An updated review of miRNA and its uses in diagnosis and therapy

2. Micro-RNA :en route to the clinic
Prof.: Ekbal M. Abo Hashem
Professor of Clinical Pathology
Mansoura University
As Next-Generation Medicine

3. AGENDA :-Introduction
• Historical perspectives
• Nomenclature
• Role in diseases
• miRNAs as biomarkers in plasma or serum
• miRNA detection methods
• Therapeutic implications
• Future perspectives
• literature sources

4. INTRODUCTION
• MicroRNAs (miRNAs) are small non-coding RNAs
(ncRNAs) approximately 20 nucleotides (nt) in length,
that regulate gene expression post-transcriptionally
by binding to 3 untranslated regions (UTR), coding
sequences or 5UTR of target messenger RNAs
(mRNAs), and leading to inhibition of translation or
mRNA degradation.
• It is estimated that miRNAs regulate approximately
30% of the human protein-coding genome.

5. • Lin-4 was the first miRNA to be discovered, in 1993, by the joint
efforts of Ambros’s and Ruvkun’s laboratories.
• In the nematode Caenorhabditis elegans, heterochronic genes
control the temporal development pattern of all larval stages.
• One of these genes is lin-4.
• Lin-4 activity is required for the transition from the L1 to L2 stage of
larval development
5
Discovery of the first miRNA: lin-4

6. Discovery of a second miRNA: Let-7
• Likewise lin-4, let-7 is a heterochronic gene of C. elegans and was
the second miRNA to be discovered, in 2000, seven years after the
finding of the first miRNA.
• Reinhart et al. at Ruvkun’s laboratory reported that let-7 was a 21-nt
RNA controlling the L4-to-adult transition of larval development .
• Unlike lin-4, the let-7 sequence is conserved across species from
flies to humans .
• In humans, it was detected at different expression levels in the
majority of the tissues, including brain, heart, kidney, liver, lung,
trachea, colon, small intestine spleen,stomach, and thymus .

7. • The discovery that let-7 is conserved across species triggered a
revolution in the research of a new class of small ncRNAs, called
miRNAs.
Currently, thousands of miRNAs have been identified in humans
and other species, and miRNA online sequences repositories,
such as the miRbase database, are available.
Furthermore, current tools and software developed for miRNA
target prediction facilitate studies of miRNAs functional network.

8. • RNA polymerase II and appropriate transcription factors stimulate
transcription of the microRNA gene (A) into a primary long transcript with a
stem loop structure called a primary microRNA transcript (pri-miR).
• The primary transcript (B) is then processed by Drosha, an RNAase III
enzyme, to produce a small precursor hairpin microRNA (pre-miR).
• Pre-miRNAs are transported to the cytoplasm by Exportin 5 (C) for further
processing .
• In the cytoplasm the precursor microRNA is then processed into a mature
19-24 nucleotide duplex (D) by another RNAase enzyme Dicer into
miRNA:miRNA* duplexes.
Processing of microRNA

9. • Dicer also processes long dsRNA molecules into small interfering RNA
(siRNA) duplexes.
Next, the duplex is split into a primary and secondary strand (E); then the
primary strand is loaded into the RNA-induced silencing complex (RISC).
Next the microRNA with RISC targets specific messenger RNA (mRNA)
transcripts (F) at the seed region to induce either mRNA degradation (left)
or block translation (right).
depending, at least in part, on the level of complementarity between the
small RNA and its target

12. ▪ Knowledge of miRNA nomenclature is beneficial for ordering and
interpreting miRNA diagnostic tests. miRNAs are named using a
conventional prefix-name-number-suffix format (e.g. hsa-miR-1-1 or
hsa-miR-133a).
▪ The prefix hsa- (Homo sapiens) is used for all human miRNAs. The
names “miR” or “mir” refer to the mature miRNA and precursor
miRNA gene or stem-loop structure, respectively.
miRNA nomenclature

