Delivering RNAi Therapeutics - Drug Discovery and Development

Active investigation of several different RNAi delivery mechanisms is underway, but the challenge still remains.

The phenomenon of RNA interference, a fundamental biological process by which cells regulate gene expression, was only discovered in the late 1990s. Since then, the development of RNAi from a basic scientific discovery to, first, a powerful research tool and, more recently, a promising therapeutic approach, has occurred very rapidly.^1-3 RNAi is now routinely used to assess gene function, both in vitro and in vivo, and many innovative screens have been reported that use RNAi to identify potential drug targets. As a therapeutic approach, the specific gene knockdown that can be achieved using RNAi, and the ability to silence previously undruggable targets, have made such therapies very attractive for many disease areas.

Delivery vehicle	Route of administration	Target tissue/cell type	Target	Reference
Simple excipients
saline	intravenous	multiple tumor models	multiple	(9) for review
saline	intravitreal	ocular vasculature	VEGF/VEGF R1	(10) (11)
saline	intranasal	lung tissue in different disease models	HMOX1, Angpt2, MIP2, DDR-1	(12-15)
D5W, surfactant	intranasal	SARS infection	SARS	(16)
saline	stereotactic injection	brain	AGRP	(17)
saline	intraventricular	brain	Dopamine Transporter	(18)
saline	intrathecal	brain	P2X3	(19)
Liposomes/Lipoplexes
Liposome	intravenous	liver	ApoB	(5, 20)
Liposome	intravenous	liver	HBV	(21)
Liposome	intravenous	lung (vascular permeability)	Caveolin-1	(22)
Lipoplex	intravenous	tumor endothelium	CD31	(23)
Lipoplex	intravaginal	vaginal mucosa	Herpes Virus	(24)

Many of the key challenges related to using siRNAs as drugs have been addressed already. For example, methods for the discovery of potent and specific siRNA sequences to virtually any gene are now well established. The instability of siRNAs in biological fluids has been addressed through stabilizing chemical modifications (for a review see⁴). Avoidance of immune stimulation by siRNAs has also been achieved through appropriate modifications of the basic siRNA structure.⁵ As a result of these advances, siRNA-based therapies have quickly reached the clinic in several cases, where initial results suggest that siRNAs themselves are well-tolerated from a safety perspective.

Despite these advances, however, there remains the challenge of drug delivery for siRNA-based therapeutics. Before the full potential of RNAi can be realized, methods for delivering siRNA safely, effectively, and conveniently to the right tissues and cell types are needed. This review summarizes recent progress in siRNA-based drug delivery, and discusses some of the promising approaches that are currently being explored.

The RNAi mechanism has two important implications for drug discovery. First, since RNAi-based gene silencing occurs in the cytoplasm, where the RISC (RNA-induced silencing complex) brings together the antisense strand of an siRNA duplex and the corresponding mRNA molecule,⁶ any method for delivery of siRNA-based therapeutics must be capable of releasing the siRNA into the cytoplasm of relevant cells. This is the key challenge of siRNA delivery. All other aspects of delivery, such as targeting to particular cell types, must be compatible with the basic requirement that cells be able to take up the siRNA and release it into the cytoplasm in an active form. Second, since the process that leads to cleavage of the mRNA strand does not consume the antisense strand of the siRNA, the siRNA is able to act catalytically, and thus can persist in silencing its gene for a significant period of time without interfering with the genetic information. In vivo experiments have shown that a single administration of a formulated siRNA can lead to gene silencing lasting several weeks.

Several types of siRNA delivery are under active investigation, including direct delivery of naked siRNA; encapsulation into liposomes and lipoplexes; conjugation to antibodies, peptides, aptamers, and other molecules; and formation of complexes with chemical and biological polymers. Each of these approaches has potential advantages and particular challenges. Delivery approaches that have been reported in the literature are summarized in the tables in this article. Each general category of delivery is discussed below.

