Pharm Res

DOI 10.1007/s11095-017-2134-2

EXPERT REVIEW

The Race of 10 Synthetic RNAi-Based Drugs

to the Pharmaceutical Market
Ricardo Titze-de-Almeida 1 & Catherine David 2 & Simoneide Souza Titze-de-Almeida 1

Received: 22 November 2016 / Accepted: 27 February 2017

# Springer Science+Business Media New York 2017

ABSTRACT Ten years after Fire and Melo’s Nobel Prize for biological barriers), RNAi definitively opens a wide avenue for
discovery of gene silencing by double-stranded RNA, a re- drug development.
markable progress was achieved in RNA interference
(RNAi). Changes in the chemical structure of synthetic oligo- KEY WORDS miRNA . pharmaceutical market . RNA
nucleotides make them more stable and specific, and new interference . RNAi . siRNA
delivery strategies became progressively available. The atten-
tion of pharmaceutical industry rapidly turned to RNAi, as an
opportunity to explore new drug targets. This review ad- ABBREVIATIONS
dresses nine small-interfering RNAs (siRNAs) and one unique LNA Locked nucleic acid
microRNA (miRNA) inhibitor, which entered the phase 2–3 LNP Lipid nanoparticle
clinical trials. The siRNAs in focus are PF-04523655, TKM- LODER LOcal Drug EluteR
080301, Atu027, SYL040012, SYL1001, siG12D-LODER PEG Polyethylene glycol
(phase 2), QPI-1002, QPI-1007, and patisiran (phase 3). PLGA poly(lactic-co-glycolic) acid
Regarding miRNAs, their content can be down- or up-regu- RISC RNA-induced silencing complex
lated, by using miRNA inhibitors (AntimiRs) or miRNA RNAi RNA interference
mimics. Miravirsen is an AntimiR-122 for hepatitis C virus SNALPs Stable nucleic acid lipid particles
infection. The flexibility of RNAi technology is easily under-
stood taking into account: (i) the different drug targets (i.e.
p53, caspase 2, PKN3, β2-adrenergic receptor, mutated INTRODUCTION
KRAS, microRNAs); (ii) therapeutic conditions, including
ophthalmic diseases, kidney injury, amyloidosis, pancreatic RNA interference (RNAi) therapeutics comprise a relatively
cancer, viral hepatitis; and (iii) routes of administration (ocu- new class of drugs that are about to reach the pharmaceutical
lar, intravenous, subcutaneous, intratumoral). Although some market. All these drugs present a chemical structure of short
issues are still matters of concern (delivery, toxicity, cost, and chains of nucleotides that essentially differ in their sequence of
puric and pyrimidic bases (1).
While proteins are the most common targets for traditional
medicines like β-adrenergic antagonists for glaucoma treat-
* Ricardo Titze-de-Almeida ment, the messenger RNA (mRNA) is the target of RNAi-
ricardotitze.unb@gmail.com based drugs. In this sense, siRNA SYL040012 will counteract
β2-adrenergic activity by cleaving its mRNA, thus reducing
Catherine David
http://biotika.com.br
receptors that stimulate aqueous humor production in the
eye (2). RNAi, post-transcriptional gene silencing or knock
1
Technology for Gene Therapy Laboratory, ASS 128, ICC Sul, University
down are synonymous for a process of mRNA downregula-
of Brasília–UnB, Campus Darcy Ribeiro, FAV., Brasília, DF 70910-970, tion that directly affects the respective protein level (Box I) (3,
Brazil 4). Proteins comprise the vast majority of pharmacological
2
Av. Pedro Severino Junior, 366, sala 45 e 46 – Vila Guarani, São drug targets, meaning that RNAi holds an inherent broad
Paulo, SP 04310-060, Brazil therapeutic potential. Finding the correct target for knock

down is the first technological challenge; delivery is the sec-
ond, not the less important.
Many aspects of RNAi, including origins, cell pathways,
and biological roles are issues revised elsewhere (5–8). This
review focuses on ten synthetic molecules that take advantage
of RNAi pathways, which are shortly described in Fig. 1. Nine
of them are small-interfering RNAs (siRNAs) that are du-
plexes of 19–23 ribonucleotides with sense (passenger) and
antisense (guide) strands (Fig. 2a). The duplex is incorporated
into a complex of proteins named RNA-induced silencing
complex (RISC) that will first remove the passenger strand.
A seed region of the remaining guide strand will look for
complementary nucleotides in mRNA sequence and bind to
them by hydrogen bonds (9, 10). Then RISC complex will
execute cleavage of the mRNA found by the guide strand
(Fig. 1a) (11, 12). Finally, a single RNAi drug is a microRNA
inhibitor to miR-122 (AntimiR-122). AntimiRs are antisense
oligodeoxynucleotides targeted to specific miRNAs, thus
abolishing its role in disease pathogenesis (see Fig. 1b).
The present work reviews preclinical and clinical studies on
ten synthetic RNAi-based drugs that entered phase 2–3 of
human clinical trials registered in U.S. National Institutes of
Health - NIH (Table I). The small-interfering RNAs (siRNAs)
are PF-04523655, QPI-1002, QPI-1007, TKM-080301,
Atu027, SYL040012, SYL1001, patisiran, and siG12D-
LODER. A microRNA inhibitor for hepatitis C virus infec-
tion named miravirsen is also part of this review. These oligo-
nucleotides are candidates to inaugurate a new class of thera-
peutic agents in the pharmaceutical market.
SIRNAS IN CLINICAL STUDIES REGISTERED IN
NIH
Pf-04523655 (Pf-655)
PF-04523655 is a siRNA designed to silence RTP801 in indi-
viduals with neovascular age-related macular degeneration
(AMD). Patients with choroidal neovascularization secondary
to AMD and diabetic retinopathy overexpress RTP801 as an
hypoxia-inducible gene, which plays a role in disease patho-
genesis (13–15). Regarding siRNA structure, PF-04523655 is
a duplex of 19 ribonucleotides, stabilized by 2′-O-methylation
that introduces a 2′-methoxy group in ribose (2′-O-Me), as
shown in Fig. 2b (16, 17). Studies of RNA silencing were
performed by intravitreal (IVT) injection (18). It is the same
procedure as for ranibizumab (Lucentis®), a standard therapy
for AMD based on the inhibition of vascular endothelial
growth factor (VEGF) (19). Even being a naked siRNA, PF-
04523655 showed the ability to penetrate retinal cells without
help of any transfection reagent and to silence RTP801 in
assays in vitro and in vivo (20). Also in a pharmacokinetic study,
Titze-de-Almeida, David and Titze-de-Almeida
PF-04523655 injected by IVT route shows plasmatic levels
within a few hours (18).
PF-04523655 was well-tolerated in preclinical tests with
rats and cynomolgus monkeys (18). Local adverse effects were
only detected at the highest IVT injection of 3 mg/eye in
monkeys. The first phase 1 human clinical trial
(NTC00725686) included subjects that received increasing
doses in single IVT injection of PF 04523655 and were follow-
ed up for 24 months. This dose-escalation study evaluated the
safety, tolerability, pharmacokinetics, and dose-limiting toxic-
ities. For safety reasons, the study was organized in two strata
with specific end points. In the first one, the drug was admin-
istered to individuals legally blind (21) at doses of 0.05–3 mg
(n = 3 individuals per dose). They presented neovascular
AMD unresponsive to prior treatment and a best corrected
visual acuity (BCVA) ≤ 20/200. After the first safety results of
PF-04523655 in phase 1, regulatory authorities approved fur-
ther trials and the sponsor tested efficacy in phase 2. It includ-
ed 27 patients with potential to benefit from therapy using
doses ≥1 mg from end point 1; BCVA between 20/200 and
20/800. For dose-escalation, individuals received the next
higher dose of PF-04523655 provided no dose-limiting toxic-
ity had been detected. The study found at least one adverse
event in ≈ 70% of individuals, but no dose-limiting toxicity.
Undesired reactions were mild to moderate and not related to
PF-04523655. Only one report of retinal pigment epithelial
detachment could possibly be related to PF-04523655
(1.5 mg), in a patient that started receiving ranibizumab in
the study eye. Part of the drug injected into the eye was
absorbed into systemic circulation. IVT injection of PF-
04523655 in doses ≥0.4 mg produced detected plasma levels
at 1, 4, and 24 h post injection. However, all tested doses were
below the lowest limit of quantification by day 14. The max-
imal concentration in plasma was between 1–4 h after injec-
tion. The highest median plasma concentration was 2.86 ng/
mL at 4 h after IVT injection of 2.25 mg of PF-04523655. In
summary, this study shows that a single IVT injection of PF-
04523655 ≤ 3 mg was safe and well-tolerated in patients
with AMD. Although the main focus of this phase 1
study was toxicity, it also evaluated a possible efficacy
of PF-04523655 in the end point 2. Unfortunately, a
single IVT injection of PF-04523655 had no benefit in
visual acuity or retinal thickness (18).
The first phase 2 clinical trial on PF-04523655, the Monet
study (NCT00713518), evaluated the efficacy of PF-
04523655 in monotherapy and in association with
ranibizumab for AMD treatment (16). All individuals first re-
ceived ranibizumab 0.5 mg at a baseline. The study tested the
following conditions: 1. Two doses of PF-04523655 in mono-
therapy, every 4 weeks for 8 weeks (1 mg or 3 mg); 2. PF-
04523655 in monotherapy, every 2 weeks for 8 weeks
(3 mg); 3. An association of PF-04523655 + ranibizumab
(0.5 mg), every 4 weeks for 8 weeks (1 mg or 3 mg). The
RNAi-based drugs in phase II – III clinical trials