13. • Numbers (i.e. numerical identifiers) are assigned sequentially to
miRNAs in order of discovery. The suffixes -1, -2 and -a, -b
respectively indicate occasions when identical or near-identical
mature miRNA sequences are generated from two distinct
precursor loci.
Two emerging prefixes are also of interest; sequence families (sf-)
are groups of miRNAs that share the same seed sequence for
mRNA targeting and genomic clusters (cluster-) indicate groups of
miRNAs that are co-transcribed.
Knowledge of sequence families and genomic clustering is helpful
for evaluating miRNA targeting and function

14. MicroRNAs in cancer
▪ The first report suggesting a role of miRNAs in cancer was published
in 2002. MiR-15 and miR-16 were found to be located at chromosome
13q14, a region frequently deleted in chronic lymphocytic leukemia
(CLL).
▪ Both genes were deleted or downregulated in greater than 60% of B-
cell human CLL, indicating that these genes behave as tumor
suppressors in CLL
MicroRNAs in disease: a historical perspective

15. • Oligonucleotide miRNA microarrays and, more recently, deep sequencing
(next generation sequencing) have permitted the analysis of the entire
known miRNAome.
In addition, other methods such as bead-flow cytometry, quantitative real-
time polymerase chain reaction, and high-throughput array-based
enzyme assay have been used to assess miRNA expression in tumors and
other diseases.
To date, altered miRNA expression had been reported in almost all
types of cancer.
MicroRNAs can act as oncogenes(oncomirs) or tumor
suppressors and are involved in a variety of pathways deregulated
in cancer

16. Examples of oncogenic miRNAs
miR-9 AML Specifically overexpressed in MLL-rearranged
AML and promotes leukemia progression
miR-17-92 AML Up-regulated in MLL-rearranged AML and
targets p21 and RASSF2
miR-21 Breast cancer Overexpression of miR-21 contributes to proliferation and
metastasis
miR-27a NSCLC Promotes proliferation in NSCLC cells
miR-30a/c RCC Downregulation leads to increased expression of HIF2a

17. Examples of oncogenic miRNAs (cont)
miR-126 AML Up-regulated in core-binding factor (CBF)
leukemia
miR-181a/b Breast, liver and colon cancers -Promote tumorigenesis and tumor
progression
miR-196a Gastric cancer Promoted EMT, migration and invasion
miR-196b AML Upregulated in MLL-rearranged AML and targets Fas
miR-421 Gastric cancer Marker of circulating tumor cells
AML, Acute Myeloid Leukemia; NSCLC, Non-Small Cell Lung Cancer; RCC, Renal Cell
Carcinomas

18. Examples of tumor-suppressor miRNAs
miR-29b AML Represses Sp1 which resulted in c-KIT inhibition
miR-34b/c Lung cancer A positive feedback between p53 and miR-34 mediates
tumor suppression in human lung cancer
miR-126 Breast, lung, and colon cancers
plays a critical tumor-suppressor role in tumor
initiation and metastasis
miR-150 AML A critical tumor-suppressor gatekeeper in AML by targeting FLT3
and Myb
miR-155 Breast cancer Downregulates RAD51 and sensitizes cancer cells to
irradiation

19. Examples of tumor-suppressor miRNAs (cont.)
miR-181a/b AML Their increased expression is associated with good prognosis
and hinders tumor cell growth
miR-375 Breast cancer Forced expression re-sensitizes cells to tamoxifen
treatment
miR-494 Lung cancer Regulated by ERK1/2 it modulates proliferation and
apoptosis response
miR-495 AML; gastric cancer Specifically down-regulated in MLL-rearranged
AML; Shown to block migration and invasion
miR-551a Gastric cancer Forced expression leads to a block in migration and
invasion

20. • MicroRNAs deregulation can be caused by several mechanisms
including deletion, amplification, mutation, or dysregulation of
transcription factors that target specific miRNAs.
In addition, miRNAs can be controlled by epigenetic mechanisms :
DNA methylation and histone modifications.
In cancer patients, metastasis is the principal cause of death.
The metastatic process involves multiple steps: cell motility, invasion of
adjacent stroma, intravasation, systemic dissemination (through either the
blood or lymph), extravasation into the parenchyma of distant tissues, and
finally proliferation at a new site, giving rise to secondary tumor.
In this process, miRNAs have a dual role as they can promote or inhibit
metastasis

21. MicroRNAs in cardiovascular diseases.
More than 12 miRNAs were identified as deregulated during cardiac
hypertrophy and heart failure.

