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MicroRNA Therapeutics
Perspectives in
MicroRNA Therapeutics
Kevin Steffy, Charles Allerson, and Balkrishen Bhat
Decades of research and development have
produced a rich, deep pipeline of preclinical
and clinical programs based on oligonucleotide
therapeutics. In particular, anti-miR therapeutics
represent an exciting opportunity in the field of
microRNA drug discovery. The authors provide
further insight into microRNA biology, and the
simplicity of anti-miR oligonucleotide drug
delivery, which can restore balance and function
to dysregulated microRNA pathways of gene
expression.
R
NA-based therapeutics hold significant potential as promising treatment options for human disease. In the past 20 years,
advances in the RNA field have identified several novel RNAbased therapies that are currently under clinical investigation,
including antisense oligonucleotides, small interfering RNA (siRNA),
and microRNA. By targeting RNA and modulating human biology at
the molecular level, these new technologies have allowed drug-discovery
efforts to focus on a broad range of disease targets once deemed to be
“undruggable.”
Leading RNA biotechnology companies have since expanded the
target space and generated multiple clinical candidates characterized
by improved target specificity, improved drug safety, and demonstrated
efficacy in patients. These companies have traditionally focused on targeting specific genes relevant to the disease indication through the control of protein synthesis at the RNA level. More recently, drug discovery
researchers are attempting to regulate entire networks of genes through
the modulation of a single microRNA. Targeting microRNAs with either
oligonucleotide inhibitors, namely anti-miRs, or miR-mimics (doublestranded oligonucleotides that replace microRNA function), provides a
novel class of therapeutics and a unique approach to treating disease by
modulating entire biological pathways (see Figure 1).
Targeting specific genes using
antisense oligonucleotides and siRNA
Kevin Steffy, PhD,* is the global alliance manager,
Charles Allerson, PhD, is the associate director
of chemistry, and Balkrishen Bhat, PhD, is the
senior director of chemistry, all at Regulus Therapeutics,
3545 John Hopkins Ct., San Diego, CA 92121, tel.
858.202.6321, [email protected].
*To whom all correspondence should be addressed.
Antisense oligonucleotides and siRNA have great potential to become
mainstream therapeutic entities. This is due, in part, to their high specificity and wide therapeutic target space in the genome. The antisense
approach targets a specific gene and interrupts the translation phase of
the protein production process by preventing the mRNA from reaching the ribosome (1). Antisense drugs are short (15–23mer) chemically
modified nucleotide chains that hybridize to a specific complementary
area of mRNA. On hybridization, the mRNA is recognized as a RNADNA hybrid and degraded through an RNase H cleavage mechanism
and not translated by the ribosome into a functional protein (see Figure
2). By inhibiting the production of proteins involved in disease, antisense drugs can create pharmacologic benefit for patients.
MicroRNA Therapeutics
RNA interference (RNAi) is a highly conserved sequence-dependent eukaryotic process for regulating gene expression. Small
stretches of double-stranded RNA ranging from 19 to 25 base pairs,
and known as siRNA, utilize the RNA induced silencing complex
(RISC) pathway to target a specific gene and bind to its homologous
mRNA. This results in site-specific mRNA cleavage and protein
degradation (see Table I) (2). The presence of the RNAi cellular
components, combined with silencing, specificity, and efficacy
makes it an attractive mechanism for targeting dysregulated gene
expression in human disease.
Targeting gene pathways using
microRNA therapeutics
More than 750 microRNAs have been identified to date, regulating
an estimated one-third of all human genes (3). Using sophisticated
bioinformatics analyses and enhanced detection methodologies,
scientists demonstrated that a single microRNA may be capable of
regulating hundreds of messenger RNAs that function in the same
or related pathways. Because microRNAs have functions in multiple
biological pathways, a change in expression or function of microRNAs might give rise to diseases, such as cancer, fibrosis, metabolic
disorders and inflammatory disorders. The demonstration that several microRNAs are up-regulated in a particular disease phenotype
provides the rationale to use anti-miR technology to restore the balance of normal gene regulation inside the cell (see Table I) (4).