Fig. 1 Synthetic RNAi-based drugs modulate RNAi pathways. (a) siRNA pathway. First, double-stranded RNA (dsRNA) molecules from infecting pathogens or
cellular genes are processed by a ribonuclease enzyme Dicer, which interacts with TRBP and PACT, forming short dsRNA with ≈ 22 nucleotides and two
nucleotide overhangs at 3' end – siRNAs. Synthetic siRNAs can also be introduced into cells (red box). Regardless their origin, siRNAs will be incorporated into a
complex of proteins: RNA-induced silencing complex (RISC). The endonuclease Argonaute 2 (AGO) removes passenger (sense) strand of the siRNA, allowing
antisense or guide strand to bind to fully complementary sequences in the mRNA coding region (CDS). In summary, siRNAs conduct the active RISC to specific
mRNA for cleavage by AGO enzyme. (b) miRNA pathway. MicroRNAs (miRNA) are coded by genes present in mammalian DNA. At the top, RNA polymerase
II (Pol II, in brown) will transcribe miRNA genes in long RNA sequences of hundreds of nucleotides. Transcripts will form a peculiar shape of a hairpin. Each hairpin
is a duplex of ≈ 70 partially complementary ribonucleotides that ends with a short non-complementary sequence (loop). Except for the hairpin, miRNA has an
mRNA shape. miRNA is processed in cell nucleus where it is 7-methyl-guanosine capped (m7G, filled red circle) in 5' end and polyadenylated at 3' end, forming
the primary miRNA (Pri-miRNA). Drosha and DGCR8 enzymes will cleave RNA sequences outside the hairpin to form miRNA precursor (Pre-miRNA), further
exported to cytoplasm by Exportin 5. As for siRNA pathway above described, Dicer enzyme will process the Pre-miRNA into a miRNA with 18 – 25 nucleotides.
The miRNA duplex associates with the RISC (miRISC), sense strand is removed, and the guide strand binds complementary sequences in target mRNAs. The
difference with siRNA is that miRNAs binds to several mRNAs with partially complementary pairing, and target sequences are placed in 3'-UTR region. The
absence of a full complementarity between miRNA and mRNA sequences compromises AGO endonuclease activity. In consequence, effect is a translational
repression or mRNA degradation instead of cleavage of mRNA by AGO. Synthetic miRNAs (miRNA mimic) or antisense sequences that inhibit specific miRNAs
(AntimiR) will artificially modulate the miRNA content (orange boxes). AGO Argonaute 2, CDS Coding sequence region of mRNA, DGCR8 DiGeorge syndrome
critical region gene 8, Dicer a ribonuclease enzyme, Drosha a ribonuclease enzyme, miRISC miRNA duplex associated with the RISC complex, PACT Protein
activator of the interferon-induced protein kinase (PKR), Pol II RNA polymerase II, RISC RNA-induced silencing complex, TRBP HIV-1 Trans-activation response
(TAR) RNA-binding protein.

control group was treated with only ranibizumab (0.5 mg). All
monotherapy protocols with PF-04523655 were less effective
than ranibizumab. Although not statistically significant
(P = 0.33), treatment with PF-04523655 + ranibizumab
showed a higher improvement of BCVA values (9.5 letters)
in comparison with ranibizumab alone (6.8 letters). This asso-
ciation, however, was not superior to ranibizumab in reducing
the central subfield retinal thickness and total subfoveal cho-
roidal neovascularization (CNV) area. The same occurred for
decreases in lesion thickness. Finally, some adverse reactions
 Titze-de-Almeida, David and Titze-de-Almeida

Fig. 2 Schematic structure of a siRNA duplex and chemical modifications in oligonucleotides. (a) A typical 21-mer siRNA with its annealed sense and antisense
strands, and two overhangs nucleotides. RNA duplex is dismounted in cell cytoplasm by RISC complex, and the sense strand is eliminated. The antisense strand
will guide the RISC complex to the target mRNA for cleavage. (b) Schematic changes in RNA-backbone. At the center, an unmodified ribose for comparisons.
Letter B represents a nucleotide base. Ribose modifications ring include fluorine (2'-F), methoxy (2'-OMe), locked nucleic acids (LNA) and unlocked nucleic acids
(UNA). Phosphodiester bond can be substituted by phosphorothioate. Chemical modifications of siRNA nucleotides improves nucleases resistance and reduces
immunogenicity and off-target silencing, not affecting efficacy.

not related to PF-04523655, mild to moderate in severity,
occurred in the study eye (4).
Matisse study was the third phase 2 clinical trial and fo-
cused on Diabetic Macular Edema - DME (NCT01445899).
This study evaluated the efficacy and safety of PF-04523655,
in monotherapy or in association with ranibizumab. It was
organized in two strata. The first analyzed safety, dose-
limiting toxicity, and pharmacokinetics of a single IVT injec-
tion of PF-04523655 in subjects with low vision. The second
evaluated the safety, tolerability, and ability to improve visual
acuity. For the latter, patients with DME received 6 monthly
IVT injections of PF-04523655 alone or associated with
ranibizumab. Anatomical changes in the retina and retinal
nerve fiber layer morphology were also examined. This study
is completed, but no results have been published yet.
also reduces costs. A significant limitation of PF-04523655 is
efficacy as the siRNA was not superior to the reference drug
ranibizumab (anti-VEGF) in patients with AMD. PF-
04523655 has only shown a tendency to increase the visual
acuity when administered in combination with ranibizumab,
without improving eye disease histopathology. Despite the
interest of Pfizer that get a license for the technology, it may
be difficult to convince reimbursement organizations to pay
for, as a current standard treatment for AMD gives reasonably
good results. However, the use of PF-04523655 can be fa-
vored whether it is confirmed that anti-VEGF agents present
a potential risk for geographic atrophy progression in patients
with AMD (22–24). Finally, there is an expectation about a
phase 2 study on PF-04523655 for patients with another eye
disorder, DME.

Critical Analysis QPI-1002 (I5NP)

