A review of recent patents concerning therapy of respiratory diseases using gene silencing by RNAi (RISC) and EGS (RNAse P).

Abstract

This article will review recent developments in the field of gene silencing as a therapy for respiratory and related inflammatory and immunologic diseases. The respiratory epithelium offers an attractive target for therapies derived from nucleic acids since the respiratory epithelium contains endogenous lipids that can facilitate uptake of polar nucleic acids and related compounds. Both RNAi (RNA Interference) in which a messenger RNA (mRNA) is targeted by an endogenous enzyme complex termed RISC (RNA Interference Silencing Complex, also previously termed RNA Induced Silencing Complex in earlier references) and also gene silencing using EGS (External Guide Sequences) in which a messenger RNA (mRNA) is targeted by an endogenous RNA enzyme termed RNase P are summarized including selected patents. The strengths and limitations of these technologies such as problems of delivery to specific tissues and potential for non-specific inflammatory response and off-targeting are compared. The possibility of therapy designed exploit synergies between both RISC and RNAse P and therapeutic benefits of inhibiting either or both pathways are also considered.

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Recent Patents on Inflammation & Allergy Drug Discovery 2007, 1, 49-55 49
1872-213X/07 $100.00+.00 © 2007 Bentham Science Publishers Ltd.
A Review of Recent Patents Concerning Therapy of Respiratory Diseases
Using Gene Silencing by RNAi (RISC) and EGS (RNAse P)
David H. Dreyfus1,* and Lucy Ghoda2
1Pediatrics, Yale School of Medicine and Medical Director, Founder Keren Pharmaceutical Inc., 488 Norton Parkway,
New Haven CT 06511, USA; 2University of Colorado School of Medicine and Webb-Waring Institute, Denver CO, and
Scientific Director, Keren Pharmaceutical Inc., 488 Norton Parkway, New Haven CT 06511, USA
Received: December 18, 2006; Accepted: December 22, 2006; Revised: December 27, 2006
Abstract: This article will review recent developments in the field of gene silencing as a therapy for respiratory and
related inflammatory and immunologic diseases. The respiratory epithelium offers an attractive target for therapies
derived from nucleic acids since the respiratory epithelium contains endogenous lipids that can facilitate uptake of polar
nucleic acids and related compounds. Both RNAi (RNA Interference) in which a messenger RNA (mRNA) is targeted by
an endogenous enzyme complex termed RISC (RNA Interference Silencing Complex, also previously termed RNA
Induced Silencing Complex in earlier references) and also gene silencing using EGS (External Guide Sequences) in which
a messenger RNA (mRNA) is targeted by an endogenous RNA enzyme termed RNase P are summarized including
selected patents. The strengths and limitations of these technologies such as problems of delivery to specific tissues and
potential for non-specific inflammatory response and off-targeting are compared. The possibility of therapy designed
exploit synergies between both RISC and RNAse P and therapeutic benefits of inhibiting either or both pathways are also
considered.
Key Words: RNAi, EGS, RISC, RNAse P, gene expression, stem cells, mRNA, gene silencing.
INTRODUCTION
This review will review recent developments in the
therapy of respiratory diseases with gene silencing using the
new technologies of RNAi and External Guide Sequences
(EGS). To facilitate the reader’s understanding of the current
state of this technology, a particular disease of clinical
importance, influenza, is discussed in detail as a means of
illustrating the current state of the art and problems
remaining. In such a rapidly developing field it is impossible
to refer to all relevant patents and references available. The
approach of this review will be to utilize the selected patents
as the basis of a discussion of two different methods of gene
silencing RNAi and EGS applied to respiratory disease,
rather than a comprehensive review of many rapidly
expanding intellectual developments in this area.
The review will cite patents selected by the editors of this
series as representative of recent patents in the subject area
of gene silencing of respiratory diseases [1-10], as well as an
additional patent obtained by the author of relevance to the
subject [11]. Relevant respiratory diseases include inflam-
matory diseases such as asthma [1,3] as well as infectious
diseases affecting the lung and upper respiratory tract such as
influenza and other respiratory viruses [3,5]. Reference is
made also to related therapy of other inflammatory diseases
such as those of the eye [4] and malignancy [2] as well as
therapy directed at altering the growth and properties of cells
[7], viral infection [6] and the nature of lipid compounds
enhancing the delivery of silencing constructs to tissues [10].