Simple excipients
Local delivery, without systemic exposure, is sometimes a desirable approach. Compared to systemic administration, drug doses are typically lower and the risk of systemic side effects is reduced. Direct delivery of siRNAs has been demonstrated in animal models to the lung,^12-15 eye,^{10, 11} and central nervous system^17-19 using naked siRNAs formulated only in saline or other simple excipients. Clinical programs are underway that use direct intravitreal injection for the treatment of age-related macular degeneration, and intranasal administration for pulmonary viral infection. However, local delivery is only possible in limited cases, and it is not clear what drives the uptake of the siRNA into the cytoplasm in these cases. Furthermore, a recent report suggests that the observed anti-angiogenic effect of naked siRNA administration via intravitreal injection is actually due to activation of toll-like receptors and not to specific knockdown of targeted genes.⁷

Delivery vehicle	Route of administration	Target tissue/ cell type	Target	Reference
Polymer Carriers/Nanoparticles
PEI	intravenous	lung (influenza A infection)	lnfluenza-NP and – PA	(25)
low molecular weight PEI	intraperetoneal	tumor xenograft	Her-2	(26)
Chitosan	intranasal	lung	GFP	(27)
Atelocollagen	intratumoral	tumor xenograft	VEGF	(28)
Transferrin targeted nanoparticles	intravenous	tumor xenograft	RRM2	(29)
Liquid-targeted stabilized nanoparticles	intravenous	tumor xenograft	VEGF-R2	(30)
Dynamic Polyconjugates	intravenous	liver	ApoB	(31)

Liposomes and lipoplexes
Liposomes and lipoplexes have been used extensively for siRNA delivery. Introduction of siRNA into cultured cells is routinely accomplished using lipid-based transfection reagents. Delivery in vivo, both locally and by systemic administration, has been accomplished in model systems by formulating siRNAs in lipid nanoparticles. Such delivery vehicles typically contain a cationic or fusogenic lipid, a polyethylene-glycosylated lipid, and cholesterol. These particles either fuse with cell membranes to deliver their siRNA cargo to cells or are endocytosed with a subsequent release of the liposomal payload from the endosome into the cytoplasm. When delivered systemically, this approach is capable of effective and long-lasting silencing of genes in the liver, at doses that are reasonable for intravenous injection.²⁰ Local delivery of lipoplexed siRNA has been demonstrated by intravaginal and intracolonic administration in animal models of viral and inflammatory diseases.²⁴

Several challenges are associated with liposomes and lipoplexes. First, some cells are refractory to this approach. Even in cell culture, some cell types, such as lymphocytes, are not amenable to lipid-based transfection. Second, when administered systemically, most of the formulated siRNA goes to the liver. Delivery to a broader range of tissues, or targeted delivery to particular cell types, may prove difficult. Antibody-decorated liposomes represent one interesting possibility.38 A recent report uses vitamin A-coupled liposomes to deliver siRNA to hepatic stellate cells.⁸ And finally, toxicity is a concern. Many cationic lipids show toxicity, both in vitro and in vivo. The challenge is to find liposomal reagents that can efficiently deliver siRNA within an acceptable therapeutic window.

Polymer carriers/nanoparticles
Nanoparticles can also be formed by complexing polymers, such as polyethylenimine (PEI), with siRNAs via electrostatic interactions.^{25, 26} Pharmacokinetic properties are often improved by incorporating polyethylene glycol (PEG) to stabilize the particle and prevent aggregation. When presented to cells, PEI-based nanoparticles associate with the cell surface electrostatically and then are endocytosed. PEI appears to lead to an increase in the pH of the endosomal compartment, leading to release into the cytoplasm. Incorporating pH-sensitive moieties into the nanoparticle can further enhance endosomal release.³¹

A potential advantage of polymers is that they can incorporate targeting molecules such as transferrin, folate, antibodies and sugars.^29-31 Successful delivery of siRNAs has been demonstrated in several disease models. Delivery has been shown for lung, liver, and tumor tissue. Toxicity is observed at higher doses; however, modifications to the polymer structure may improve the safety profile.
In addition to PEI, other polymers show promise for siRNA delivery. Included in this category are natural polymers such as chitosan and atelocollagen.^{27, 28} Chitosan has been used for delivery to lung epithelial cells following intranasal administration, and to implanted breast tumor cells after intravenous dosing. Atelocollagen has shown promise in xenograft tumor models.