PF-04523655 uses the intravitreal route of administration, a
standard procedure as for ranibizumab. This route will re-
quire an ophthalmologist assistance for the dosing regimen,
every 2–4 weeks for eight weeks, or 6 monthly injections.
Regional administration typically brings a significant advan-
tage: the dose is reduced making the treatment cheaper. PF-
04523655 in fact required 1 or 3 mg per administration, an
amount relatively small compared to other drugs of this re-
view. Indeed, it was not formulated in nanoparticles which
QPI-1002 was designed to protect proximal tubule cells in
kidney injury associated with ischemia or nephrotoxicity.
Acute kidney injury (AKI) is a new term for acute renal failure,
a clinical syndrome characterized by a rapid decrease in renal
excretory function (25). AKI is an important health problem
due the incidence, mortality rate, and risk for patients to de-
velop a chronic and dialysis dependent disease (26, 27). The
death of renal cells by apoptosis plays an important role in
AKI pathogenesis, regardless its etiology was ischemia, sepsis,
RNAi-based drugs in phase II – III clinical trials
or a nephrotoxic injury (28). Thus, the inhibition of apoptotic
pathways in injured renal cells would be of value in AKI. The
siRNA in focus, QPI-1002, was designed to silence the pro-
apoptotic p53 protein in proximal tubule cells (PTC), thus
reducing the ischemia-reperfusion injury. It is a naked,
blunt-ended 19-base-pair duplex, and 2′-O methylated (29).
QPI-1002 was the first siRNA systemically administered to
humans, given by the intravenous route (I.V.). Intravenously
injected siRNAs have a peculiar pharmacokinetics: they are
internalized mainly by kidney and liver cells (30). This pattern
of distribution was confirmed in various studies with RNA
duplexes and, in addition, with single-stranded antisense
DNA oligonucleotides (31–35). As the kidney is a natural tar-
get for I.V. injected oligonucleotides, kidney diseases caught
the attention for siRNA-based therapeutics.
Molitoris et al. (2009) executed the first preclinical study on
QPI-1002, testing its efficacy in ischemic and chemotoxic
models of AKI. First, the potential reduction of ischemic inju-
ry of kidneys was evaluated in rats submitted to bilateral renal-
clamps. Animals received I.V. injections of 1 mg/Kg each of
QPI-1002, before (−2 h, −30 min) and after (+4 h, + 8 h) the
injury (29). Following the expected pattern of distribution,
QPI-1002 concentrated in PTC cells. Serum creatinine levels
of ischemic rats decreased from 3.7 mg/dL to 1.9 mg/dL in
the siRNA-treated group, showing the improvement of renal
function (p < 0.01). Apoptosis of PTC cells caused by ischemia
was reduced. In addition, this p53-targeted siRNA attenuated
kidney injury in the model of aortic clamp. Rats have received
QPI-1002 via I.V. 4 h after a 60 min of 90% suprarenal aortic
clamp. QPI-1002 also protected renal function from the inju-
ry caused by the chemotoxic agent cisplatin (7.5 mg/Kg, in-
traperitoneal route). For that, the animals received three
siRNA injections (12 mg/ Kg), 30 min before and on
days two and three after cisplatin administration (29).
Indeed, QPI-1002 showed efficacy in rats subjected to
renal transplantation (36).
An extensive pharmacokinetic and toxicological testing on
QPI-1002 was executed in rat and nonhuman primates to
support the first-in-man clinical trial (37). The study first used
naive male cynomolgus monkeys. They received 4 separate
I.V. doses of QPI-1002, at Day 4 (5 mg/Kg/dose) and Day 11
(25 mg/Kg/dose). The authors opted for 4 doses to provide
flexibility for further tests in human clinical trials. Animals
were examined for general health, food intake, cardiovascu-
lar-, respiratory-, and nervous system activity, as well as serum
chemistry and hematology. For sub-acute toxicity, monkeys
received 3, 12, or 48 mg/Kg/dose by I.V. route (brachial
veins). For acute toxicity, animals received a single injection
of QPI-1002 (200, 500, or 1000 mg/Kg). This study also
tested QPI-1002 in rats, as a second species, for sub-acute
and acute toxicity and pharmacokinetics. In sub-acute toxicity
tests, the animals received QPI-1002 (3, 15, or 60 mg/Kg/
dose) or a rat analogous named QM5 (15 or 60 mg/Kg/dose)
via tail vein. Sub-acute toxicity of QPI-1002 (10 mg/Kg) was
also tested in nephrectomized rats, as a model of renal impair-
ment. Acute toxicity test in rats included single I.V. injections
of QPI-1002 (200, 800, 1200 or 2000 mg/Kg) or QM5
(1200 mg/Kg). Finally, the study evaluated QPI-1002 in ge-
netic toxicity assays. Plasma concentration of QPI-1002 after
I.V. injection decreased rapidly, >90% in 30 min post-dosing
(200 mg/Kg). The drug was predominantly distributed to the
kidney, as expected. This finding agrees with in vivo imaging by
two-photon microscopy that showed fluorescent labeled
siRNAs in kidney tissue. About renal cells, QPI-1002 accumu-
lates mainly in PTC, with a peak at 5–30 min post-injection.
The levels decreased in 2 h, and became undetectable after
24 h. This data agreed with a previous work that found a
decrease of p53 mRNA content in PTC for 24–48 h post-
injection of QPI-1002 (29). The temporary and site-specific
effect of this siRNA is a valuable strategy to control the apo-
ptosis of tubular cells soon after an acute injury. Afterwards,
p53 expression will return to the basal level and cells with
irreversible damage could be removed. QPI-1002 caused no
adverse effects on the cardiovascular, respiratory and central
nervous systems. Toxicity was found only for doses ≥800 mg/
Kg in rats and ≥1000 mg/Kg in monkeys. QPI-1002 was not
genotoxic.
The first human phase 1 clinical trial on QPI-1002
(NCT00554359) was carried on patients undergoing cardio-
vascular surgery. Those individuals received a single I.V. in-
jection of QPI-1002 at 4 h after removal of the cardiopulmo-
nary bypass machine. No results are available but the drug
had to be well-tolerated, as the study advanced to a phase 2
clinical trial (NCT02610283). The study will evaluate the ef-
ficacy and safety of QPI-1002 in patients (n = 340) who are at
high risk for AKI following cardiac surgery. This clinical trial
is now recruiting participants.
QPI-1002 could also protect renal cells in patients that
receive kidney transplants. Those patients can develop a form
of AKI related to the transplant and named delayed graft
function (DGF) (38). It occurs due the ischemia-reperfusion
injury, and may result in renal damage. Patients will show
post-transplantation oliguria, with an increased risk for allo-
graft rejection and lower long-term survival (39). Thus, Quark
performed a clinical trial phase 1/2 with patients undergoing
deceased donor kidney transplantation (NCT00802347).
Besides drug safety evaluation in phase 1, the study also ex-
amined whether QPI-1002 would protect transplanted pa-
tients from DGF. The clinical trial had two main arms. Part
A was a dose-escalation study to find out the maximum toler-
ated dose of QPI-1002 (0.5 mg/Kg, 1.5 mg/Kg, 5 mg/Kg, or
10 mg/Kg). Part B evaluated safety and efficacy of QPI-1002,
at the dose 10 mg/Kg identified in part A. In this phase 2
study (NCT00802347, QRK.006B), the drug was adminis-
tered in a single I.V. dose, at 30 min after reperfusion.
Individuals were organized in groups, according to the donor

and method for tissue preservation. Patients (n = 331) were
from 52 transplant centers in North America and Europe (40).
The relative risk of DGF was defined as the need for dialysis
within the first 7 days post-transplant, excluding dialysis in the
first 24 h because of hyperkalemia/hypervolemia. As cited
above, patients received a single I.V. injection of QPI 1002
(10 mg/Kg) at 30 min after reperfusion. The intended end
point of a 30% decrease in relative risk of DGF in QPI-1002-
treated individuals was not achieved for the total population,
as it reached 15%. However, QPI-1002 could reduce by
30.5% this relative risk of DGF in patients of the group named
ECD/CS (expanded criteria donor/cold-stored) (n = 177). A
similar effect occurred in patients that received kidney grafts
from older donors (˃ 35 or 45 years old). Positive changes in
dialysis after transplant was another benefit of QPI-1002,
found in ECD/CS group. It extended the time for the first
dialysis in the first post-transplant month. QPI-1002-treated
patients also showed a lower number of dialysis sessions (6.0 vs.
11.2) and a shorter first course of dialysis (13.4 vs. 25.3 days).
Finally, patients treated with QPI-1002 showed a tendency to
need 1.5 fold less dialysis sections in the 6-month study period
of follow up (375 vs. 561, p = 0.059). Regarding safety, Quark
reported a similar safety profile between QPI-1002-treated
and placebo groups (40). In 2010, the company established
an agreement with Novartis for the development and com-
mercialization of QPI-1002 (41).
Quark is recruiting patients for a phase III clinical trial on
QPI-1002 (NCT02610296, QRK306), with an estimated en-
rollment of 634 individuals. The ReGIFT study will evaluate
whether this drug reduces DGF in patients that receive kidney
allografts from donors ˃ 45 years after brain death.
Critical Analysis
QPI-1002 addresses AKI, a severe renal disease that may
require dialysis - a significant constraint and expensive pro-
cess. The therapeutic rationale is to suppress the pro-apoptotic
p53 protein to reduce the death of renal cells, thus preserving
the kidneys physiology. The intended endpoint of 30% reduc-
tion in DGF - a form of AKI related to transplants, was met
for a subgroup of individuals and not the entire population.
Indeed, QPI-1002 showed a tendency to reduce the need for
dialysis in patients with kidney transplants. Thus efficacy is a
critical point for QPI-1002. In contrast, this siRNA holds
many positive properties: (i) as a naked siRNA, it can be a
relatively cheaper drug; (ii) treatment is via I.V. route, com-
monly used in hospitals; (iii) I.V. route favors the distribution
of QPI-1002 to the target renal cells; (iv) only a single admin-
istration of QPI-1002 (10 mg/Kg) is needed, resulting in a
total amount of ≈ 700 mg for a patient with 70 Kg. If ongoing
phase III gives positive results, the orphan drug status can
speed-up access to the pharmaceutical market. In this sense,
Titze-de-Almeida, David and Titze-de-Almeida
Novartis has invested 10 million USD for development and
commercialization of QPI-1002 (41).
QPI-1007
The loss of retinal ganglion cells (RGC) is a hallmark of
ophthalmic diseases that result in visual impairment and
blindness like nonarteritic anterior ischemic optic neu-
ropathy - NAION and glaucoma (42–44). As apoptosis
is a key event in RGC loss, proteins involved in apo-
ptotic pathways are attractive targets for therapeutics.
Caspase-2 is a protein expressed by RGC during injury,
and treatments which inhibit this pro-apoptotic protein
have provided neuroprotective effect (45–47). QPI-1007
is a caspase-2-targeted siRNA developed to protect
RGC in optic neuropathies, by IVT injection (48). It
is a naked RNA duplex of 19 nucleotides, with 2′-O-
methylated sugar nucleosides in the guide strand.
Passenger strand contains chemical changes at both
ends, as shown in Fig. 3a. One L-DNA cytidine nucle-
otide placed before the last nucleotide at 3´ end, and an
inverted deoxyabasic moiety at 5′ end, represented by
the letters BY^ and Bia^, respectively (49). QPI-1007
guide strand will form Watson & Crick’s base pairs with
nucleotides 240–258 of human caspase-2 mRNA se-
quence (transcript variant 2, NM_001224.4; Fig. 3b), a
key step in the siRNA pathway explained in Fig. 1. The
rat orthologous mRNA has an identical 5′-
gccagaatgtggaactcct −3′ target sequence in positions
251–269 (NM_022522.2; Fig. 3b). It allowed the use
of identical siRNAs in both species.
QPI-1007, then named siCASP2, has shown ability to si-
lence caspase-2 expression in cells in culture (48). It caused
80% and 65% knock-down of caspase-2 mRNA in human
Hela cells and rat PC12 cells, respectively. siCASP2 resisted
to nuclease attack in serum and vitreous humor during 24 h at
37°C, without causing immune or off target effects. Most
RGC had internalized siCASP2 in few hours, as revealed by
Cy3-labelled siRNAs. Finally, the most important result –
siCASP2 reduced RGC apoptosis in two rat models of acute
optic nerve (ON) injury. In the model named ONC (optic
nerve crushed), the ON of both eyes are crushed infraorbital
to transect all RGC axons, causing a high cell loss at 7 days.
siCASP2 caused a significant dose-dependent protection of
RGC at doses of 5, 10 and 20 μg per eye. Also, doses of 20
and 35 μg keep RGC density in injured eyes to values close to
intact retinas RGC density (≈98%). Single IVT dose of
siCASP2 is able to protect RGC cells during 7 days post-
ONC. The duration of effects lasted for up to 30 days in
animals that received 2 IVT injections of 20 μg siCASP2, at
Days 0 and 10 after ONC. This study also used the model of
optic nerve transection (ONT), based on the axotomy of all
RGCs of the left eye. The procedure causes a more aggressive
RNAi-based drugs in phase II – III clinical trials