The special conditions and problems of therapy of
*Address correspondence to this author at the Pediatrics, Yale School of
Medicine and Medical Director, Founder Keren Pharmaceutical Inc., 488
Norton Parkway, New Haven CT 06511, USA; Tel./Fax: 203-777-6726;
E-mail: dhdreyfus@pol.net
respiratory diseases with gene silencing as well as
advantages of this particular organ system as a target for
gene silencing technology have been reviewed previously
and will be summarized here [3,12].
The recent discovery of gene silencing double stranded
RNA molecules termed RNAi (RNA interference or
interfering RNA) that activate a host enzyme complex
termed RISC (RNA Interference Silencing Complex also
previously termed RNA Induced Silencing Complex) has
generated tremendous scientific interest as well as meriting
the Nobel prize [13-17]. RNAi can serve as a research tool
and also may provide a new technology for translational
research and therapy of human diseases such as influenza
[18,19]. In gene silencing by RNAi a small double stranded
RNA termed siRNA (Small interfering RNA) generated
either by processing of a larger double stranded RNA
transcript or introduced as a synthetic molecule into
eukaryotic cells activates the endogenous RISC complex to
cause cleavage or other transcriptional inactivation of a
target mRNA similar or identical in sequence to the RNAi.
The concept of using endogenous enzyme complex to
silence genes is not unique or novel to RNAi however, and
in fact was first proposed as a strategy using the endogenous
RNA enzyme RNAse P and small RNA sequences termed
EGS (External Guide Sequences) as depicted in Fig. 1
[20,21]. Potential targets for EGS therapy involving the
respiratory tract have included atopic diseases such as
asthma [12] and influenza [22]. The discovery of RNase P, a
molecule that is itself composed of RNA and thus termed an
RNA enzyme also was rewarded with the Nobel Prize. Both
the discovery of RNAi and RNAse P were in turn proceeded
by studies of conventional anti-sense DNA for therapy of
respiratory and other diseases, although this approach has

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50 Recent Patents on Inflammation & Allergy Drug Discovery 2007, Vol. 1, No. 1Dreyfus and Ghoda
been limited by the low potency and specificity of
conventional anti-sense (a review of conventional antisense
technology is beyond the scope of this article and the reader
is directed to cited references) [12,23].
PRINCIPLES OF RNAi AND EGS THERAPY
The authors of pivotal studies in the discovery of RNAi
and RISC demonstrated that double stranded RNA appears
to silence genes through a mechanism distinct from conven-
tional antisense [13]. Unexpectedly, while some decrease in
gene expression of a target messenger RNA (mRNA)
resulted from antisense single-stranded RNA binding to the
target messenger RNA, much greater inhibition of gene
expression of a target mRNA was evident with double
stranded RNA containing both messenger RNA sequences
and complementary sequences. This led to the subsequent
discovery that an enzyme complex termed RISC and
associated proteins imports and unwinds short mRNA
sequences and their complementary sequences and uses the
complementary sequence as a template to cleave multiple
other copies of mRNA [17]. In invertebrates and plants other
host proteins can amplify the double stranded RNA but
RNAi amplification does not appear to occur in vertebrates
[16].
In contrast to conventional antisense technology in which
a small complementary RNA or DNA binds and inactivates
mRNA consuming both the complementary RNA and the
target mRNA, the RISC complex can cycle through many
cleavage reactions of mRNA targets without being destroyed
or consumed. Thus, RNAi is much more potent than
conventional antisense technology. A single small double
stranded RNA molecule introduced into a target eukaryotic
cell could in theory destroy all complementary target mRNA
or nearly complementary mRNA expressed in the cell for a
prolonged period of time. Because of the potential for highly
potent and specific gene inactivation a number of
translational research studies and research programs were
immediately initiated to capitalize on the potential of RNAi.