Introduction to microRNAs
MicroRNAs are small noncoding RNAs that are approximately
20–25 nucleotides in length. They regulate expression of multiple
target genes through sequence-specific hybridization to the 3’
untranslated region (UTR) of messenger RNAs and block either
translation or direct degradation of their target messenger RNAs
(5). MicroRNA genes are expressed in the cell nucleus as a precursor called the primary microRNA which, upon further processing
by an enzyme called Drosha, lead to pre-microRNA (see Figure 2).
Once exported into the cytoplasm, the pre-microRNA is cleaved
2
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Figure 2: MicroRNAs are key regulators of the genome. Hybridization of
microRNAs (red) to their target seed sequence in mRNAs regulates and
directs the expression of an entire network of genes. AGO is Argonaute
protein, DGCR8 is DiGeorge critical region 8, miR is microRNA, RISC is RNA
induced silencing complex.
by the Dicer enzyme into a 20–25 nucleotide-long double-stranded
RNA that is then loaded into RISC. This process is followed by the
unwinding of the two RNA strands, the degradation of the passenger
strand, and the retention of the mature microRNA. Through the
RISC, the microRNA guides and targets messenger RNAs through
direct base pairing. The 5’ region of microRNA, also known as the
“seed” region (nucleotides 1 through 8 or 2 through 9), is the most
critical sequence for targeting and function (6). The microRNA
target sites, located in the 3’ UTR of messenger RNAs, are often
imperfectly matched to the microRNA sequence.
MicroRNAs do not require perfect complementarity for target recognition, so a single microRNA is able to regulate multiple messenger
RNAs. Although microRNAs exert subtle effects on each individual
messenger RNA target, the combined effect is significant and produces measurable phenotypic results. The ability of microRNAs to
influence an entire network of genes involved in a common cellular
process provides tremendous therapeutic potential and differs from
the specificity of today’s drugs, which act on specific cellular targets.
MicroRNAs play integral roles in several biological processes, including immune modulation, metabolic control, neuronal development,
cell cycle, muscle differentiation, and stem-cell differentiation. Most
microRNAs are conserved across multiple animal species, indicating
the evolutionary importance of these molecules as modulators of critical biological pathways and processes (3).
Anti-miR therapeutics
The association of microRNA dysfunction with disease has created enormous potential for selective modulation of microRNAs
using anti-miR oligonucleotides, which are rationally designed and
chemically modified to enhance target affinity, stability, and tissue
uptake. Aberrantly expressed or mutated microRNAs that cause
significant changes in critical biological pathways represent poten-
ALL FIGURES COURTESY OF THE AUTHORS
Figure 1: The RNA therapeutics opportunity. MicroRNAs represent
a new set of drug targets capable of regulating an entire network of
related genes.
Table I: Overview of the current RNA-based drug-discovery platform.
Technology
Compound
Target
Delivery
Mechanism
Regulus microRNA
platform
•S
ingle stranded
anti-miRs (15-19nt)
•D
ouble-stranded
miR-mimics (21-23bp)
microRNAs
•N
o DDS for anti-miRs
•D
DS required for mimics
(single strand mimic in
process)
microRNA targeting leads to
pathway modulation
siRNA
Double-stranded RNAs
(22bp)
messenger RNA
DDS required
Cleavage of a single mRNA by
RISC/AGO2
ASO
Single-stranded oligos
(15-20nt, gapmer)
messenger RNA
No DDS
Cleavage of a single mRNA in
nucleus by RNase H
tial targets whose selective modulation could alter the course of disease. From a mechanistic view, the inhibition of the microRNA target is based on the specific annealing of the anti-miR (see Figure 3).
A stable, high-affinity bond between the anti-miR and the microRNA will compete with binding to the 3’ UTR target region.
Studies by Regulus Therapeutics and others have demonstrated
that modulating microRNAs through anti-miR oligonucleotides can
effectively regulate biological processes and produce therapeutically
beneficial results in murine models of cardiac dysfunction; reducing
cancer metastases in murine tumor models; and reducing viral load
in the chimpanzee model of hepatitis C virus infection (7,8,9). Most
recently, advances in oligonucleotide chemistry have improved potency
and stability by modification with novel 2’,4’-constrained 2’O-ethyl (cEt)
nucleotides (10). The ability to achieve increased inhibitory potency with
this next generation of bicyclic nucleic acid chemistry could make a
significant positive impact on the design of anti-miR inhibitors for a
vast array of microRNA disease targets.