22. MicroRNAs in autoimmune diseases
miR-203, which is expressed in keratinocytes, is upregulated in psoriasis-
affected skin compared with healthy human skin or other chronic inflammatory
skin disease.
MiR-146 is the other miRNA that is also upregulated in psoriasis.
Increased expression of miR-155 and miR-146 was found in rheumatoid
arthritis (RA) synovial fibroblasts and RA synovial tissue,and can be used as
biomarkers for RA.
In addition, several other miRNAs have been implicated in RA, such as miR-132,
miR16 , miR-346 , and miR-223
In systemic lupus erythematosis (SLE) , 16 miRNAs were differently
expressed ,and 66 were described in lupus nephritis

23. MicroRNAs in neurodegenerative diseases
A significant number of miRNAs is specifically expressed in the central nervous
system and plays a role in neuronal development .
So , miRNAs have been linked to neurodegenerative diseases such as
Alzheimer’s, Parkinson’s and Huntington’s disease, which are caused by
excessive neuronal death in the diseased brain.
miR-9, miR-25b, and miR-128 were upregulated, and miR-124a was
downregulated in Alzheimer’s disease brain (hippocampal region) samples
miRNA modifications are linked to regulation of proteins involved in these
diseases.

24. MicroRNAs as biomarkers in plasma or serum
▪ Circulating nucleic acids can be found in blood serum/plasma, including
miRNAs
▪ These small non-coding RNAs in the blood are incorporated into
microparticles and exosomes (50- to 90-nm membrane vesicles) that
prevent their degradation, conferring an advantage to the use of miRNAs as
markers in serum.
▪ In addition, detection of miRNAs in serum is easy owing to highly sensitive
PCR detection methods, the lack of post-processing modifications of
miRNAs, and simple methods of miRNAs extraction from serum.

25. MicroRNAs as biomarkers in plasma or serum
▪ To date, miRNAs deregulation in serum of cancer patients have been
described for several cancers, including leukemia, lymphoma, and
gastric, colorectal, lung, oral and squamous cell, breast, ovarian,
prostate, pancreatic, and hepatocellular cancers.

26. “As this increasingly powerful molecular approach
matures, it is expected that miRNA-guided diagnostics
will greatly assist clinical decision-making through
quantitative detection of novel tissue-based and/or
minimally invasive biomarkers.

27. Expression profiling showed that some miRNAs, such as miR-21, are ubiquitously expressed whereas
others are specifically expressed, a key point supporting the utility of miRNA-guided diagnostics.

28. miRNAs have been considered a top candidate for
the next generation of biomarker as they possess a
few advantages over other candidates such as
proteins and metabolites .
First, miRNA biomarkers can lead to early diagnosis due to their upstream
positions in regulation cascades.
Second, novel miRNA biomarkers would be more readily discovered by
genomic tools such as oligonucleotide microarrays and deep sequencing which
deliver higher throughput than mass spectrometry, the primary tool for protein
and metabolite biomarker identification.

29. •Third, low abundant miRNA biomarkers can be amplified and then
detected in a clinical setting by real-time quantitative PCR (qPCR),
an approach used in FDA-approved clinical tests already; whereas,
no equivalent approach is available in detecting low abundant
proteins or metabolites.
The adoption of the locked-nucleic acid (LNA) technology in miRNA
probe design could improve the sensitivity and specificity of miRNA
qPCR assays even further

30. •miRNA stability in solid and liquid clinical samples should be fully investigated to
unlock their potential as highly informative tissue biomarkers and minimally
invasive biomarkers.
miRNAs are present in a wide range of fresh tissues and cells and their
expression is stable following artificial degradation (incubation at 80 C for 240
min) or prolonged storage in physiological salt solution (incubation at 4 C for 14
d) despite dramatic decreases in RNA integrity.
miRNAs are also detectable in formalin-fixed paraffin-embedded (FFPE) tissues
blocks , even after a decade of storage , with excellent correlation between
miRNA profiles generated from fresh and FFPE tissues .