Virtually, any mRNA can be also be targeted to RNase P
by designing an EGS complementary to the mRNA target
and containing conserved transfer RNA loop sequences [20,
21] (Fig. 1). The RNase P enzyme recognizes the structure of
a transfer RNA in addition to certain highly conserved
sequences in the transfer RNA loop regions. Like RNAi,
small chemically modified EGS can be introduced into cells
using lipids as a carrier to avoid the use of retroviral
expression vectors [24]. As with RNAi the result is a
catalytic inactivation of mRNA rather than a one-to-one
inactivation typical of conventional antisense technology.
RNAi and EGS technology are variations on a common
theme of using an endogenous RNA processing pathway for
a novel use namely catalytic degradation of a target mRNA
sequence (Fig. 2).
As with all nucleic acids including conventional
antisense nucleotides, delivery of the highly charged double
stranded RNAi molecules may be problematic. Therefore
many initial translational therapies and patents involving
RNAi have focused on topical applications for example
therapy of respiratory diseases and diseases of the eye. All of
these target organs are relatively exposed for topical therapy
although in the future better delivery systems may permit
delivery of RNAi, EGS and related small nucleotides to
internal organs and tissues. In the case of respiratory tissues,
the endogenous lipids of the lung, termed surfactant are
capable of binding to nucleic acids such as RNAi and
possibly permitting their spontaneous uptake into the
respiratory epithelium and other tissues [12]. In other
epithelial tissues or tissues of the eye, specialized lipids can
be optimized for local delivery of the RNAi sequences
although this is not a trivial problem [25].
Fig. (1). As shown in the left side of the figure, the RNA enzyme RNAse P recognizes the folded structure of a tRNA molecule and certain
conserved tRNA sequences in the tRNA t-loop. RNAse P then cleaves the precursor 5' tRNA leader sequence to generate an active tRNA
shown in the conventional “cloverleaf” orientation. Cleavage of the 5' leader is required to generate mature tRNA. As shown in the right side
of the figure, an External Guide Sequence (EGS) designed to bind to a target mRNA forms a structure resembling a tRNA precursor
“cloverleaf” upon binding to a target mRNA and thus can cause site specific cleavage of the target mRNA by RNAse P. Following cleavage
of the target mRNA the EGS can diffuse to another target mRNA permitting multiple enzymatic cleavage of target mRNA per EGS.

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Recent Therapy of Respiratory DiseasesRecent Patents on Inflammation & Allergy Drug Discovery 2007, Vol. 1, No. 1 51
SIMILAR PROBLEMS OF RNAi AND EGS RELATED
TO NON-SPECIFIC EFFECTS SUCH AS INTER-
FERON-MEDIATED INATE
RESPONSES TO OLIGONUCLEOTIDES AND OFF-
TARGETING
INFLAMMATORY
Adding to the complexity of translational therapy with
RNAi, and related therapies involving small nucleic acids,
all eukaryotic cells contain receptors specific for small single
and double stranded RNA termed Toll receptors. Toll
receptors trigger an interferon-mediated inflammatory
response as part of the innate immune system. Experimental
observations have led to multiple at times conflicting
observations regarding whether therapy with RNAi might
associated with acceptable levels of nonspecific or
inflammatory responses [26-30]. In the absence of data from
clinical studies in humans these issues remain unresolved.
Possibly, these concerns may be overcome by using a correct
choice of RNAi molecule highly specific for the target
mRNA and also limiting the dose of the RNAi as well as
improving the chemistry of lipid or other carrier molecules,
as noted in the introduction as a source of patents and
potential issues of intellectual property. Clearly, the future of
RNAi as a translational therapy will depend on both the
ability to deliver the small double stranded RNA molecules
to target cells effectively while limiting nonspecific and
inflammatory effects of the therapy and compounds
facilitating delivery of small nucleic acids have been
described [10].
Conventional antisense therapies have been handicapped
by relatively low potency relative to the required inactivation
of target mRNA and nonspecific binding and irreversible
effects or toxicity due to high concentrations of conventional
antisense required to achieve measurable differences in
target mRNA expression. The specificity of RNAi in terms
of off-target effects is also complex however since slight or
even significant mismatching between mRNA targets and
other mRNA can still cause gene silencing through effects
on protein translation of mRNA as well as degradation of
mRNA by RISC [31-35].
