RNA interference (RNAi) is a powerful strategy for suppressing gene expression in a sequence-specific manner. This strategy offers new potential opportunities for treating various diseases by addressing otherwise 'undruggable' targets. Currently, the most promising type of RNAi-based advancing therapeutics in preclinical and clinical trials is incorporating small interfering RNAs fragments (siRNAs), synthetic 21-23 base pairs double-stranded RNA molecules, into the cell cytoplasm1,2.
Despite advantages such as eliminating clinical safety concerns associated with viral vectors and a lesser interruption to endogenous gene regulation machineries, translation of siRNAs into clinical practice faces some major hurdles, like low efficacy of crossing the plasma membrane and entering the cytoplasm, stimulation of the immune system that often causes global suppression of gene expression, rapid renal clearance and degradation by RNases. Therefore, generating nanocarriers for targeted delivery of siRNAs is necessary. Devising such systems enforces dealing with the challenges of developing fully degradable particles targeted to the appropriate cell-type, evading the stimulation of the immune system, and utilizing cellular mechanisms for internalization and releasing the siRNAs into the cell cytoplasm2,3.
Unlike systemic delivery to solid tumors and the liver, systemic delivery to leukocytes (immune cells), due to their resistance to conventional transfection methods and to their dispersing in the body, remains challenging4. We have developed antibody-protamine (a positively charged protein) fusion proteins directed to the lymphocyte function associated antigen-1 (LFA-1) integrin, a cell surface adhesion molecule that is expressed in all leukocytes' subtypes. Those fusion proteins selectively delivered siRNAs into leukocytes, both in vitro and in vivo. Furthermore, by targeting these fusion proteins to the high affinity conformation of LFA-1 that characterizes activated lymphocytes, we demonstrated selective gene silencing, which unlike most immunosuppressive therapies, could provide a way to overcome the unwanted immune stimulation without global immunosuppressive effects on bystander immune cells5.
In order to increase payload and achieve robust targeted gene silencing, we have generated integrin-targeted stabilized nanoparticles (I-tsNP), which have been covalently coated with anti-B7 integrin (highly expressed in gut mononuclear leukocytes) antibody, and demonstrated that those particles can selectively deliver siRNAs to leukocytes involved in gut inflammation (see illustration). Made from natural biomaterials, these nanoparticles offer a safe platform for siRNAs delivery, avoiding cytokine induction and liver damage6. Using this system, we identified cyclin D1, a regulator protein of the entry into, and the progression throughout the cell cycle7, as a potential new target for treating inflammation6.
|Integrin-targeted stabilized nanoparticles (I-tsNP) . The particles have been developed as ~80nm liposomes, formed from natural phospholipids, hence avoiding the potential toxicity of cationic lipids and polymers. Hyaluronan (HA), a naturally accruing glycosaminoglycan, was attached to the surface of the liposomes, stabilizing the particles during siRNA entrapment and systemic circulation in vivo. Then, a monoclonal antibody against the integrin was attached to HA. The particles were loaded with siRNAs condensed with protamine while maintaining their nanodimentions6.
Utilizing siRNAs to manipulate gene expression in leukocytes holds great promise for the drug discovery field, as well as for facilitating the development of new therapies platforms for leukocytes implicated diseases such as inflammation, blood cancers, and leukocytes-tropic viral infections. Additionally, siRNA delivery to leukocytes could serve as a powerful tool for understanding leukocytes' biology. Moreover, since one can easily change the payloads inside the nanoparticles (by using different sequences of siRNAs, or other drugs) or the targeting agent (by replacing the antibody or the ligand decorating the nanoparticle's surface), it is reasonable that this platform might be applicable to other types of diseases outside the hematopoietic system.
1. Sledz CA and Williams BR. RNA interference in biology and disease. Blood, 106, 787-794 (2005).
2. de Fougerolles A, Vornlocher HP, Maraganore J, Lieberman J. Interfering with disease: a progress report on siRNA-based therapeutics. Nature reviews, 6(6), 443-453 (2007).
3. Peer D, Karp JM, Hong S, Farokhzad OC, Margalit R, Langer R. Nanocarriers as an emerging platform for cancer therapy. Nature nanotechnology, 2(12), 751-760 (2007).
4. Goffinet C, Keppler OT. Efficient nonviral gene delivery into primary lymphocytes from rats and mice. Faseb J, 20(3), 500-502 (2006).
5. Peer D, Zhu P, Carman CV, Lieberman J, Shimaoka M. Selective gene silencing in activated leukocytes by targeting siRNAs to the integrin lymphocyte function-associated antigen-1. Proceedings of the National Academy of Sciences of the United States of America, 104(10), 4095-4100 (2007).
6. Peer D, Park EJ, Morishita Y, Carman CV, Shimaoka M. Systemic leukocyte-directed siRNA delivery revealing cyclin D1 as an anti-inflammatory target. Science, 319(5863), 627-630 (2008).
7. Stacey DW. Cyclin D1 serves as a cell cycle regulatory switch in actively proliferating cells. Curr Opin Cell Biol, 15, 158-163 (2003).
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