Targeted Delivery of Small Interfering RNA: Approaching Effective Cancer Therapies

Abstract

Three of the primary requirements for the development of effective dual-targeting therapeutic modalities for the treatment of cancer are the tumor-targeted delivery of the therapeutic molecules of interest to the tumor site(s) in the body (both primary and metastatic), passage of the molecular therapeutic through the cell membrane, and targeting specifically a growth or apoptotic pathway. However, lack of efficient targeted delivery, low transfection efficiency, instability to nucleases, poor tissue penetration, and nonspecific immune stimulation have hindered the translation of small interfering RNA (siRNA) into clinical applications. The development of a systemically administered, tumor-specific immunoliposome nanocomplex with high transfection efficiency could overcome these limitations and thus realize the potential of siRNAs to become effective anticancer clinical modalities.

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2008;68:1247-1250. Cancer Res
Kathleen F. Pirollo and Esther H. Chang
Effective Cancer Therapies
Targeted Delivery of Small Interfering RNA: Approaching
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Targeted Delivery of Small Interfering RNA:
Approaching Effective Cancer Therapies
Kathleen F. Pirollo and Esther H. Chang
Department of Oncology, Georgetown University Medical Center, Washington, District of Columbia
Abstract
Three of the primary requirements for the development of
effective dual-targeting therapeutic modalities for the treat-
ment of cancer are the tumor-targeted delivery of the
therapeutic molecules of interest to the tumor site(s) in the
body (both primary and metastatic), passage of the molecular
therapeutic through the cell membrane, and targeting
specifically a growth or apoptotic pathway. However, lack of
efficient targeted delivery, low transfection efficiency, insta-
bility to nucleases, poor tissue penetration, and nonspecific
immune stimulation have hindered the translation of small
interfering RNA (siRNA) into clinical applications. The
development of a systemically administered, tumor-specific
immunoliposome nanocomplex with high transfection effi-
ciency could overcome these limitations and thus realize the
potential of siRNAs to become effective anticancer clinical
modalities. [Cancer Res 2008;68(5):1247–50]
Although the use of potent, sequence-specific small interfering
RNAs (siRNA) to suppress expression of specific transcripts was
originally a useful technique for probing gene function in vitro,
their successful application in vivo in animal models against a
spectrum of diseases, including cancer (1–5), has spurred interest
in developing this approach for siRNA-based therapeutics.
However, there are still significant obstacles to be overcome before
these molecules can be used in the clinic as anticancer agents,
the focus of this minireview. Perhaps foremost among these is the
issue of delivery. The in vivo use of siRNAs effectively against
cancer hinges on the availability of a delivery vehicle that can be
systemically administered to reach both primary and metastatic
tumor cells. Moreover, because sufficient intact, functional siRNA
must be delivered into the target cell to reach an effective
intracellular concentration, and to limit potential side effects due
to randomized, general transfection of normal, nontarget tissues, it
is also crucial to develop means of directing such a siRNA delivery
vehicle specifically to the target cells.
Naked siRNAs, delivered into the bloodstream, even when
chemically modified, have extremely short half-lives (seconds to
minutes) due to renal clearance (because of their small size; ref. 6).
In addition, whereas most RNases are inactive against double-
stranded RNA (dsRNA), some serum RNases can degrade siRNA (6).
Cellular uptake of naked siRNA is also limited. To overcome some
of these challenges, siRNAs have been complexed to a variety of
nonviral lipids or protein carriers, including cholesterol, liposomes,
antibody protomer fusions, cyclodextrin nanoparticles, fusogenic
peptides, aptamers, biodegradable polylactide copolymers, and
polymers (4–7). Positively charged cationic liposomes and poly-
mers, such as polyethyleneimine, are currently the two major
carriers used to complex with negatively charged siRNA for
systemic delivery (3, 8). Although most of the reports in the
literature use delivery approaches that are not systemically
administered and/or specifically targeted to the tumor, there are
a few reports of targeted i.v. delivery of siRNA in animal models of
cancer.
The RGD peptide and transferrin (Tf), as well as antibodies
and antibody fragments [such as anti-Tf receptor (TfR) and anti–
epidermal growth factor receptor], have been used as targeting
ligands for i.v. siRNA delivery against tumors (reviewed in ref. 8).