These concerns are also relevant to the alternative
technology of gene silencing using EGS that activate a host
RNA processing complex termed RNase P [20,21]. Because
both technologies use small nucleic acids complementary to
host mRNA they are expected to have related problems
related to tissue delivery and nonspecific inflammatory
effects and off targeting. Without direct experimental
evidence it is impossible to predict which technology, RNAi
or EGS, will have less nonspecific inflammatory effects.
Synthetic EGS contain different modified bases than nucleus
Fig. (2). In this figure cleavage of the target mRNA is illustrated by a jagged arrow with the steps for cleavage by EGS and RNAse P shown
on the left and cleavage by RNAi and RISC on the right side of the figure respectively. The cleavage of target mRNA by RNAse P following
binding of EGS to the target mRNA (left side) is analogous to targeted cleavage by the RISC complex of target mRNA following binding of
the RNAi (right side). In both cases an endogenous RNA processing pathway RNAse P or RISC respectively is directed by a template
sequence to cleave and inactivate a target mRNA through design of a complementary template sequence termed an EGS or RNAi
respectively. As discussed in the text, in both cases the cleavage of mRNA targets can cycle through multiple cleavage reactions of the target
mRNA since EGS and RNAi are not consumed or inactivated by cleavage of the target. Since EGS and RNAi utilize different enzymes and
are active in different locations in the cell the potential exists for synergy or additive effects in target inactivation.

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52 Recent Patents on Inflammation & Allergy Drug Discovery 2007, Vol. 1, No. 1Dreyfus and Ghoda
resistant RNAi [24]. In addition, upon introduction into the
cell RNAi have a defined secondary structure as a double
stranded RNA, whereas the structure of an individual EGS is
not defined initially. EGS only assume a biologically defined
structure upon combining with the target mRNA and exist
prior to binding target mRNA as single stranded RNA mole-
cule that may remain single-stranded or base pair to itself
unpredictably [20]. EGS are slightly smaller than RNAi
(approximately 30 nucleotides single stranded minimal EGS
sequence) versus RNAi with a minimal size of approxi-
mately two 23 nucleotide sequences bound to each other (for
a total of at least 46 nucleotides) and the size difference
might, or might not, be important in terms of triggering
endogenous innate immune receptors for nucleic acids.
Since RNase recognizes a target of approximately 11
complementary nucleotides [20] with some cleavage of
target even with one or two mismatches in this region one
might suppose a priori that EGS will have different patterns
of target site mismatch and off targeting relative to RNAi
that recognizes a target of 22 or 23 nucleotides in the target
mRNA with also some tolerance of mismatches. However a
number of factors such as target site accessibility and degree
of mismatch tolerated preclude a conclusion without
experimental evidence as to which technology will have less
off targeting effects as well as less nonspecific inflammatory
effects [20]. It is also likely that the method of delivery such
as formation of lipid carriers will affect all of these variables
in a manner that must be determined experimentally as noted
previously.
Delivery to target tissues using viral expression vectors is
possible with both RNAi and EGS [36] in contrast to
conventional antisense thus allowing regulation of gene
silencing to turn on and off genes as needed for various
therapy applications. This could be a particularly important
application of both of these technologies for example in
design of stem cells induced to differentiate or and therapy
of malignant tissues to permit very high levels of gene
silencing RNA expression if toxic effects of RNAi or EGS
expression can be limited. The optimism surrounding RNAi
and EGS as a translational therapy, however, results from the
very high potency of this approach to gene silencing and also
the potential for reversible targeting of gene expression
which may overcome the disappointments associated with
conventional antisense therapy.
COMPARISON OF RNAi AND EGS AND POTENTIAL
FOR SYNERGY IN THERAPY
There are important differences between the biology of
the RISC complex required for RNAi and RNase P required
for processing of transfer RNA. For example all cells are
constantly making transfer RNA (tRNA) resulting in very
high levels of RNase P present in all cells, in contrast to the
RISC complex that is induced by inflammatory stimuli and
may be present at low levels or not present in some cells.
Thus it is possible that the RISC complex may be saturated
or rapidly saturated with RNAi and thus unable to target high
levels of mRNA or multiple targets as suggested in
preliminary studies in some cell types [37]. In contrast it
seems unlikely that RNase P would be saturated by multiple
target mRNA.