Fig. 3 QPI-1007 and mRNA target sequences. (a) A schematic representation of QPI-1007 duplex. Passenger (5'→3') and guide (3'→5'). Uppercase letters
represent unmodified nucleotides. Chemical changes are: (i) 2'-O-methyl sugar modified RNA nucleosides, represented by lowercase letters; (ii) L-DNA cytidine
nucleotide, represented by Y; (iii) inverted deoxyabasic moiety, represented by ia. (b) Complementarity between QPI-1007 guide strand and the human and rat
caspase-2 mRNAs NCBI reference sequences, NM_001224.4 and NM_022522.2, respectivelly. Target sequences for QPI-1007 in caspase-2 mRNA are
represented in red (5'→3'), while QPI-1007 guide strands are written in orange (3'→5'). QPI-1007 molecular sequence was previously published.

loss of cells. Animals received 2 doses of siCASP2 (10 μg), the
first immediately after ONT and the second at Day 7 post-
ONT. SiCASP2 caused a 15% increase in RGC survival
compared to the control siRNA at 2 weeks after injury (48).
Indeed, Vigneswara and Ahmed (2016) recently found that
siCASP2 IVT injected every 8 days could protect RGC cells
for 12 weeks after ONC (50).
A preclinical study evaluated the pharmacokinetic and tox-
icological properties of QPI-1007, to support drug testing in
human trials (49). Studies were carried on rabbits and rats.
Preclinical studies on rabbits evaluated ocular and systemic
effects of single or repeated bilateral IVT injections. Doses
and intervals for QPI-1007 IVT injections varied according
to the experimental assays. For pharmacokinetics, single doses
of 0.3 mg/eye or 3 mg/eye, and time points from 1 to 336 h
post QPI-1007. Single-dose or repeated-doses of 0.3, 1, or
3 mg/eye were tested for toxicity. In rats, QPI-1007 was ad-
ministered by I.V. route via tail vein at single-doses (4, 20, or
100 mg/Kg) or repeated ones (10 or 75 mg/Kg/day, for
28 days). Genotoxicity tests, bacterial mutation assays, chro-
mosome aberration test, and micronucleus evaluation were
carried on. QPI-1007 presented a half-life (t1/2) of 2 days in
vitreous and retina/choroid in the rabbit. Repeated IVT in-
jections every 2 or 4 weeks for up to 11 doses caused no
accumulation. QPI-1007 was chemically stable in vitre-
ous, and was eliminated by a slow egress to blood,
following by a rapid clearance via renal filtration. No
systemic or genetic toxicity was observed. The QPI-
1007-related adverse effect found was only a mild inflam-
matory cell infiltration in vitreous (49).
In 2010, Quark started the first clinical trial on QPI-1007
(NCT01064505). A phase 1 clinical study on safety, tolerabil-
ity, dose-limiting toxicities, and the pharmacokinetics of a sin-
gle IVT injection was executed. Recruited patients presented
either a chronic optic nerve atrophy - Blegally blind^ in the
study eye (stratum I) or a recent onset of NAION disease, with
diagnosis at ≤28 days (stratum II). As a secondary end point,
the study also evaluated if QPI-1007 causes anatomical
changes in the optic nerve head and retina or affects visual
acuity and visual field. Quark reported no dose-limiting tox-
icity in 5 of 6 cohorts of patients with chronic optic nerve
atrophy (51). QPI-1007 was well tolerated also in patients with
NAION. No serious adverse event was found, no dose limiting
toxicities and no discontinuation due to adverse effects oc-
curred. Patients treated with QPI-1007 (51.7%, n = 29)
showed improvements in visual acuity, as defined by an in-
crease in BCVA values ≥3 lines (≥ 15 letters) at month 3.
Indeed, none of the NAION patients treated with QPI-1007
lost 3 or 2 lines of visual acuity at months 3 and 6, compared to
9.1% and 14.8% in historical controls. Drug was able to
change the natural history of disease (52). The company is
now recruiting participants for a phase 2/3 clinical trial on
 Titze-de-Almeida, David and Titze-de-Almeida

Fig. 4 Schematic representation of a stable-nucleic-acid-lipid particle (SNALP). These particles of < 200 nm in size have provided an efficient delivery system for
siRNAs administered by intravenous route. SNALPs resemble the well-known liposomes, as they are vesicles with an outer lipid bilayer enclosing a water
compartment inside. SNALPs present some valuable improvements for in vivo studies. Negatively-charged siRNA molecules (the small double-stranded
structures, in orange) are incorporated into the aqueous inner part with the help of cationic lipids (1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane,
DlinDMA). Helper lipids with a yellow head (1,2-distear-oyl-sn-glycero-3-phosphocholine, DSPC) promote the release of siRNAs from SNALPs in cell
cytoplasm. Polyethylene glycosylated lipids (PEG–C–DMA) offers an external surface more hydrophilic and neutral, for stability and crossing barriers.
Cholesterol helps to stabilize the lipid particle formulation. Reprinted with permission from Reference (56). Copyright (2012), Elsevier. DSPC 1,2-distear-oyl-
sn-glycero-3-phosphocholine, PEG–C–DMA Polyethylene glycosylated lipids, PEG–C–DMA Polyethylene glycosylated lipids.

efficacy and safety of QPI-1007 in ≈530 patients with recent-
onset of NAION (NCT02341560).
Finally, QPI-1007 was tested in subjects with acute attack
of primary angle-closure glaucoma (APACG)
(NCT01965106). This phase 2 study evaluated the safety,
tolerability, and pharmacokinetics of a single IVT injection
of 1.5 mg QPI-1007. The clinical trial also examined the
prevention of damage to nerve tissue in the glaucomatous
eye and changes in visual function.The study finished in
July 2015 and no results are available yet.
check whether the benefits were statistically significant. From
a market point of view, QPI-1007 may be of value for other
eye diseases that result in loss of visual acuity and blindness (as
glaucoma, for example), as it prevents apoptosis of retinal
cells. Indeed, Quark was granted a patent from the United
States Patent Trademark Office on the use of QPI-1007 for
treatment of patients with NAION (53). The protective effects
of this siRNA on retinal cells and consequent ability to im-
prove visual function must be confirmed in a phase 2/3 trial in
patients with NAION.