Anti-miR oligonucleotide drug delivery
Up until nearly a decade ago, insufficient in vivo stability, limited methods of delivery and tissue distribution of oligonucleotides hampered
successful clinical development for several promising oligonucleotide
therapeutic agents. As high molecular weight, highly charged polyanionic molecules, oligonucleotides faced many hurdles in reaching their
target organ or target cell type. First-generation antisense phosphorothiolated oligodeoxynucleotide clinical candidates administered into the
bloodstream had a low affinity for their target, poor stability because of
nuclease degradation, unfavorable immunostimulatory properties, and
rapid excretion by renal clearance, resulting in shortened half-lives (11).
To increase their metabolic stability and tissue half-life, antisense and
anti-miR oligonucleotides from second-generation nucleoside chemistries were developed that dramatically altered the pharmacokinetic
properties of these molecules (10,12).
The introduction of chemical modifications such as 2’ methoxyethyl
(MOE) and 2’, 4’-constrained 2’O-ethyl (cEt) into the ribose sugar ring
significantly improved both the pharmacokinetic and safety profile
of antisense oligonucleotides. Once delivered systemically, these second-generation compounds rapidly partition from the plasma and are
taken up by cells of multiple tissues without the need of formulation
or a delivery vehicle. Benefitting from nearly 20 years of oligonucleotide chemistry advances at companies such as Isis Pharmaceuticals,
leading developers of anti-miR therapies have garnered a tremendous
advantage in improved delivery strategies. The high water solubility of
anti-miR oligonucleotides due to their polyanionic chemical structure
has allowed anti-miR formulation in simple aqueous solutions such as
buffered saline (13). The only limiting factor is the viscosity of the solution, which is generally concentration-dependent for single-stranded
oligonucleotides (13). This simple anti-miR formulation is in contrast
to the requirements for double-stranded siRNA drug delivery, which
must fully encapsulate the siRNA in a lipid nanoparticle to systemically deliver its contents to a target tissue (14).
Anti-miR route of administration and tissue distribution
Bioavailability and tissue distribution of anti-miRs have been studied extensively in rodents and nonhuman primates. The preferred
route of administration for most therapeutic anti-miR compounds
is subcutaneous systemic delivery, because it provides efficient dissemination of the drug to different tissues including the liver, kidney
and adipose tissue without the need of a drug delivery system. Additionally, the biodistribution of anti-miRs in multiple animal species
following subcutaneous administration provides valuable information
Figure 3: Single-stranded oligonucleotide anti-miRs pharmacologically
modulate dysregulated microRNAs. The anti-miR oligonucleotide (black)
binds and hybridizes to the abnormally expressed microRNA (red), blocking
its function within the cell. DGCR8 is DiGeorge critical region, and miR is
microRNA.
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MicroRNA Therapeutics
Anti-miR delivery and function
mRNA expression profiling methods coupled with statistical techniques that can measure small changes in the expression of many
genes have become powerful tools to further our understanding
of the biological role and function of microRNAs. Relying on the
scientific findings that some microRNAs are capable of regulating
hundreds of messenger RNAs, studies were performed in mice
to determine anti-miR delivery to different cell types. Mice were
treated with a specific anti-miR (intraperitoneal injection) and
multiple cell types were harvested to for mRNA expression studies using Sylamer enrichment analysis (15). Anti-miR oligonucleotides are distributed to peritoneal macrophages as evidenced by
flow cytometry analysis and target gene up-regulation (see Figure
6). An analysis identifying an overrepresented set of genes associated with a specific anti-miR biological effect was conducted
and a data plot from the isolated macrophages was generated that
demonstrated the most up-regulated sets of genes after anti-miR
treatment. P values generated for this dataset suggest statistically
significant preferential up-regulation of genes matched to their
target sequence after anti-miR treatment.