Remarkably, even if the delivery problems to various
tissues can be solved, the RISC complex is part of a much
larger family of enzymes termed DDE recombinases
including retroviral integrases and RNase H as well as the
RAG (Recombination Activating Gene proteins) required for
generation of immunoglobulin molecules [38]. All of these
other DDE enzymes are either present in viral pathogens
such as retroviruses or are components of the innate review
response to viral pathogens that responds to the inflam-
matory cytokines termed interferons. Thus, RNAi cannot be
viewed in isolation, but rather is part of a complex series of
enzymes involved in innate immunity and interferon-
mediated inflammatory responses. It is likely that activation
of RISC to target mRNA will have complex effects on the
innate immune system, and conversely therapy targeting the
innate immune system or antiviral drugs may also interact
with RNAi [38].
Differences in the biology of the two RNA processing
pathways may also explain the observation that the onset of
gene silencing by RNase P occurred within 24 hours of EGS
expression versus 48 hours for RNAi in one comparative
system where the two gene silencing technologies were
compared directly using an identical mRNA target [39]. In
this study both EGS and RNAi expressed from an identical
retroviral vector were equally effective in decreasing target
mRNA expression and were also both non-toxic to cells over
the several day period studied.
A problem with direct comparisons of EGS and RNAi
potency is that the target site size and consensus sequences
of the two enzymes are very different, and thus the sites are
not equivalent in terms of other factors such as accessibility
to the targeting nucleotides and intra-target base pairing [40].
RISC also forms a complex with other proteins contributing
other enzymatic capabilities such helicases that can provide
the ability to unwind intra-molecular double stranded
regions, while helicase activity is not present in RNAse P.
Like RNAi, EGS can be optimized by selection protocols to
increase potency and hence reduce non-specific effects [41-
44].
A clear advantage of RNAi relative to EGS is that EGS
can only target genes that are expressed in the cell nucleus
where RNAse P resides, probably resulting in an inability to
target some respiratory pathogens such as Respiratory Synci-
tial Virus (RSV) that do not enter the cell nucleus, while
RNAi, active in the cell cytoplasm can target RSV mRNA.
This problem could however be averted however by
designing EGS specific for the viral receptors for pathogens
such as RSV that are not accessible to RNAse P directly.
Because gene silencing with RNAi is specifically designed
for inactivation of viral pathogens and host genes RNAi
might be more potent than gene silencing with RNase P as
suggested by one study in which potency of RNAi was
compared directly with RNase P [40]. It is likely that there
may be some genes that are particularly accessible to
silencing by RISC and other genes more accessible to
silencing by RNase P related to unpredictable structural
features of target mRNA, tissue expression and tissue
specific binding factors as well as differing levels of the
silencing complexes. Thus, comparative studies need to be
repeated with other specific targets for any generalizations

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Recent Therapy of Respiratory DiseasesRecent Patents on Inflammation & Allergy Drug Discovery 2007, Vol. 1, No. 1 53
can be made regarding comparison of the two technologies
for a particular application to human disease.
Another interesting possibility is that since both RNAi
and EGS can be expressed from viral vectors or introduced
using similar lipids sequences or other delivery devices into
cells, both forms of gene silencing could be delivered
simultaneously [12]. In this way synergy between the two
technologies, the possibly higher potency of RNAi versus
the more rapid onset and higher levels of the enzyme
complex of RNase p might become evident. One could
imagine a scenario in which a mixture of RNAi and EGS
were both introduced into the pulmonary epithelium for
therapy of respiratory diseases such as influenza or asthma to
maximize the benefits of both modalities.
STATE OF THE ART CURRENTLY WITH SPECIFIC
REFERENCE TO THERAPY OF INFLUENZA USING
RNAi AND EGS
As noted above, there are many theoretical observations
suggesting that gene silencing with either RNAi or EGS may
have advantages over conventional antisense or as a novel
form of therapy in cases where existing small molecule
therapies are limited by side effects or unavailable. It will be
helpful to review the case of influenza as a respiratory
disease targeted by both silencing technologies in experi-
mental systems to illustrate the current state of the art and
results of pre-clinical studies. Current therapy for influenza
is limited to agents that either block the viral neuraminidase
such as Oseltamivir (Tamiflu, Roche, Tamiflu.com) or
amantidine and related compounds that block viral entry into
cells. Available therapeutic agents for influenza are currently
effective particularly for prophylaxis but have limitations
such as the development of resistant flu strains, and side
effects beyond the scope of this review. Vaccines for
influenza are also beyond the scope of this review but are
only effective against known strains of virus, not rapidly
emerging strains and currently must be grown in eggs, a
process requiring months. Most importantly, vaccines
require 2 or more weeks for an immune response and often a
second dose to achieve protective immunity, and thus would
be ineffective for a rapidly emerging epidemic of a novel
influenza strain.