Critical Analysis
QPI-1007 followed the same strategy of QPI-1002, as it sup-
presses an apoptotic protein involved in cell death. Compared
to QPI-1002, QPI-1007 is a siRNA with much more chemical
changes for improving its knocking-down action. The target is
caspase 2, an enzyme that triggers apoptosis of retinal cells in
ischemic optic neuropathies like NAION. The drug is injected
by IVT route, like PF-04523655, thus requiring an ophthal-
mologic service. Although QPI-1007 improved visual acuity
and stabilized the disease progression, the data was compared
only with historical controls. A phase 2 controlled study must
TKM-080301 (TKM-PLK1)
TKM-080301 targets polo-like kinase 1 (PLK1), a protein
overexpressed in cancer cells promoting an uncontrolled cell
proliferation (54). Some PLK-1 inhibitors are already in hu-
man clinical trials for anticancer therapy (55). Regarding
TKM-080301, it is a siRNA for I.V. administration formulat-
ed in stable nucleic acid lipid particles (SNALPs, as shown in
Fig. 4) (56, 57). The drug is developed by Tekmira (BTKM^
from siRNA designation TKM-080301), a company that
changed name to Arbutus Biopharma Corp. in 2015.
RNAi-based drugs in phase II – III clinical trials
Preclinical characterization of TKM-080301 included
in vitro and in vivo pharmacological activity studies, effects on
immune system and toxicity (58). The drug showed antipro-
liferative activity on cancer cell lines and ability to silence
PLK-1. TKM-080301 also presented antitumor activity in
xenograft models of human cancer. Effects were confirmed
by histopathology and molecular methods, showing PLK1
mRNA cleavage products in samples from treated animals.
A single dose of TKM-080301 caused PLK1 silencing that
persisted for 7–10 days. No measurable immune activation
was found. Toxicity was restricted to liver and spleen, without
myelosuppressive effects. Preclinical data highlighted PLK1 as
a promising anticancer target and supported the clinical eval-
uation of TKM-080301.
The first phase 1/2 clinical trial of TKM-080301
(NCT02191878) evaluated safety and toxicity. Primary out-
come measures were the maximum tolerated dose (MTD) and
dose-limiting toxicities (DLT), as determined in a dose escala-
tion study. Sequential cohorts of 3 to 6 patients, with either
advanced solid tumors (i.e. advanced hepatocellular carcino-
ma - HCC) or lymphomas, received escalating doses of TKM-
080301 (0.15 to 0.90 mg/Kg) in cycles of 28 days of treatment
(59). In each cycle, the siRNA was administered as a 30-min
IV infusion once a week for three consecutive weeks (at days 1,
8, and 15) followed by a 1-week rest period. Common adverse
effects were rigors (33%) and fever (25%) (60, 61). Mild to
moderate delayed infusion-related reactions were observed.
Some cytokines were upregulated, like interleukins (IL) IL-6,
IL-8, and monocyte chemoattractant protein-1 (MCP-1) at
doses ≤0.75 mg/Kg/week (59). DLT occurred in two patients
at 0.9 mg/Kg/week with adverse grade 3 reactions. Thus
MTD was defined as 0.75 mg/Kg/week, and this dose was
adopted for subsequent studies. The authors concluded the
siRNA was generally well-tolerated. Secondary outcomes in-
cluded evaluation of pharmacokinetic parameters and a pre-
liminary antitumor activity. Pharmacokinetic studies in cycles
1 and 2 revealed a dose proportional Cmax (maximum plasma
concentration) and AUC (area under the curve) and no accu-
mulation. Even 6–8 cycles of TKM-080301 has caused no
cumulative toxicity in two individuals. TKM-080301 present-
ed a preliminary antitumor efficacy (59). Clinical benefits were
found in 4 of 9 (44%) patients treated with ≥0.6 mg/Kg
TKM-080301 (61). An expansion cohort (n = 10 patients)
was enrolled in 2013 aimed to explore pharmacodynamic
data and antitumor activity from biopsy samples at previously
determined phase 2 dose, the MTD of 0.75 mg/Kg/week
[(61); NCT01262235]. Authors compared biopsy specimens
(for example, same lesion of the right lobe liver) pre- and two
days post-TKM-080301 dose 1. They found a 476-bp PLK1
mRNA cleavage product after treatment, by using 5′-RACE
(rapid amplification of cDNA ends) polymerase chain reaction
(PCR). Predicted cleavage PLK-1 amplicons were observed in
agarose gel electrophoresis and confirmed by sequencing (61).
Another clinical trial evaluated safety and antitumor activity
of TKM-080301 in patients with refractory adrenocortical
cancer (ACC). The study used treatment cycles described
above, and the anticancer activity was evaluated by
RECIST 1.1 (Response Evaluation Criteria in Solid Tumors
1.1) every two cycles (62). The study enrolled 16 subjects that
received 0.6 or 0.75 mg/kg/week TKM-080301 for up to
18 cycles. Four out of eight patients that received at least
two cycles of treatment showed an improvement of stable
disease. One of them presented a 13% decrease in tumor
diameter. Indeed, a metastatic intraperitoneal non-
functional ACC showed a tumor reduction of 19% after cycle
2 and 49% after cycle 14. Some serious adverse effects caused
by TKM-080301 were ECG T-wave inversion, musculoskel-
etal pain and infusion reaction. Despite adverse reactions, this
study revealed that: 1- TKM-080301 was generally well tol-
erated at 0.6–0.75 mg/Kg for up to 18 cycles and; 2- the
siRNA presented an antitumor activity in patients with refrac-
tory ACC (62).
Critical Analysis
Proposed regimen for TKM-080301 (18 cycles X 3 injections
per cycle X 0.75 mg/Kg) results in a high amount of siRNA of
≈ 2800 mg, but it shows antitumor activity in about 50% of
patients with advanced hepatocellular carcinoma or refracto-
ry adrenocortical cancer. As PLK1 is deregulated in various
types of cancer, this siRNA may have an increased market in
the future. However, PLK1 is a druggable target. PLK inhib-
itors already in clinical trials (like volasertib in phase 3) would
be potential competitors of TKM-080301 (63). Regarding
adverse effects, patients showed mild to moderate infusion-
related reactions with cytokines upregulated - a common con-
sequence of lipid-carried siRNAs administered by I.V. route.
The cost of therapy could be acceptable if phase 2 and 3
confirms impressive preliminary results.
ATU027
Atu027 is a chemically synthesized siRNA designed to silence
protein kinase N3 (PKN3) mRNA. PKN3 is a downstream
effector of the phosphoinositide 3-kinase (PI3) signaling cas-
cade in highly vascularized tissues (64, 65). Acting as an
Bendothelium-modulating^ drug, it stabilizes vessel integrity,
counteracts inflammation in tumor region, and reduces the
spread of metastatic cells (66, 67). The target is the PKN3-
mRNA sequence 5′-agacuugaggacuuccuggacaa-3′ (65, 68). It
is a double-stranded 23-mer nucleotide, blunt-ended, and
chemically stabilized by alternating 2′-O-methyl-nucleotides.
Atu027 is formulated in lipid particles (siRNA-lipoplex,
Atuplex®) for systemic administration and delivers PKN3-
targeted siRNAs into the cytoplasm of vascular endothelial
cells in vivo. The lipoplex contains positively charged liposomes