Figure 4: Similar pattern of tissue distribution for chemically modified
anti-miRs in mouse and monkey. Anti-miR oligonucleotide quantitation
was performed on tissues by either mass spectrometry analysis or
capillary gel electrophoresis. IP is Intraperitoneal, SC is subcutaneous.
Mouse
ug Anti-miR/g tissue (μm)
400
3000
300
200
2000
100
1000
15
10
5
4
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Bone marrow
Lung
Lymph nodes
Heart
Spleen
Liver
34 mg/kg/week delivered IP
for 3 weeks
kidney
Lymph
Lung
Muscle
Heart
Spleen
Intestine
Liver
kidney
0
25 mg/kg/week delivered SC
for 6 weeks
Figure 5: The distributions of anti-miRs in mice and monkey are
highly correlated. Quantitative analysis of drug concentration by mass
spectrometry revealed a good correlation of drug tissue distribution
across multiple species.
Monkey Vs. Mouse
10000
Kidney
1000
Liver
Heart
Lymph
Spleen
100
Lung
10
1
10
100
1000
Mouse drug levels
(μg/g)
Anti-mR 1
Conclusion
Targeting pathways of human disease with microRNA-based drugs
represents a novel and potentially powerful therapeutic approach.
Recent data demonstrate not only that dysregulated microRNAs
are associated with and can cause human disease, but that selective
modulation through anti-miR intervention can provide therapeutic benefits. Anti-miR oligonucleotides can be easily administered
through local or parenteral injection routes with sufficient uptake
of the agent to achieve sustained target inhibition in tissues and
organs without the need of formulation. Improvements in antimiR chemical design and pharmacokinetic properties will allow
further exploration of microRNA biology and broaden the utility
of microRNA therapeutics.
Monkey
4000
Monkey drug levels
(μg/g)
regarding organs that may be successfully treated, as well as those
organs unlikely to be affected. Multiple studies were performed in
mice and monkeys with second generation anti-miR 1 and anti-miR 2
compounds given subcutaneously once weekly over several weeks. A
quantitative analysis of tissues demonstrated broad biodistribution of
modified anti-miRs among multiple tissue types including the kidney,
liver, lymph nodes, adipose tissue, and spleen, as demonstrated by
mass spectrometry analysis (see Figure 4). These organs have been previously shown to be target sites for oligonucleotide distribution after
parenteral administration (13). Additionally, the similar pharmacokinetics and correlated tissue distribution of each anti-miR in different
preclinical animal models provide important guidance for selection
of different disease indications and may assist in better clinical trial
designs with anti-miR therapies (see Figure 5). Effective delivery of
anti-miR oligonucleotides has also been demonstrated in different
species through multiple routes of administration including: intravenous, intraperitoneal, intratracheal, intranasal, and intracerebral. A
more detailed analysis of anti-miR tissue distribution using quantitative whole body autoradiography to provide additional quantitative
information is in progress.
Anti-mR 2
References
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2. S.M. Elbashir et al., Nature 411 (6386), 494–498 (2001).
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8. L. Ma et al., Nature 28 (4), 341–347 (2010).
Figure 6: Functional drug delivery of anti-miRs in mouse peritoneal macrophages. Flow-cytometry studies (a) and gene-regulation studies (b)
demonstrate the internalization of anti-miR and target engagement in macrophages (Sylamer analysis). Potential targets containing heptamer 1-7
(GCATTAA) or heptamer 2-8 (AGCATTA) are enriched. X-axis is ranked genes by fold change. Y axis is -log (P-value enrichment). PBS is Phosphatebuffered saline.
PBS
CD11b
78.17
2.67
30.98
103
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100 19.14
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100 13.24
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0.02
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(b)
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3’ - UGGGGAUAGUGUUAAUCGUAAUU-5’
54.47
1.31
102
103
MFI
Cy3
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(a)
20
GCATTAA
AGCATTA
15
10
5
0
-5
-10
0
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20000
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Messenger RNA
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Boca Raton, 2007), pp. 183–215.
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Properties of Phosphorothioate 2’-O-(2-Methoxyethyl)-Modified Antisense Oligonucleotides in Animals and Man,” in Antisense Drug Technology: Principles, Strategies, and Applications, S.T. Crooke, Ed. (CRC Press,
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