Delivery vehicle	Route of administration	Target tissue/cell type	Target	Reference
Conjugates
cholesterol	intravenous	liver, jejunum	ApoB	(32)
a-tocopherol	intravenous	liver/hepatocytes	ApoB	(33)
Peptides and proteins
gp120 Fab-antibody-protamine fusion	intravenous	tumor	c-myc, MDM2, VEGF	(34)
RVG-ARG(9) peptide	intravenous	neuronal cells	GFP, SOD1	(35)
chol-(Arg9)-peptide	intratumoral	tumor tissue	VEGF	(36)
Others
PSMA aptamer-siRNA	intratumoral	prostate tumor	PLK1	(37)
Ab-decorated liposome	intravenous	leukocytes	Ku70, CyclinD1	(38)

Conjugates
Conjugation of siRNA molecules to a targeting molecule is a promising means of delivery. The conjugate is usually formed through a covalent attachment of the targeting molecule to the sense strand of the siRNA, so as not to disrupt silencing activity. Conjugation is an attractive approach because of the wide variety of potential targeting moieties, including proteins, peptides, and aptamers, as well as the possibility of using natural compounds such as cholesterol that would be expected to show low toxicity.

Conjugation to cholesterol was used to silence apoB effectively in liver and jejunum following intravenous administration in mice.³² Fairly high doses were required, but pre-complexing to serum lipoproteins led to more efficient silencing. Conjugation to ?-tocopherol has also been used to silence apoB in hepatocytes.³³

Peptides and proteins
Peptides and proteins can serve as delivery vehicles as well, and have the potential to be safe and to target to particular cell types. Conjugation to siRNAs is generally achieved by electrostatic interaction. In the case of peptides, a basic region, such as a poly-Arg stretch, is used.^{35, 36} For antibodies, conjugation to a protamine fusion protein has been used.³⁴ In the latter case, delivery has been demonstrated following intravenous administration to a number of cell types, including tumors. If generally applicable, the antibody approach could provide remarkable delivery specificity to a wide variety of cell types.

Summary
In general, the main challenges related to therapeutic siRNA delivery are finding safe ways of getting siRNAs to relevant cells and promoting effective cellular uptake and siRNA release into the cytoplasm. A single solution to the delivery problem is unlikely. A more likely scenario involves many delivery methods, each tailored to a particular cell type relevant to a particular indication using delivery systems of a modular nature with elements responsible for cell type specificity, cellular uptake/endocytosis and endosomal escape.

RNAi is among the most exciting biological discoveries of the last several decades, both from the perspective of understanding fundamental biology and, potentially, from the standpoint of creating a revolution in the treatment of disease. Advances in drug delivery are critical to fulfilling the tremendous therapeutic promise of RNA interference.

About the Author
John F. Reidhaar-Olson, PhD, is a Research Leader in RNA Therapeutics at Roche in Nutley, N.J. He received his Ph.D. from the Massachusetts Institute of Technology, and performed postdoctoral research at the University of California, San Francisco.
Hans-Peter Vornlocher, PhD, is Managing Director at Roche Kulmbach GmbH in Kulmbach, Germany, focusing on the development of RNAi Therapeutics. He obtained his Ph.D. in Biochemistry from the University of Bayreuth in Germany.

This article was published in Drug Discovery & Development magazine: Vol. 11, No. 6, June, 2008, pp. 22-25.

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