to which the negatively charged siRNAs are attracted and
form complexes (69). The four components of Atu027 are
the following: 1- PKN3-targeted siRNA described above; 2-
cationic lipid AtuFECT01; 3- neutral helper lipid; 4- a
pegylated lipid (65, 69).
Preclinical studies examined the antitumor efficacy of
Atu027. This siRNA was intravenously administered to mice,
rats and cynomolgus monkeys, causing an RNAi-mediated
knock-down of PKN3. Regarding anticancer effects, it re-
duced tumor growth and lymph node metastasis in orthotopic
mouse models (65). The authors found a significant reduction
in lymph vessel density, but not in microvascular density.
Atu027 also prevented the formation of lung metastasis, as
found in two experimental mice models (66).
The first-in-human phase 1 clinical trial evaluated the safe-
ty, tolerability, pharmacokinetics, pharmacodynamics, and
anticancer activity of Atu027 on single and repeated infusions
in patients with advanced refractory solid tumors
(NCT00938574) (67). Patients first received the drug as a sin-
gle treatment, followed by a 3-week safety period for clinical
examinations. Then eight infusions twice per week over a 28-
day cycle were administered. Each lyophilized Atu027 dose
was diluted in 5% xylitol, and was administered via I.V. route
over four hours. Cohorts of patients (n = 3 to 7) were exam-
ined for drug safety before dose escalation, i.e., three patients
per dose level extended by at least another three for definition
of MTD (the B3 + 3^ design scheme). The study enrolled
thirty-four patients with advanced refractory cancer for testing
10 escalating doses of Atu027 (0.001 – 0.336 mg/kg) (67).
Regarding pharmacokinetics, I.V. administration of Atu027
caused a rapid increase in siRNA plasma concentration dur-
ing the 4 h infusion, followed by a steep decrease. Maximal
siRNA level (Cmax) was 180 ng/mL, achieved for Atu027 dose
of 0.336 mg/Kg at 2 h post injection (Tmax). Half-life values
for single and eighth repeated siRNA injections were 16 h and
10 h, respectively. Regarding toxicity, MTD was not achieved
as only one patient showed a grade 3 adverse reaction possible
related to Atu027 at the highest dose (0.336 mg/Kg). This
siRNA was well-tolerated; less than 10% adverse events were
grade 3 or greater, and most of them resulted in complete
recovery. They were not related to escalating dosage. About
immune response commonly found for particulate com-
pounds, this study showed a dose-dependent increase in C3
protein that is a marker of complement activation. No clini-
cally significant changes were found for interferon alfa, IL-1β,
IL-6, tumor necrosis factor α, and MCP-1. Also, no antibodies
against siRNA Atu027 molecule were detected. Finally,
Atu027 was effective in 52% patients (14 of 27). They showed
disease stabilization after repeated treatment (week 8), ac-
cording to RECIST criteria. Eight of them still had a
stable disease at week 12. Some patients also showed
less metastatic lesions (i.e. lung, lymph node, liver, bone)
after Atu027 treatment (67).
Titze-de-Almeida, David and Titze-de-Almeida
Another study with Atu027 was a phase 1/2 clinical trial
(NCT01808638). This work evaluated whether Atu027 could
be administered in association with the standard chemother-
apeutic gemcitabine for patients with advanced or metastatic
pancreatic adenocarcinoma. The clinical trial evaluated two
dose regimens of Atu027. In arm 1, the 28-day cycle was
composed by weekly injections of Atu027 and gemcitabine
for three consecutive weeks (days 1, 8, and 15) followed by a
week with no treatment. Patients received a total of six admin-
istrations of this siRNA in 8 weeks. Cycles were repeated,
except if toxicity or disease progression occurred. Patients en-
rolled in arm 2 also received gemcitabine once weekly (days 4,
11, and 18), but Atu027 was given twice weekly (days 1, 4, 8,
11, 15, 18, 22, and 25). Thus they received eight doses (33%
more Atu027 than patients of study arm 1). In this arm 2, the
combination cycle was followed by a 28-day treatment with
only gemcitabine. The company reported Atu027 was well
tolerated in combination with gemcitabine for patients with
pancreatic cancer. A higher dose of Atu027 tested in study
Arm 2 provided a longer duration of progression-free survival
(PFS) rate (5.33 months versus 1.81). The difference was statis-
tically significant for patients with metastatic pancreatic can-
cer. Overall survival (OS) was also higher in patients of study
arm 2 in comparison with those of arm 1 (70).
Critical Analysis
Treatment of pancreatic cancer remains a great challenge in
oncology (71). Atu027 is also a formulated anticancer siRNA,
with anti-angiogenic and anti-metastatic properties. It re-
quires a smaller dose (0.336 mg/Kg) and fewer injections
(n = 8) than TKM-080301, resulting in a lower total dose of
≈ 190 mg (8 injections X 0.336 mg/Kg X 70 Kg). I.V. injec-
tion increased C3 proteins that indicate an immune activa-
tion. Pancreatic cancer is a very aggressive and metastatic
disease (72). Thus Atu027 was associated with gemcitabine,
which improved the progression-free survival (PFS) rate in
patients with advanced- and metastatic pancreatic adenocar-
cinoma. This siRNA is a promising alternative for patients
with incurable pancreatic carcinoma to be examined in a
further phase 3 clinical study.
SYL040012 (Bamosiran)
SYL040012 is a siRNA targeting β2-adrenergic receptor, de-
signed for glaucoma treatment (2). Glaucoma is an ophthal-
mic disease that causes visual impairment, leading to blindness
as a result of progressive optic nerve damage (73). The main
risk factors for glaucoma are elevated intraocular pressure
(IOP), genetic and ethnic backgrounds, and age (74). IOP is
caused by an unbalance between production and drainage of
the aqueous humor (AH) (75). Drugs that reduce AH produc-
tion and control IOP are valuable for patients with glaucoma.
RNAi-based drugs in phase II – III clinical trials
β-blockers produce this effect and can be prescribed for this
disease (76). Instead of blocking β-receptors proteins, another
strategy is to knock-down the corresponding mRNA by RNAi.
SYL040012 is a naked 21-base synthetic siRNA targeting the
β2-adrenergic receptor mRNA.
In preclinical testing, SYL040012 (100 nmol/l) reduced
50% of β2 receptor mRNA content in cells in culture at
24 h – 48 h after transfection (2). siRNA showed no significant
effects on other adrenergic receptors, β1 or α1B, revealing its
strict specificity. This naked siRNA rapidly enters the eye and
reaches various structures, including producing HA like ciliary
body. Fortunately, only low levels of SYL040012 duplex (not
biologically active) reached the plasma and systemic organs.
The ocular route is advantageous for administering siRNAs;
the first RNAi drugs entering clinical trials attended ocular
diseases (77–79). Eyes are anatomical structures placed exter-
nal and apart from the body. Administrating drugs topically
by eye drop solutions reduces their distribution to the systemic
circulation. The amount of drug administered is lower com-
pared to other routes of administration. Finally, the immune
responses are relatively lower in the eyes, reducing the risk of
undesired reactions against therapeutic nucleotides (80).
The study cited above also measured the efficacy of
SYL040012 for IOP reduction in New Zeland White
Rabbits (2). The authors first tested the model with two drugs
used for glaucoma treatment, dorzolamide and latanoprost.
Both successfully reduced IOP for six hours. These drugs,
however, are not β-blockers, limiting the informative val-
ue of this test. Rabbits present β2-receptors widely distrib-
uted in ocular tissues, mainly in conjunctival, corneal, and
ciliary process (81).
In SYL040012 preclinical testing, a topical dose of
20 nmol/day for four consecutive days caused ≈ 22%
decrease in IOP in healthy animals. The drug also showed
efficacy in a glaucoma rabbit model named Boral water
overloading^. Animals first received 40 nmol/eye/day also
for four consecutive days, and then ocular hypertension
was induced by water loading. SYL040012 caused a sig-
nificant reduction in IOP compared to the PBS-control
group. As expected, the content of β2 receptor mRNA
was reduced in lacrimal glands and ciliary body at 24 h
after the last SYL040012 dose. Safety was tested in
cynomolgus monkeys. They received topical ocular instilla-
tion of SYL040012 (72, 144, and 288 nmol/eye/day) dur-
ing a period of 28 days. Clinical and histopathological
examination revealed the drug was safe and well tolerated
(2).
The first human phase 1 clinical trial on SYL040012
(NCT00990743) evaluated the safety, tolerability, and bio-
availability in thirty healthy volunteers with IOP below
21 mmHg (82). The effect of different siRNA doses on IOP
was also followed. The drug was instilled as eye drops in one
eye, and the untreated fellow eye served as a control. In the
first study interval, Interval 1 (n = 6 individuals), SYL040012
was instilled in a single dose of 600 μg/eye/day. Only after the
tolerability criterion (i.e. absence of grade 3 or higher toxicity)
was meet at 72 h, the next individual was tested. In the
Interval 2, the drug was given in daily instillations over seven
consecutive days. Two doses tested were 600 and 900 μg/
eye/day (n = 12 individuals each dose). Ophthalmic tolerance,
adverse effects, vital signs (cardiovascular and respiratory pa-
rameters), clinical biochemistry, pharmacokinetic from blood
samples, and IOP at various time points post instillation were
measured. As a result, no ocular pathological changes were
observed at any time. Single and repeated administrations of
SYL040012 were safe and well tolerated. Six mild adverse
events were observed, and half of them considered unrelated
to the drug. SYL040012 was not detected in blood samples
after a single or repeated administration. For IOP, the repeat-
ed dose schedule of Interval 2 reduced IOP in 15 of 24 healthy
subjects. After four days of administration of 600 μg/eye/day,
SYL040012 caused an overall statistically significant reduc-
tion in IOP. The effect was more prominent in individuals
(n = 5) with higher basal IOP values.
Three other clinical trials were also completed, as follows:
(i) Phase 1/2 evaluated tolerance and drug efficacy in unmed-
icated patients with elevated IOP (NCT01227291). (ii) Phase
2 study of tolerability and effects of SYL040012 on IOP of
patients with ocular hypertension or open-angle glaucoma
(NCT01739244). SYL040012 was instilled daily over 14 days
to 89 patients, organized in groups receiving the following
doses: 80 μg/eye/day (0.2%), 300 μg/eye/day (0.75%),
900 μg/eye/day (2.25%), or placebo (83). The drug was well
tolerated. Adverse reactions occurred in 14.6% of the pa-
tients, and most of them were mild. SYL040012 caused a
significant reduction in IOP at the dose 300 μg/eye/day, in
comparison with placebo group or the basal values (i.e. IOP
curve obtained in screening period). (iii) Phase 2 clinical trial
named SYLTAG evaluated four doses of SYL040012 eye
drops, in comparison with timolol maleate 0.5%, for lowering
IOP in patients with open-angle glaucoma (NCT02250612)
(84). Effects of each siRNA concentration on IOP and the
local and systemic safety were evaluated after a 28-day treat-
ment. This multicenter study enrolled 184 patients, that were
randomized into groups receiving 150 – 600 μg of
SYL040012 daily and 0.5% timolol twice a day. All treat-
ments reduced IOP at Day 28 with the highest effect - com-
pared to the baseline – occurring for SYL040012 300 μg. In
the total population, SYL040012 had not achieved the objec-
tive of non-inferiority compared to timolol (85). However, in a
subgroup of patients with IOP >25 mmHg, SYL040012
450 μg was non-inferior to timolol. Regarding toxicity, no
grade 3 or 4 adverse reactions occurred in the 28-day treat-
ment period. Furthermore, adverse events were less frequent
in all SYL040012 groups (8% - 12%) than in timolol group
(24%) (84).

Critical Analysis
SYL040012 addresses glaucoma, a disease currently treated
with eye drops containing β-receptor antagonists, prostaglan-
dins, and others. Prostaglandin analogs have been preferen-
tially prescribed compared to β-blockers due to the safety pro-
file and patient compliance (76). The rationale of SYL040012
as a selective β2 suppressive drug is interesting regarding safety
since non-selective blockers like timolol are relatively contra-
indicated for patients with cardiac problems (86). There is a
risk of systemic absorption via the nasolacrimal system and
following side effects from blockade of β1 heart receptors
(87). However, suppression of β2 receptors by SYL0400012
might cause bronchoconstriction in patients with asthma and
chronic obstructive pulmonary diseases (88). Fortunately, the
phase 1 study did not detect the drug in blood samples, prob-
ably due to the rapid attack of plasmatic ribonucleases on a
siRNA molecule that was not protected either by chemical
changes (i.e. phosphorothioate linkages) or a nanoparticle. In
fact, SYL0400012 caused less adverse events than timolol.
This siRNA showed activity in comparison with placebo.
However, it was not equivalent to timolol except in patients
with high IOP, which reduces the potential market for a pro-
spective drug. The total amount of 13 mg (28 daily doses X
450 μg per dose) is relatively high for an eye drops siRNA.
Thus the cost-benefit relation is problematic. From a general
view, SYL0400012 was the first siRNA tested in humans for
topical use in eyes, bringing a contribution for the biotechnol-
ogy of RNAi. At that time, chemical changes in siRNA mol-
ecules that increase both duration of activity
(phosphorothioated backbones that avoid ribonuclease attack)
and target specificity (LNA, locked-nuclei acids) were not yet
available. Even nanocarriers for in vivo use were in their infan-
cy. The question is how much this biotechnological platform
could improve in terms of efficacy and dosing regimen wheth-
er it received new layers of technology, like: (i) chemical
changes in siRNA structure; (ii) nanoparticles delivery; (iii)
siRNA structural changes to single-stranded antisense oligo-
nucleotide, like a GapmeR for example, that can be formu-
lated in particles or administered naked.
SYL1001
SYL1001 is a siRNA designed for prevention and treatment of
ocular pain and Dry Eye Syndrome, formulated for topical
administration by eye drops. The target of SYL1001 is the
vanilloid receptor TRPV1 (transient receptor potential cation
channel, subfamily V, member 1), a nociceptor found in cor-
nea and its respective nerve terminals (89).
Preclinical testing of SYL1001 used the rabbit model of
ocular irritation induced by the capsaicin, a natural agonist
of TRPV1 (90). This study first validated different TRPV1-
targeted siRNAs by in vitro assays and tested them in New
Titze-de-Almeida, David and Titze-de-Almeida
Zealand white rabbits. SYL1001 showed efficacy for ocular
irritation, reducing TRPV levels and ocular pain caused by
capsaicin. Analgesia was comparable to that obtained by the
reference analgesic capsazepine. For toxicological analyses,
two different species - dogs and rabbits - received ocular in-
stillations of SYL1001 during 28 days. No toxic effect was
found. Regarding pharmacokinetics, the drug was not detect-
ed above the limit of detection of 40 ng/ml in all time points
and doses administered. In summary, SYL1001 presented
safety and efficacy in the model of ocular pain induced by
capsaicin (90).
A pharmacokinetic study evaluated the biodistribution of
SYL1001 in New Zealand rabbits (91). Cornea, conjunctiva,
and lachrymal gland were harvested at 5 min after eye drop
instillation. Also, the assay collected trigeminal/semilunar
ganglion to examine whether SYL1001 had access to neurons
innervating the cornea. To disclose systemic exposure, sam-
ples of lung, liver, kidney, and plasma were analyzed.
SYL1001 was detected in all ocular structures and trigeminal
ganglion, and also in plasma. Regarding human clinical trials,
a phase 1 study evaluated safety and tolerability of this siRNA
(NCT01438281). Thirty healthy individuals received
SYL1001 by eye drops. For an initial evaluation of safety,
the drug was administered to six subjects in a single dose.
Then escalating doses were given to 24 individuals, as a single
daily dose, in one eye, for seven days. The drug was well
tolerated, without causing changes in ocular surface or severe
adverse effects. Regarding pharmacokinetic analysis,
SYL1001 was not detected in plasma above the limit of quan-
tification of 40 ng/ml (92).
Two phase 2 clinical trials evaluated safety and efficacy of
SYL1001 in patients with ocular pain associated with Dry Eye
Syndrome (NCT01776658 and NCT02455999). Analgesic
effect and ocular tolerance of four doses of SYL1001
(0.375%, 0.75%, 1.125% and 2.25%), including a placebo
group, were evaluated after ten days of treatment of 127 pa-
tients (93). The drug caused analgesia in Visual Analogue
Scale (VAS), which was significantly better than placebo
for dose 1.125%. The analgesic effect measured by
Ocular Surface Disease Index (OSDI) scale, however,
showed no difference between treated and control groups.
No serious adverse events occurred, either locally in the
eye or systemically (93).
Critical Analysis
The treatment of Dry Eye Disease is still a challenge in oph-
thalmology, including cyclosporine, anti-inflammatory drugs,
artificial tears, and other drugs (94, 95). SYL1001 does not act
in the disease pathogenesis. But it is an attractive analgesic for
the ocular pain faced by patients, as it blocks the neuronal
signaling at its origin in TRPV1 nociceptors. Administered
dose of SYL1001 is moderate, making possible an affordable
RNAi-based drugs in phase II – III clinical trials