31. •Remarkably, miRNAs are stable in plasma and serum .
In plasma, miRNAs are resistant to endogenous RNase activity and detectable
following extended storage or multiple freeze -thawing cycles .
In serum, miRNAs are similarly resistant to RNase digestion, boiling, low/high pH
extended storage, and multiple freeze-thawing cycles .

32. “Cell-free (cf-miRNA ) were found within
microvesicles/exosomes, apoptotic bodies (AB),HDL
structures, or complexed with AGOproteins (that
constitute the miRNA-induced silencing complex,
miRISC)), which protect them by the action of RNAses.

33. Pre-analytical variables in miRNA testing
▪ Pre-analytical variables are potential sources of inconsistency in
miRNA testing, resulting from differences in sample collection,
handling and processing, nucleic acid extraction and quality control,
and physiological variations between individuals .
▪ These pre-analytical variables can be considered as common to all
samples or specific to solid or liquid clinical specimens .

34. Common variables include differences in RNA extraction and
quality control, miRNA stability, individual variance (e.g. age and
race), and concurrent drug or medication use or chemical
exposure.
Tissue-specific variables include: specimen time ex vivo, type of
fixative, and storage conditions and inherent stability.
Liquid sample-specific variables include: specimen collection (use
of additives such as heparin), storage and stability, blood cell
count, hemolysis, plasma volume, and plasma components.

35. Optimized RNA extraction methods
▪ are required to derive maximal miRNA information content from
clinical samples.
▪ Currently, miRNAs can be extracted from a wide range of fresh and
archived, solid and liquid clinical specimens using organic extraction
(e.g. guanidium thiocyanate-phenolchloroform), filter-based spin
basket formats, magnetic particle methods, or direct lysis methods.

36. RNA quality control
▪ is essential for understanding the amount, purity, and integrity of RNA that
can be used in miRNA clinical testing.
▪ Typically, RNA quality is assessed through a combination of UV
spectrophotometry and agarose/polyacrylamide gel electrophoresis or
fluorescent dye-based quantitation.
▪ Each approach has respective shortcomings that can impact miRNA testing,
including: (i) generation of misleading absorbance readings due to the
presence of DNA or extraction reagents, and (ii) photo-degradation, binding
of the fluorescent dye to the side of the tube or to DNA in solution, or
damage to the RNA standards through repeated freeze-thawing cycles.

37. miRNA detection methods
▪ Accurate miRNA detection and/or visualization in clinical samples are core
activities in miRNAguided diagnostics.
▪ To enable these activities, sequencing-based, amplification-based, and
hybridization-based approaches have been designed, leveraging or working
around the small size sequence heterogeneity, and 50-P and 30-OH termini
of miRNA molecules.
▪ Common approaches to miRNA clinical testing include small RNA
sequencing , quantitative miRNA real-time reverse -transcription
PCR (qRT-PCR) , miRNA microarray , multiplexed miRNA detection
with color coded probe pairs , and miRNA in situ hybridization .