Since influenza unlike many other respiratory viruses
such as SARS or rhinovirus replicates in the cell nucleus it
can be targeted by EGS or RNAi using these agents directly
to block expression of viral proteins. Another possibility that
has not been tested experimentally is that therapy of both
influenza and related respiratory pathogens that are not
directly targeted by EGS or not effectively targeted by RNAi
could be targeted by eliminating the inflammatory response
to the viral pathogen which in many cases resembles the
cytokine profile of posts viral asthma [5, 12].
EGS targeting conserved viral proteins were first shown
to block replication of the influenza virus using retroviral
vectors to express EGS stably in cell lines [22]. Genes
encoding polymerase binding
nucleoprotein (NP) were chosen for targeting because these
genes and the proteins they encode are highly conserved
among many influenza strains in contrast to the rapidly
protein (PB2) and
changing hemaglutinin (HA) and neuraminidase (NA) genes
and proteins targeted by vaccines and small molecule
therapies. The authors showed that cell lines stably
expressing EGS targeting either of these two proteins
exhibited significantly decreased replication of the influenza
virus and that cell lines expressing EGS targeting both
proteins almost completely abolished viral replication. No
adverse effects appeared on the cells relative to control. A
limitation of the studies were that results were not extended
to preclinical studies in animals or using nuclease resistant
small EGS that do not require retroviral vectors [24].
Currently, Keren pharmaceutical has constructed retro-
viral vectors expressing EGS demonstrated to be effective
against influenza replication in cell culture for further studies
including preclinical studies in animals. These retroviral
vectors are based on a lentivirus expression system that does
not require replicating cells in contrast to the vectors used
previously [22] and thus may more readily infect epithelial
cells in vivo. EGS expressed from lentivirus might be consi-
dered as a prophylactic therapy in veterinary applications
such as poultry, the natural host of influenza, if they are
shown to be safe and effective. Also we anticipate that if
EGS expressed from retroviruses are safe and effective in
animal models such as mouse for prophylaxis and therapy of
influenza then further studies would be justified using small
nuclease resistant EGS in preclinical studies for eventual
therapy in human subjects.
Following the discovery of RNAi, influenza was also
targeted in a series of studies [18]. Two proteins were
targeted [18] including the nucleoprotein (NP) and PA
protein (alternative nomenclature for PB protein targeted in
[22]). Following demonstration of RNAi efficacy and safety
in preliminary studies in cell culture, RNAi were
administered to mice both before and after viral challenge.
Efficacy and safety of the therapy was confirmed against a
variety of viral strains both as a prophylactic therapy and as a
post-infection therapy in mice [1,18]. Therapeutic effects of
the RNAi were also not related to nonspecific effects of
interferon and appeared to be additive because targeting both
proteins was more effective than targeting either protein
singly. Unfortunately, the administration protocol in mice
was complicated and included both hydrodynamic and nasal
administration of small nuclease resistant RNAi constructs
as well as a retroviral vector expressing the constructs thus it
is not possible to determine which of these forms of therapy
was effective.
As noted previously it would be desirable to use small
nuclease resistant RNAi or EGS constructs administered
directly to the respiratory tract for therapy in humans and it
is unlikely that humans will tolerate either hydrodynamic
therapy that involves injection of a large quantity of
nucleotide and carrier under pressure intravascularly, or
retroviral therapy with the possibility of permanent genetic
alterations. Thus these results both for EGS and RNAi must
be reviewed as preliminary although promising. These
studies also both identify the mode of delivery of the EGS or
RNAi therapy as a concern currently limiting applications in
clinical settings. Another potential area of promise is the
finding that RNAi can be expressed in the functional form