Schiffelers et al. (9) linked siRNA against vascular endothelial
growth factor (VEGF) receptor 2 to polyethyleneimine that was
PEGylated with an RGD peptide ligand at the distal end as a
means to target tumor neovasculature. They reported inhibition
of both tumor angiogenesis and growth rate in mice bearing
murine neuroblastoma N2A tumor xenografts. Similarly, anti-
angiogenic effects were also observed in ocular neovasculariza-
tion in the herpes simplex virus disease model (10). Hu-
Lieskovan et al. (11), using a Tf-targeted, cyclodextrin-based
polycation for delivery of siRNA to tumors in a mouse model of
Ewing’s sarcoma, saw transient reduction of tumor growth.
Recently, Bartlett et al. (12) used a Tf-targeting,
64
Cu-labeled,
cyclodextrin-containing polycation to systemically deliver an anti-
luciferase siRNA molecule to Neuro2A-Luc tumor cells. Using
simultaneous positron emission tomography/computed tomogra-
phy to monitor siRNA whole-body biodistribution kinetics and
tumor localization to correlate biodistribution data with
functional efficacy, they concluded that the primary advantage
of the targeting molecule is associated with cellular uptake rather
than tumor localization. This same group had previously used Tf-
targeted polyplexes carrying an anti-luciferase siRNA to examine
the kinetics of siRNA-mediated silencing in mice bearing
Neuro2A-Luc tumors (13).
In a proof-of-principal study, Song et al. (14) reported that a
protamine-antibody fusion protein using the Fab fragment of HIV-1
envelope antibody (F105-P) as the targeting molecule i.v. delivered
FITC-labeled siRNA only to B-16 melanoma tumors modified to
express HIV env and not to normal tissues. They also used F105-P
to deliver a cocktail of siRNAs against c-myc, MDM-2, and VEGF to
mice bearing s.c. B-16 HIV env–expressing xenografts resulting in
tumor growth inhibition.
Recently, Pirollo et al. reported the development and use of a
tumor-specific, nanosized immunoliposome complex for systemic
delivery of siRNA (the scL delivery system; refs. 15–17). This
complex [TfR single-chain antibody fragment (TfRscFv)/liposome/
siRNA] is composed of an anti-HER-2 siRNA encapsulated by
a cationic [1,2-dioleoyl-trimethylammonium-propone (DOTAP)
Requests for reprints: Esther H. Chang, Department of Oncology, Georgetown
University Medical Center, TRB/E420, 3970 Reservoir Road Northwest, Washington,
DC 20057-1469. Phone: 202-687-8418; Fax: 202-687-8434; E-mail: change@
georgetown.edu.
I2008 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-07-5810
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dioleoyl phosphatidylethanolamine (DOPE)] liposome, the surface
of which is decorated with a targeting moiety, an anti-TfRscFv
(15–17). The TfRscFv contains the complete antibody binding site
for the epitope of the TfR recognized by the monoclonal antibody
(mAb) 5E9 (18, 19). TfR levels are elevated in various types of
cancer cells (20). Elevated TfR levels also correlate with the
aggressive or proliferative ability of tumor cells (20). The TfR
also recycles during internalization in rapidly dividing cells,
such as cancer cells (20), thus contributing to the uptake of
TfR-targeted nanocomplexes even in cancer cells where the
actual level of the TfR may not be elevated compared with
normal cells. Although we have also used Tf as a targeting ligand
(21), TfRscFv has certain advantages over the Tf molecule itself or
an entire mAb in targeting liposomes to cancer cells with elevated
TfR levels. (a) The size of the scFv (f28 kDa) is much smaller
than the Tf molecule (80 kDa) or the parental mAb (155 kDa).
The scFv-liposome-DNA complex may thus exhibit better
penetration into small capillaries characteristic of solid tumors.
(b) The smaller scFv has a practical advantage related to the
scaled-up production necessary for the clinical use. (c) Unlike Tf,
the scFv is a recombinant molecule and is not isolated from
blood.