38. miRNA diagnostic workflow
▪ Typically, miRNAs are extracted from solid and liquid clinical specimens
using guanidium thiocyanate-phenol-chloroform, commercial filter-based
spin baskets, magnetic particles, or direct lysis methods; this step is
bypassed for miRNA visualization in tissue sections using miRNA in situ
hybridization.
▪ RNA quality control is subsequently performed using UV spectrophotometry
and agarose/polyacrylamide gel electrophoresis or fluorescent dye-based
quantitation to assess RNA yield, purity, and integrity.
▪ Next, miRNAs are detected in clinical samples using sequencing-based,
amplification-based, or hybridization-based methods followed by method-
specific data acquisition and statistical analysis

40. Comparison of miRNA detection methods commonly used in
clinical practice.
▪ Small RNA sequencing :
Sequencing-based detection Comprehensive profiling
▪ Multiplexed
▪ Quantitative
▪ High sensitivity
▪ High specificity
▪ Highly labor intensive
▪ 30 adapter ligation bias
▪ Requires bioinformatics analysis
▪ Comparatively expensive
▪ ng-mg

41. Comparison of miRNA detection methods commonly used in
clinical practice.(cont.)
▪ Quantitative miRNA real-time reverse-transcription PCR
Amplification-based detection
Limited profiling
▪ Multiplexed
▪ Quantitative
▪ High sensitivity
▪ High specificity
▪ Comparatively inexpensive
▪ Moderately labor intensive
▪ Sensitive to contaminants
▪ <ng

42. Comparison of miRNA detection methods commonly used in
clinical practice.(cont.)
▪ miRNA microarray
Hybridization-based detection
▪ Comprehensive profiling
▪ Multiplexed
▪ Relative quantitation
▪ Low sensitivity
▪ Low specificity
▪ Comparatively expensive
▪ ng-mg

43. Comparison of miRNA detection methods commonly used in
clinical practice.(cont.)
▪ NanoString nCounter expression system
Limited profiling
▪ Multiplexed
▪ Quantitative
▪ High sensitivity
▪ High specificity
▪ Comparatively expensive
▪ Emerging methodology
▪ ng

44. Comparison of miRNA detection methods commonly used in
clinical practice.(cont.)
▪ miRNA in situ hybridization
Hybridization-based detection Preserves tissue architecture
▪ Multiplexed
▪ Quantitative
▪ Labor intensive
▪ Comparatively expensive
▪ Emerging methodology
▪ N/A

45. Therapeutic implications
MiRNAs are aberrantly expressed in several diseases; therefore, these
small ncRNAs represent potential therapeutic targets for the diseases
they are functionally associated with.
MiRNAs that are upregulated in diseases should be targeted using anti-
miRNAs, which are antisense oligonucleotides with specific
modifications.
For instance, antagomirs, a class of anti-miRNAs that is cholesterol-
conjugated to facilitate cellular intake and serum protein binding, could
be used to block oncomirs in cancer .

46. In 2005, Krützfeldt et al. reported for the first time the use of antagomirs
in vivo in mammals .
Using a mouse model, Krützfeldt and colleagues systemically delivered
via intravenous injection antagomirs against miR-16, miR-122, miR-192,
and miR-194 that specifically downregulated the corresponding
miRNAs.
Silencing of miRNAs using antagomirs was long lasting, and miR-16-
antagomir effects were detected in multiple tissues, except in the brain,
possibly due to the blood-brain barrier ..

47. Other approaches to efficiently inhibiting miRNAs in vivo include
the use of locked nucleic acid (LNA) oligos or 2-O-methoxyethyl
phosphorothioate (MOE) modification.
Elmén et al. evaluated for the first time the effect of an LNA-anti-
miRNA in non-human primates, with surprising results.
These authors intravenously injected an LNA-anti-miRNA-122 into
African green monkeys and were able to efficiently silence the
mature miR-122.
The effect was long-lasting and safe .

48. MiR-122 is a liver expressed miRNA essential for hepatitis C virus (HCV)
replication.
Using an LNA-anti-miR-122, the HCV viremia was suppressed in
chronically HCV-infected chimpanzees.
Moreover, this therapy generated a high barrier to resistance, and no
side effects were detected.
In addition to these direct-inhibitory methodologies, an indirect
technology can be used through downregulation of miRNA biogenesis
pathway components.
Tetracycline-inducible shRNAs could be used to downregulate Dicer or
Drosha, key components of the miRNA-biogenesis pathway; however,
this mechanism should be tightly controlled, as downregulation of this
pathway will have an effect on all miRNAs .

