Sunday, February 3, 2008

Journal Club: Targeted Systemic Delivery of Stabilized Immunoliposomes to Leukocytes

In the latest issue of the premier scientific journal Science, Dan Peer and colleagues from Harvard Medical School report on the successful development of an RNAi delivery system that allows for targeted gene knockdown in leukocytes following systemic (intravenous) administration (Peer et al. Systemic Leukocyte-Directed siRNA Delivery Revealing Cyclin D1 as an Anti-Inflammatory Target. Science 319: 627). This research may be an important milestone towards opening up many more targets for systemic RNAi Therapeutics beyond the liver, lung, and tumors.

Until now, most systemic RNAi applications in pre-clinical animal models involved the passive delivery of various nanoparticle formulations which proved particularly effective in highly vascularized organs such as the liver. Other organs, however, are more difficult to reach mainly because most RNAi delivery formulations will either have been metabolized or excreted through the kidney, before they can reach the less accessible places in the body or enter some of the more difficult-to-transfect cell types of the blood. The present paper represents one of the more promising reports on achieving efficient gene knockdown in these organs through formulations that are actively targeted to the organ of interest through a range of peptides, antibodies, aptamers, and small molecules.

In order to combine high RNAi loading capacity, stability with consequently favorable circulation times, and cell targeting, the authors started with simple ~80nm (neutral) liposomes and added to their outside stabilizing hyaluronan (similar principle to the cationic liposome-based SNALPs- stable nucleic acids lipid particle- which typically carry stabilizing PEG on their outside). In a second step, they covalently attached an antibody specific for an integrin highly expressed on leukocytes that traffic to the gut and play a central role in autoimmune inflammatory bowel diseases such as Crohn’s. In a last step, the immunoliposome particles were loaded with the siRNA cargo that had been condensed with the highly basic protamine so as to achieve 80% loading efficiency (~4000 siRNAs per particle). That the siRNA-protamine condensation step should facilitate such efficient loading into the neutral liposomes is quite notable and may solve the poor nucleic acid loading typically associated with neutral liposomes (note: neutral liposomes may be advantageous over certain cationic liposomes in reducing unwanted interactions in the body).

The nanoparticles were then tested for gene silencing in the notoriously difficult-to-transfect leukocytes. Strikingly, sequence-specific, integrin-dependent, and antibody-dependent silencing was obtained both in vitro and in mice. The silencing efficiency was quite remarkable, typically ranging between 70-85%. When an siRNA was targeted to cyclin D1, this resulted in a reduced Th-1 response while the Th-2 response was unaffected. This is thought to be beneficial in treating chronic inflammatory diseases like Crohn’s, and sure enough, Cyclin D1 suppression by RNAi in a mouse model for intestinal inflammation almost completely reversed the disease phenotype. Hence, this paper not only demonstrates systemic gene silencing in leukocytes, but also validates Cyclin D1 as a potential drug target for Crohn’s and other inflammatory diseases.

Two aims underlie the targeted delivery paradigm: one is to deliver the RNAi trigger in sufficient amounts to the tissue of interest to achieve therapeutic levels of gene silencing; the other is to reduce potentially harmful exposure of non-target tissues to the siRNA, e.g. in instances where uptake of the siRNA in macrophages or dendritic cells may increase the risk of an unwanted immune response and render dosing less predictable, or where silencing of a gene itself in a non-target tissue may have adverse consequences. So far, most reports on targeted delivery satisfied mainly aim 1 while leaving aim 2 largely unaddressed. Quite impressively, the authors demonstrate in a biodistribution experiment truly targeted delivery by showing that the siRNA-nanoparticle were quantitatively re-directed to the gut in the mouse disease model (an almost 100-fold increase) and depleted from the blood and liver which would otherwise take up a good fraction of siRNA-nanoparticle studded with control antibody.

Of course, more time will have to be spent characterizing this system before it may move into the clinic. Safety is just one aspect that needs to be looked at in more detail, but at least in terms of cytokine activation and body weight was found to be satisfactory in the present study. Although some experiments involved repeat administration, it will further be important to determine whether there is immune recognition to any of the components of the particles which may interfere with repeat dosing. This also relates to the comparative complexity of these multifunctional particles which may also mean that the total costs of manufacturing them could considerably exceed that of the siRNA effector alone.

Surely, there will be a number of diseases for which such expenses are more than justified. I personally am not a great believer in blanketing large populations more or less indiscriminately with statins or aspirin, but rather like to see more targeted therapies that show clear benefits in well-defined patient populations. Since I expect many of the future RNAi Therapeutics to fall into the latter category and ultimately represent a bigger bang for the healthcare dollar, it is therefore important that the healthcare and patent systems facilitate rather than antagonize such innovative drug development efforts.

4 comments:

Nika said...

Dirk: Your blog is well done and a fantastic resource!

I have some questions re: RNAi and the coming year in terms of hot topics, etc.

I was wondering if you could respond to my email address and then I will explain further.

nika.boyce@gmail.com

Fellow Scientist,

Nika

zimstock said...

Interesting post! What is the difference between this technology and the one that Silence has developed? happy to correspond directly too

Dirk said...

Zimstock,
Unfortunately, I have little insight into Silence's delivery efforts except that they appear to work on various formulations (see quote below). There is some public information out there on a delivery system developed together with Genzyme, which like in the present invention involves liposomes, but I very much doubt that they or their licensors are currently using antibody-mediated targeting in late pre-clinical or clinical programs.

"At Silence Therapeutics, despite the important progress that we have made, we
do not believe there will be `one delivery solution to fit all challenges' and
therefore throughout 2007 we continued to explore a wide range of other
delivery approaches in conjunction with partners and academic collaborators. In
particular during the year we established and progressed several delivery
partnerships and collaborations, which we have chosen not to publicise until we
have secured our proprietary position."

Dirk Haussecker said...

Nika,

If you want, you can send me an email to dirkh@stanford.edu.

By Dirk Haussecker. All rights reserved.

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