHHH
(where single-underlined sequences are PTD sequences from TAT, the underlined and italicized sequence is an HA-tag, and the boldface sequence is the DRBD from RNA-dependent eIF-2 alpha protein kinase (PKR) protein). The DRBD sequence non-covalently complexes with siRNA in a way that is distinct from the PTD sequences, which remain competent to mediate transit of the complex through the plasma membrane.
Image obtained from Traversa Therapeutics website.
These complexes were tested in several systems, including human H1299 lung adenocarcinoma cells expressing green fluorescent protein (GFP) and red fluorescent protein, which were used to show specific RNA interference of GFP that was obtained at greater levels than with conventional lipofection techniques. These results were confirmed by single cell flow cytometry that showed that the entirety of the cell population contained siRNA with no alteration of cellular viability (in contrast with significant cytotoxicity associated with siRNA lipofection). These results were also confirmed with other cell types, including "primary human fibroblasts, keratinocytes, macrophages and melanoma and glioma cells containing integrated GFP reporter genes." Substantially similar results were obtained with these cells using siRNAs for glyceraldehyde phosphate dehydrogenase (GAPDH), a housekeeping enzyme. In these experiments, the effects of the GAPDH siRNAs on the cellular transcriptome were assessed, and were shown to be significantly smaller than the effects of these same siRNAs introduced using lipofection. In particular, the complexed siRNAs did not activate cellular genes associated with immunogenicity, including interferon-regulated genes, in contrast to lipofected siRNAs. (siRNA introduction using these complexes did not induce interferon or tumor necrosis factor expression in peripheral blood mononuclear cells, an effect observed with siRNA lipofection.)
In another set of experiments, GFP expression was suppressed in Jurkett T cells, primary murine T cells, and human umbilical vein endothelial cells (HUVEC). The primary T-cell experiments used siRNAs for CD4 and CD8, and showed specific suppression of each gene by its cognate siRNA and not by the non-cognate siRNA. (The experiments also showed that expression of an unrelated cell surface marker, CD90, was not affected by siRNA introduction.) HUVEC cells were treated with GAPDH siRNA and showed significantly lower (essentially undetectable) cytotoxicity and greater delivery than when siRNA were introduced into these cells by lipofection. Finally, human embryonic stem cells were treated with a number of siRNAs that affected proliferation and differentiation. Specifically, introduction of complexes of siRNAs for OCT4 caused cell cycle exit and differentiation.
In the most dramatic series of experiments, transgenic mice expressing luciferase in nasal and tracheal passages were treated with luciferase siRNAs complexed with PTD-DRBD. These experiments showed that luciferase expression could be drastically reduced by these siRNA/PTD-DRBD complexes in vivo.
These results also provide the basis for patent applications in the U.S. and abroad. The U.S. application is Serial No. 12/278,739, filed August 7, 2008. Entitled "Transducible Delivery of siRNA by dsRNA Binding Domain Fusions to PTD/CPPS," the application published on April 9, 2009 as U.S. Patent Application Publication No. 2009/0093026, naming Steven F. Dowdy, Jehangir S. Wadia, Bryan Meade, and Akiko Eguchi as inventors. This application is a U.S. national phase application of International Application No. PCT/US07/03641, filed February 9, 2007 and in turn claiming priority to two U.S. provisional applications (USSN 60/772,787, filed February 10, 2006, and USSN 60/775,638, filed February 21, 2006). In addition to the experiments disclosed in the research paper, the application also shows EGFR-specific siRNA introduction using PTD-DRBD complexes into EGFR-expressing glioblastoma in mice. The application also discloses alternative embodiments of PTD and DRBD sequences that can be used in the complexes.
The significance of these results is expressed by the authors as follows:
siRNA delivery has become the rate-limiting barrier to efficient cell culture and preclinical and clinical usage of siRNA therapeutics . . . . Although current siRNA delivery approaches have merit, they 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 alternations in cell biology. In contrast, the PTD-DRBD siRNA delivery approach described here fulfills many of the criteria for and efficient siRNA delivery system for primary cells. . . . Because DRBDs bind to dsRNAs (siRNAs) independent of sequence composition, PTD-DRBD could in theory deliver any siRNA into cells. Lastly, the intranasal knockdown of luciferase demonstrates the in vivo potential of PTD-DRBD-mediated siRNA delivery.
The application is assigned to the Regents of the University of California, but Professor Dowdy is reported to have a start-up biotechnology company, Traversa Therapeutics of La Jolla, CA, and a researcher from Ambion is named on the research paper (but not the patent application). A serious commitment to commercial development is also evidenced by applicants' recently changing the application status to large entity.
Comments