To increase stability, the siRNA encapsulated in the immunoli-
posome complex in these studies was a modified hybrid form of
siRNA, a type of modification in which the double-stranded
molecule is composed of an unmodified antisense RNA strand and
a DNA sense strand that may or may not be modified. This
approach is different than the use of chemical modifications
(reviewed in ref. 8) to improve stability, reduce off-target effects,
and maintain efficacy of siRNAs. Three independent publications
(15, 22, 23) of RNA interference (RNAi) using these hybrid or
modified hybrid duplexes have reported that these antisense RNA/
sense DNA hybrid duplexes, which were called ‘‘siHybrids’’ (23),
were more potent (15, 22, 23) and led to longer-lasting (23) RNAi,
relative to corresponding unmodified siRNA. In contrast to the
trend for design of siRNA analogues wherein ‘‘more modification is
better,’’ the promising properties of siHybrids indicated that ‘‘less
may be more’’ (15). If so, this could translate into more cost-
effective RNAi by virtue of using a sense strand in which relatively
inexpensive DNA replaces more costly RNA having chemical
modifications. In addition, based on molecular ‘‘appearance’’ to
Toll-like receptors in the innate immune system (24), a dsRNA/
DNA siHybrid might be less immunogenic than a homologous
dsRNA/RNA siRNA and may also induce less of an IFN response
than dsRNA.
One way to increase the efficacy of the siRNA after the
complex has reached the target cell is to enhance siRNA release
from the endosome. Thus, more of the siRNA is available in the
cytoplasm for knockdown of the target gene rather than being
trapped in the endosome and undergoing lysosomal enzymatic
degradation. Endosomal compartments are generally acidic in
nature. Various methods, including incorporation of pH-sensitive
components into liposomes, have been developed to enhance the
efficiency of liposomal payload delivery by exploiting this fact
(reviewed in ref. 25). The selective destabilization of liposomes
following acidification of the surrounding medium with resultant
release of the payload has been enhanced by inclusion of specific
lipids, many based on phosphatidylethanolamine or modifica-
tions thereof (e.g., DOPE). These undergo a lamellar to hexagonal
phase transition at low pH, releasing the liposomal contents.
Several pH-sensitive synthetic peptides have also been designed
in an attempt to produce peptides that can attach to, but not
perturb, the surface of a liposome at neutral pH and
subsequently fuse adjacent bilayers at acidic pH. Chen et al.
(26), as well as others (27), have designed linear and branched
histidine-lysine (HK) polymers of varying lengths. The histidine
component is believed to buffer and disrupt the endosomes.
The inclusion of such HK copolymers significantly increased
the transfection efficiency over cationic liposomes alone (24). To
further increase the efficacy of the scL complex by facilitating
endosomal release while maintaining the small size of the
nanoparticle complex, Pirollo et al. (17) also included a small
linear pH-sensitive peptide, HoKC {K[K(H)KKK]
5
K(H)KKC; adap-
ted from that of Aoki et al. (27)}, in the targeted immunolipo-
some complex. This HoKC peptide contains a cysteine residue at
the end, enabling it to be conjugated to the liposome through a
maleimide group.
The results published by Chang and colleagues (15–17) show,
using scanning probe microscopy, that this single-chain targeted
immunoliposome siRNA complex is a nanoparticle of uniform size,
even when the HoKC peptide is included. Their findings also show
that modifying the anti-HER-2 siRNA through use of a modified
DNA sequence as the sense strand significantly improved the in
vitro efficacy of the siRNA compared with standard duplex siRNA
and that this approach could significantly sensitize (by over 80-
fold) pancreatic cancer cells to the standard chemotherapeutic
agent gemcitabine.
The most significant findings of these reports are the tumor-
targeting in vivo results. When systemically (i.v. tail vein)
administered, both forms of the complex (with and without
inclusion of the HoKC peptide) delivered the fluorescently
labeled siRNA specifically and efficiently to tumors. This was
observed in both large primary prostate tumors (16) and in two
metastasis models using human pancreatic cancer and human
melanoma MDA435/LCC6 (16, 17). The ability of this approach
to efficiently target and transfect metastatic lesions is shown in
Fig. 1. The metastasis indicated by the arrow displays a high
level of fluorescence with no significant signal in the adjacent
normal lung tissue. Moreover, micrometastases near the larger
nodule (verified by histology) are also detectable, indicating that
even tiny nodules composed of only a few tumor cells can also
be reached and transfected via this complex. These results
confirm the tumor-targeting ability and the efficient delivery of
the siRNA by the nanocomplex containing the pH-sensitive
peptide.