49. Systemic administration of miRNAs for anti-cancer therapy was used in 2009.
MiR-26a is expressed at low levels in hepatocellular carcinoma but normally
expressed in other tissues.
An adeno-associated virus was used to mediate miR-26a delivery in a mouse
model of liver cancer and was able to reduce cancer cell proliferation and
induce tumor cell apoptosis, which consequently caused tumor regression.
Since only cancer cells present miR-26a downregulation, the delivery was highly
specific and did not affect normal tissue, which was tolerant to miR-26a
restoration .

50. With the completion of many whole genome sequencing projects , thousands of
new miRNA species were identifiable by computational prediction .
Taking a variety of factors into consideration, such as sequence conservation
and thermodynamic stability of secondary structure, researchers are now able
to identify new miRNA species that failed to be discovered by cloning
approaches.
To date, the vast majority of known miRNA species have
been discovered by bioinformatics and their sequences can
be found in the Sanger miRNA registry
(http://www.miRBase.com), an open access database for
miRNA research

51. Facilitated by high-throughput genomics and bioinformatics
in conjunction with traditional molecular biology techniques
and animal models, miRNA research is now positioned to
make the transition from laboratories to clinics to deliver
profound benefits to public health.

52. miRNA online resources
miRNA-guided diagnostics relies heavily on computational tools and online
resources .
These resources have been categorized into (i) databases and repositories of
miRNA sequences and expression, (ii) miRNA target prediction algorithms, (iii)
tools for miRNA functional investigation, and (iv) online pipelines for analysis of
high throughput experiments .

53. Useful resources include: (i) miRBase e a frequently updated miRNA sequence
and expression database containing genomic locations, precursor and mature
miRNA sequences, and helpful links , (ii) TargetScan and DIANA-TarBase
websites respectively providing predicted and experimentally validated miRNA
targets for pathomechanistic insights, (iii) DIANA miRPath v.2.0 and
miR2Disease websites respectively integrating miRNA activity into pathways
analyses or linking miRNAs to phenotypes or diseases, and (iv) miRAnalyzer for
analyzing small RNA sequencing data.

54. Kozomara A, Griffiths-Jones S. miRBase: annotating high confidence microRNAs using
deep sequencing data. Nucleic Acids Res 2014;42:D68e73. Database issue.
Renwick N, Cekan P, Bognanni C, et al. Multiplexed miRNA fluorescence in situ
hybridization for formalin-fixed paraffinembedded tissues. Methods Mol Biol
2014;1211:171e87.
Garcia DM, Baek D, Shin C, et al. Weak seed-pairing stability and high target-site
abundance decrease the proficiency of lsy-6 and other microRNAs. Nat Struct Mol Biol
2011;18(10):1139e46.
Vergoulis T, Vlachos IS, Alexiou P, et al. TarBase 6.0: capturing the exponential growth of
miRNA targets with experimenta support. Nucleic Acids Res 2012;40:D222e9. Database
issue.
Vlachos IS, Kostoulas N, Vergoulis T, et al. DIANA miRPath v.2.0: investigating the
combinatorial effect of microRNAs in pathways. Nucleic Acids Res 2012;40:W498e504.
Web Server issue.
Jiang Q, Wang Y, Hao Y, et al. miR2Disease: a manually curated database for microRNA
deregulation in human disease. Nucleic Acids Res 2009;37:D98e104. Database issue.
Langmead B, Trapnell C, Pop M, et al. Ultrafast and memory-efficient alignment of short
DNA sequences to the human genome. Genome Biol 2009;10(3):R25.

55. miRNA drug development is still in its infancy with the exception of
SPC3649, a LNA-modified oligonucleotide developed by Santaris
Pharma A/S to repress the expression of miR-122, in treating chronic
HCV infection.
This miRNA drug demonstrated impressive repression efficacy on miR-
122 in mice and in African green monkeys , as well as anti-viral efficacy
in chimpanzees chronically infected by HCV.
miRNA drug development

56. miRNA and siRNA Patents
(A)Total number of keyword searches (“microRNA” and “siRNA”) performed in
both the US patent search database and the European patent office
database.
(B)Number of patents in “microRNA” and “siRNA” in case of the different
diseases.
(C)Number of patents in “microRNA” and “siRNA” in event of the different type of
cancers.