Fig. 5 Rationale on Atu027 technology for the treatment of TTR-mediated amyloidosis. (a) Patients with amyloidosis present an increased expression of TTR
gene. Atu027 is a TTR-targeted siRNA, formulated in lipid carriers for intravenous administration. (b) Lipid particles coated with ApoE bind to receptors in hepatic
cells and are internalized by endocytosis. (c) Ionizable lipids of these particles will acquire positive charges by protonation due to the acid pH of endocytic vesicles.
Positively charged nanoparticles will bind to negatively charged endosomal membrane, and the fusion of these membranes will finally deliver siRNAs in cell
cytoplasm. (d) TTR-targeted siRNAs will bind to complementary sequences in 3' end of TTR mRNA, instead of the traditional CDS region. AGO enzyme of RISC
complex will execute TTR mRNA cleavage. (e) Reducing TTR expression will prevent the accumulation of abnormal amyloid proteins in tissues. AGO Argonaute
2, ApoE apolipoprotein E, CDS Coding sequence region of mRNA, Dicer a ribonuclease enzyme PACT Protein activator of the interferon-induced protein kinase
(PKR), RISC RNA-induced silencing complex, TRBP HIV-1 Trans-activation response (TAR) RNA-binding protein, TTR transthyretin.

treatment. At 450 μg/eye/day, it causes analgesia, and a ten-
day regimen will demand 4.5 mg of siRNA per eye. Other
cycles of treatment might be necessary for these chronic eye
disorder. A phase 2 study with ≈ 130 individuals showed a
significant analgesic effect in one of two tests of analgesia at
450 μg per eye (1.125%). As pain is a clinical symptom present
in other prevalent eye diseases, the pharmaceutical market of
SYL1001 is significant. Regarding Dry Eye Disease, it is a
complex disorder for therapeutic management. The contribu-
tion of SYL1001 would be only for analgesia, not replacing
the immunosuppressive agent cyclosporine and other drugs
for symptomatic relief. For that, a multicenter phase III study
is needed to examine whether this siRNA will show efficiency
and safety in a large number of individuals.
Patisiran (ALN-TTR02)
Patisiran is a siRNA that targets transthyretin (TTR) mRNA.
Mutations in TTR gene cause a disease named transthyretin-
mediated amyloidosis (ATTR), characterized by an accumu-
lation of abnormal insoluble amyloid proteins in tissues, par-
ticularly peripheral nerves and heart (96). Prognosis for pa-
tients with TTR is poor. Current treatments are aimed to
reduce mutant TTR or to stabilize the correct conformation
of TTR protein, but these drugs are not effective in all patients
(97). The therapeutic focus of patisiran is the familial
amyloidotic polyneuropathy (FAP) related to TTR mutations.
This neurological disease is progressive, debilitating, and irre-
versible, with symptoms of sensorimotor and autonomic neu-
ropathy (98). Patisiran is a potent antitransthyretin siRNA
encapsulated in lipid nanoparticles, which targets a conserved
sequence present in TTR mRNA. Curiously, this sequence is
located in noncoding 3′-UTR region of TTR mRNA. siRNAs
usually target coding regions of mRNA while microRNAs
address noncoding 3′-UTR ones (79). The author’s argument
is that the same siRNA target sequence was present in both
the nonmutated and mutated TTR mRNA, resulting in a
more efficient gene knocking-down (99). Furthermore, the

siRNA molecules of patisiran were formulated in a lipid car-
rier previously developed by Tekmira. The benefit of this
carrier is that it vectorizes patisiran to the liver, where most
TTR proteins are synthesized. Lipid particles are coated with
the apolipoprotein E (ApoE). After I.V. injection, the carrier is
opsonized by this protein, enter the liver and bind to ApoE
receptors on hepatocytes. Within the hepatic cells, patisiran’s
siRNAs will knock-down TTR expression by the standard
post-transcriptional mechanism, using RISC complex and
Argonaute protein (Fig. 5).
Before entering phase 1 human clinical trials, patisiran
was evaluated in cynomolgus monkeys (99). TTR target
sequence is identical in this species and humans, allowing
patisiran testing without nucleotide changes in its siRNA
molecule. Patisiran caused a marked 75% - 90% TTR
knockdown in monkeys at Day 7 (0.1 mg/Kg) and Day
14 (0.3 mg/Kg), respectively. The silencing effect persisted
for 28 days. The drug also showed efficacy in a multiple
dose regimen - 0.3 mg/Kg every four weeks for five
doses.
The first human trial on patisiran was a dose-escalating
study that evaluated safety and efficacy in 17 healthy adult
volunteers (NCT01559077) (99). Subjects organized in
groups of four individuals received I.V. injected siRNAs
(0.01–0.5 mg per kilogram). Individuals also received the
following drugs to reduce the risk of infusion-related reac-
tions: dexamethasone, acetaminophen, diphenhydramine
or cetirizine, and ranitidine, as previously cited (100).
Patisiran activity was revealed by a decrease in serum
transthyretin levels, measured by enzyme-linked immuno-
sorbent assay (ELISA) (99). Doses ≥0.15 mg/Kg caused a
significant and durable TTR knock down until Day 28.
The highest siRNA dose (0.5 mg/Kg) caused the best
TTR silencing effect (93.8%). 5′ RACE assay confirmed
the specificity and RNAi-based mechanism of action of
patisiran, generating TTR mRNA cleavage products. For
pharmacokinetics, siRNA levels were measured by ELISA-
based hybridization assay. Cmax and AUC increased in a
dose-proportional manner. Finally, the study monitored
adverse effects during the 28-day period after drug admin-
istration. No serious adverse effects occurred. The overall
incidence of adverse reactions was 7.7%. A moderate
infusion-related reaction was observed in a single individ-
ual who received 0.5 mg/Kg, which was managed by a
temporary interruption in patisiran infusion, glucocorticoid
therapy, and reducing the rate of infusion. Another phase
1 clinical trial addressed safety, tolerability, and PK of
patisiran in Japanese healthy subjects (NCT02053454).
Phase 1 results above described (NCT01559077)
allowed patisiran to enter in phase 2 clinical trial
(NCT01617967). This study evaluated the safety, tolerabil-
ity, pharmacokinetics, and pharmacodynamic of multiple
escalating doses of patisiran in patients with FAP (101).
Titze-de-Almeida, David and Titze-de-Almeida
Twenty-nine adult patients with biopsy-proven ATTR am-
yloidosis and mild-to-moderate neuropathy were organized
in cohorts of individuals. They received two I.V. infusions
of patisiran, at the following dose regimens: 0.01 mg/Kg
(n = 4), 0.05 mg/Kg (n = 3), 0.15 mg/Kg (n = 3), or
0.3 mg/Kg (n = 7) every 4 weeks (Q4W), or 0.3 mg/kg
(n = 12) every 3 weeks (Q3W). To prevent infusion-related
reactions, patients were premedicated with dexamethasone,
paracetamol, and antagonists of H1 and H2 histaminergic
receptors. Most individuals presented Val30Met TTR mu-
tation, the most common form of FAP (102), and were
taking concomitant treatments with tafamidis or diflunisal.
Regarding pharmacodynamic, patisiran 0.3 mg/Kg Q3W
caused ≈ 83–87% TTR knock down after the first and
second doses, respectively, in comparison to baseline levels.
Similar results occurred for Q4W regimen at the same
dose. Patisiran caused the same effect on wild-type and
mutant TTR and showed efficacy also for patients treated
with tafamidis and diflunisal who presented an increased
baseline serum levels of TTR. Mean values of Cmax and
AUC for the first and second doses increased proportion-
ally to patisiran dose increment. Terminal half-life values
were 39–59 h for doses over 0.01 mg/Kg, at Day 0 and
Days 21/28. Regarding safety and tolerability, only two
patients treated with 0.3 mg/Kg showed serious adverse
events. Mild-to-moderate infusion-related reactions oc-
curred in 10.3% patients that received 0.3 mg/Kg
Q4W. In summary, patisiran was generally well tolerated
and caused a significant knockdown of mutant and wild-
type TTR in patients with FAP (101).
The same patients of the above-described phase 2 clinical
trial (NCT01617967) were enrolled in phase 2 open-label
extension study (phase 2 OLE study) (NCT01961921), that
evaluated the safety and tolerability of patisiran 0.3 mg/Kg
Q3W for long periods (≈ seven months). Patisiran was well
tolerated, with ≈ 15% of infusion-related reactions. No sys-
temic toxicity was observed. This siRNA caused a marked
80% TTR knockdown over nine months. Regarding thera-
peutic effects, patients were evaluated by using a modified
Neuropathy Impairment Score + 7 (mNIS + 7), and a quality
of life (QOL) measurement. Six months of patisiran stabilized
the neurological impairment and QOL in patients with FAP,
independent on other concomitant treatments (103). This on-
going study also showed positive effects on mNIS + 7 at
24 months of treatment, with only mild infusion-related ad-
verse effects (104).
Finally, patisiran entered in a phase 3 trial named
APOLLO study (NCT01960348), aimed to evaluate its safety
and efficacy in patients with FAP. Another phase 3 trial is now
enrolling participants who have completed a prior patisiran
clinical study (NCT02510261). The aim is to evaluate safety
and efficacy of patisiran in patients with FAP in a long-term
dosing, i.e. 52 weeks.
RNAi-based drugs in phase II – III clinical trials