More importantly, they were also able to show that this tumor-
specific delivery via the HoKC nanocomplex resulted not only in
virtually complete knockdown of HER-2 expression in the tumors
but also in concomitant changes in expression of pAKT, pMAPK,
Bcl-2 (down-modulation), and caspase-3 (up-regulation), all genes
involved downstream in the HER-2 signal transduction pathway
and apoptotic cell death. Furthermore, the combination of this
systemically administered, tumor-specific siRNA nanocomplex and
gemcitabine was able to inhibit significantly tumor growth of
established PANC-1 xenograft tumors.
Although the field of RNAi has made the transition from basic
research to clinical application for localized disease such as
macular degeneration in less than 10 years, a time frame perhaps
faster than that of any other approach in gene medicine, the lack of
efficient targeted delivery, low transfection efficiency, instability to
nucleases, poor tissue penetration, and nonspecific immune
stimulation have hindered siRNA from reaching its full therapeutic
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potential. This is particularly true for diseases such as cancer
where systemic delivery of targeted therapeutics is essential. In
addition to identifying the correct target gene and pathway,
effective anticancer siRNA therapies must also be able to deliver a
sufficient amount of intact, functional siRNA to the target cell.
Progress is being made to address these challenges, with several
approaches for cell-specific delivery for cancer, and to increase
transfection efficiency being reported. However, the vast majority
are still only applicable in vitro or for nonsystemic administration,
including intratumoral, i.m., and i.p. injection. The delivery system
described by Pirollo et al. (17) seems to be able to overcome the
limitations of the current technology. The complex itself is of
nanoparticle size and thus able to penetrate through the small
capillaries resulting in deeper tumor penetration. The siRNA is
a modified hybrid construct that may reduce off-target effects,
whereas the encapsulation of this siRNA within the liposome can
protect it from degradation and rapid renal clearance while in the
bloodstream. This approach has shown exquisite tumor-targeting
capabilities to primary and metastatic tumors. The TfR as a target
for tumor-specific delivery has been well documented in the
literature (20). The inclusion of the anti-TfRscFv also serves to
enhance transfection efficiency, as the receptor-bound complex is
internalized via receptor-mediated endocytosis. Once internalized,
the inclusion of the pH-sensitive, endosomal-disrupting peptide in
this nanocomplex enhances release of the payload, increasing the
effective cytoplasmic concentration of the siRNA, leading not only
to the efficient knockdown of the target as observed in the tumors
but, when used in combination with standard chemotherapeutic
agents, also to tumor growth inhibition. For maximum efficacy,
the use of this targeted siRNA delivery is envisioned not as a single
agent but as part of such a combinatorial treatment regimen.
Thus, combining all of these factors in one delivery vehicle may be
the means to advance the field beyond the current challenges.
Successful translation of this approach through clinical trials is
the next logical step toward the realization of the potential of
siRNA as anticancer therapeutics.
Acknowledgments
Received 10/9/2007; revised 10/31/2007; accepted 11/4/2007.
Grant support: SynerGene Therapeutics (K.F. Pirollo), National Foundation for
Cancer Research (E.H. Chang), and TriLink Research Award in the form of research
grade siRNAs (E.H. Chang).
We apologize to our colleagues whose outstanding publications have not been
directly cited due to space constraints.
Figure 1. In vivo tumor-specific fluorescence targeting in a metastatic mouse model. MDA435/LCC6 lung metastases were induced in female athymic nude mice by
the i.v. inoculation of 8

10
6
MDA435/LCC6 cells through the tail vein of female nude mice. Eight weeks after injection, the scL-HoKC/siRNA complex carrying
9 mg/kg of modified hybrid 6-FAM siRNA was i.v. injected into the mice. Three hours after i.v. tail vein injection, the animals were sacrificed, and tumor and other
organs were excised, photographed, and examined for fluorescence using a Nikon epifluorescence stereoscope. The identical field is shown in bright field and
fluorescence views with the arrow indicating metastases. Taken from Fig. 3B in Pirollo et al. (17).
Targeted Delivery of siRNA
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