58. List of Biopharmaceutical Companies Involved in miRNA and
siRNA Therapeutics
Different significant biopharmaceutical companies with their year of establishment since
1983 to 2017 that are involved in the development of the therapeutic miRNA and siRNA
molecules.

59. . Status of Various miRNA and siRNA Therapeutics under Clinical Trials
(Top left panel) Different siRNA molecules that are in a clinical trial and their
status of clinical trial.
(Top right panel) Different miRNAs molecules that are in preclinical and/or clinical
trial and their status of clinical trial.
(Bottom panel) Different important issues regarding development of miRNA- and
siRNA-based therapeutics molecules.

61. Significant Therapeutics siRNAs That Are in the Development Phase,
Their Indication, and their Target
ALN-RSV01
treatment of respiratory syncytial virus
(RSV) infection during lung
transplantation
RSV nucleocapsid
phase IIb clinical trial
ALN-TTR02
treatment of transthyretin-mediated
amyloidosis
transthyretin (TTR)
phase III APOLLO study
PF-04523655 (formerly
known as RTP-801i)
treatment of age-related macular
degeneration and diabetic macular
Edema
HIF-1-responsive gene

62. Significant Therapeutics siRNAs That Are in the Development Phase,
Their Indication, and their Target
QPI-1002
for the avoidance of AKI following
primary cardiovascular surgery as well as
for the prophylaxis of delayed graft
function (DGF) following deceased
donor renal transplantation
p53 Excellair
for the treatment of inflammatory
disorders like asthma
spleen tyrosine kinase (Syk)
gene
ALN-VSP for the treatment of liver cancer
VEGF gene, kinesin spindle
(KSP) protein gene

63. Significant Therapeutics siRNAs That Are in the Development Phase,
Their Indication, and their Target
Miravirsen
hepatitis C virus (HCV)
infection
phase IIa clinical trial
MRX34
for the treatment of a variety of cancers such as
colon cancer, non-small-cell lung cancer
(NSCLC), hepatocellular carcinoma, cervical
cancer, ovarian cancer, etc.
phase 1 clinical trial halted because of immune
responses
RG-101 for the treatment of HCV GalNAc-conjugated anti-miR
RG-012 for the treatment of Alport syndrome in the pipeline to initiate clinical trial phase II
MGN-1374

64. Significant Therapeutics siRNAs That Are in the Development Phase,
Their Indication, and their Target
for the treatment of post-myocardial infarction
remodeling
targets miR-15 and miR-195; it is in the preclinical
Stage
MGN-2677 for the treatment of vascular disease targets miR-143/145; it is in the pipeline
MGN-4220 for the treatment of cardiac fibrosis targets miR-29; it is in the pipeline
MGN-4893
for the treatment of disorders like abnormal red
blood cell production such as polycythemia vera
Therapeutics targets miR-451; it is in the pipeline
MGN-5804 for the treatment of cardiometabolic disease targets miR-378; it is in the pipeline
MGN-6114 for the treatment of peripheral arterial disease targets miR-92; it is in the pipeline
MGN-9103 for the treatment of chronic heart failure targets miR-208; it is in the pipeline

65. CALAA-01 to inhibit tumor and cancer therapy
M2 subunit of
ribonucleotide reductase
(RRM2) gene
Calando
Pharmaceuticals
phase 1b clinical trial; delivery system is into a
nanoparticle <100 nm in diameter
Atu-027
for the treatment of advanced solid
tumors

66. protein kinase N3 gene Silence Therapeutics phase I clinical trial
PF-655 (formerly
REDD14NP and RTP801i)
for the treatment of age-related macular
degeneration
RTP801 gene Quark Pharm phase II clinical trial
QPI-1007
treatment of nonarteritic anterior
ischemic optic neuropathy
caspase-2 gene Quark Pharm
AGN211745
treatment of age-related macular
degeneration

67. VEGFRI gene Sirna Therapeutics phase II clinical trial with 164 subjects
ApoB SNALP
for the treatment of
hypercholesterolemia
apolipoprotein B gene
Tekmira
Pharmaceuticals
phase I clinical trial concluded
RXI-109
for the treatment of fibrosis or scarring of
the skin at a post-surgical wound site or
the prevention of dermal scarring for the
management of proliferative vitro
retinopathy (PVR) and other ocular
disorders
connective tissue growth

68. factor (CTGF) gene
RXi Pharmaceuticals phase 1 clinical trial
SYL040012 ocular hypertension b2-adrenergic receptor gene Sirna
Therapeutics phase II trial completed
Bevasiranib
treatment of AMD or diabetic macular
edema
VEGF gene OPKO Health
phase III clinical trial, bevasiranib’s clinical
trial was terminated
AGN21174 age-related macular degeneration VEGFR1 gene Allergan terminated
in phase II
SPC2996 chronic lymphocytic leukemia Bcl-2 gene Santaris Pharma completed
phase II trial

69. MicroRNAs and cancer therapy
These miRNAs can enter the cell freely to inhibit downstream targets or
potentially bind to cellular receptors .
Thus, by changing miRNA expression it could change the ability of a cell to
respond to drugs either by activating resistance or bestowing sensitivity .
Through these mechanisms , miRNAs can be affected by drugs or act as a drug
themselves.

70. MicroRNAs and cancer therapy
There are multiple ways by which microRNAs can be affected by drug therapy.
Drugs can either enter through the cell membrane or bind to receptors or
cellular channels (A) to enter the cell. Once inside drugs can bind to protein
targets or transcription factors (B) to affect miRNA expression (C) or conversely
block the activation of protein targets and prevent that target from activating or
blocking a miRNA.
By driving miRNA expression this can now lead to inhibition of oncogenes (D) or
tumor suppressors (E).
Furthermore, miRNAs could potentially regulate each other (F) meaning drugs
can have multiple effects on miRNAs.

71. MicroRNAs and cancer therapy
Another possibility is that the drug can directly bind to the regulatory region of
miRNAs either inhibiting or inducing expression (G), which can then lead to
decrease of miRNA-target genes.
Drugs can also potentially bind to miRNAs themselves or to miRNA binding
partners (H) leading to a change in miRNA function.
Finally miRNAs themselves can be drugs either as modified nucleic acids or as
oligos or antisense oligos and then packaged into either viruses or microvesicles
and macrovesicles (I) .

73. Future Prospects
RNA-based therapeutics has demonstrated great promise for the treatment of
different diseases and is still evolving.
The main obstruction linked with the clinical application of RNA-based
therapeutics targeting strategies is determining how to accurately deliver
the therapeutic agents into the targeted cells.
Recent efficient delivery systems such as nanoparticle-based delivery systems
may reduce doses, which will be beneficial for treatment of different diseases
including cancer.

74. Future Prospects
Engineered nanoparticles are specially used for delivery to specific cells, which
will help to achieve this goal.
It will also help the co-delivery approach of RNA-based therapeutics with
anticancer drugs.
In addition, synchronized delivery of RNAbased therapeutics and
chemotherapy agents to the tumor cells is highly challenging.

75. Future Prospects
RNA-based therapeutics combined with conventional chemotherapy agents
might offer a new approach to treat malignant tumors in the near future and will
ultimately help to bring the RNA-based therapeutics amalgamation therapy to
the clinic for the treatment of patients.
Therapeutic miRNAs and siRNAs will enter into the clinic as next generation
drugs ,and will definitely have the potential to contribute significantly to the
future of medicine

76. The road from laboratory to clinic: the
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The hopscotch course in green is a layout of an ideal path of miRNA research
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