Fig. 6 Characterization of sig12D-LODER devices and injection procedure (a) Schematic representation of the siG12D-LODER matrix of PLGA. (b) The
picture is showing the millimetric dimension of sig12D-LODER devices. (c) Standard procedure EUS guidance for implanting devices into pancreatic tumor. (d)
Sustained release of siRNAs (10 μg) from the PLGA matrix over two months. Devices were incubated in PBS (pH = 7.4), at 37 °C. Reprinted with permission
from References (109, 112). KRAS Kirsten rat sarcoma viral oncogene homolog, KRAS-target siRNA siRNA designed to silence mutated KRAS, PLGA matrix
polymeric matrix of poly(lactic-co-glycolic) acid.

Critical Analysis
Patisiran is an excellent example of Gene Therapy. This strat-
egy to treat genetic diseases brought exciting discussions for
scientific community of the early seventies (105). The method
for gene therapy used by patisiran would be available only two
decades later (106). This formulated siRNA was designed for
ATTR, a genetic disease with a major medical need based on
its significant morbidity and mortality (107). Liver transplan-
tation and treatment with BTTR stabilizers^ tafamidis or
diflunisal are therapeutic options (108). Instead, patisiran trig-
gers RNAi on TTR gene, thus controlling ATTR at a very
fundamental step of disease pathogenesis - the mRNA of the
gene that causes amyloidosis. TTR knocking down reaches
80%. Treated patients showed stabilization of illness progres-
sion and improvement in the quality of life. Part of the success
relies on the carrier that executes a site-specific delivery of
patisiran into hepatic tissues. However, hospital assistance
for I.V. injections and drugs for controlling infusion-related
reactions are additional costs to be considered. The basic drug
costs could be an issue as treatment is expected to be for long
term, reaching ≈ 670 mg (0.3 mg/Kg, every three weeks for
24 months). Patisiran is the most advanced siRNA drug in
development, with encouraging results in phase II trial. Two
ongoing phase III studies will probably confirm the safety and
efficacy of patisiran for patients with ATTR, a genetic disease
that urgently need new therapeutic options.
siG12D-LODER
siG12D-LODER is a polymeric system complex designed for
a sustained delivery of a G12D-mutated KRAS-targeted
siRNA, developed to treat human pancreatic cancer (109,
110). As shown in Fig. 6a, b, LOcal Drug EluteR (LODER)
is a polymeric matrix of poly(lactic-co-glycolic) acid (PLGA) in
a shape of a small cylindrical rod of diameter 0.8 mm and
length 5.5 mm ± 1 mm.
Anticancer effects of this anti-KRAS drug and the ability of
LODER for long term delivery of siRNAs were evaluated
(109). First, the cylindrical shape remained unchanged after
45 days of implantation of siG12D-LODER. This molecular
device delivers siG12D siRNAs over two months when incu-
bated in phosphate buffer solution (Fig. 6d), and protects from
degradation in mouse liver tissue ex-vivo. Pancreatic carcinoma
cells exposed to siG12D-LODER showed reduced KRAS
mRNA and protein level, along with a reduced cancer cell
viability. Cells also showed a reduction in epithelial-
mesenchymal transition, represented by lower cell migration
and contact inhibition. SiG12-LODER silenced KRAS expres-
sion in subcutaneous tumors in vivo, with no toxicity in male or
Table I Synthetic RNAi-Based Drugs In Phase 2–3 Clinical Trials

Drug Target (cell role) Chemistry / formulation Route Disease Phase Status/completion Company/ Refs
collaborator

PF-04523655 RTP801 / Naked siRNA, O-methylated IVT AMD, DME II Completed/2013 Quark / Pfizer 16, 18
(PF-655) (hypoxia-inducible)
QPI-1002*1 p53 (apoptotic) Naked siRNA, O-methylated IV AKI II Recruiting / 2018*2 Quark 40
(I5NP) DGF III Recruiting / 2019*2
QPI-1007 CASP2 (apoptotic) Naked siRNA, O-methylated; IVT NAION II / III Recruiting / 2019*2 Quark 52
changes in sense strand Glaucoma II Completed / 2015
TKM-080301 PLK1 (kinase) siRNA / SNALP IV Solid tumors, HCC, NET, I / II Completed / 2015 Arbutus 59–62
(TKM-PLK1) ACC, lymphoma
Atu027 PKN3 (kinase) siRNA / LIPOPLEX IV Pancreatic cancer I / II Completed / 2016 Silence / Granzer, 67, 70
FGK
SYL040012 ADRB2 (β2 receptor) Naked siRNA Eye drops Ocular hypertension, glaucoma II Completed / 2013; 2016 Sylentis 82–85
(Bamosiran)
SYL1001 TRPV1 (nociceptor) Naked siRNA Eye drops Ocular pain in Dry Eye II Completed / 2016 Sylentis 92, 93
Syndrome
Patisiran TTR (amyloidogenic) siRNA / Lipid particle, ApoE IV TTR-mediated Amyloidosis III Active / 2017*2 Alnylam 99, 101, 103,
(ALN-TTR02) 104
siG12D-LODER KRAS (oncogene, siRNA / Miniature PLGA Intra- Pancreatic cancer*3 II Active, not yet recruiting / Silenseed 112
GTPase) device tumoral 2020*2
Miravirsen miR-122 (microRNA) AntimiR, antisense SC Hepatitis C infection II Complete / 2011 Santaris 119, 126–128
oligodeoxynucleotide,
LNA, PS

ACC adrenocortical carcinoma; ADRB2 adrenoceptor beta 2; AKI acute kidney injury after cardiac surgery; AMD Age-Related Macular Degeneration; ApoE apolipoprotein E; CASP2 Caspase 2; DGF Delayed Graft
Function in kidney transplantation; DME Diabetic Macular Edema; HCC hepatocellular carcinoma; IV intravenous injection; IVT intravitreal injection; KRAS Kirsten rat sarcoma viral oncogene homolog; LNA locked
nucleic acid; miR-122 microRNA 122; NAION Acute Non-Arteritic Anterior Ischemic Optic Neuropathy; NET neuroendocrine tumors; PKN3 protein kinase N3; PLK1 Polo-like kinase-1; PS phosphorothioated; SC
subcutaneous injection; SNALP stable-nucleic-acid-lipid particles; TRPV1 transient receptor potential vanilloid 1; TTR transthyretin. *1- drug licensed to Novartis; *2- estimated completion data; *3- siG12D-LODERs
combined with chemotherapy treatment (Gemcitabine + nab-Paclitaxel). Company names: Quark Pharmaceuticals; Pfizer; Arbutus Biopharma Corporation; Silence Therapeutics GmbH; Granzer Regulatory
Consulting and Services; FGK Clinical Research GmbH; Sylentis, S.A.; Alnylam Pharmaceuticals; Silenseed Ltd.; Santaris Pharma A/S
Titze-de-Almeida, David and Titze-de-Almeida
RNAi-based drugs in phase II – III clinical trials
female mice or rats, at up to 320 μg siG12D/Kg body weight.
At this dose, it reduced tumor growth in vivo. Rats showed larger
necrotic areas in tumor region and mice presented smaller
tumor volumes. Survival period significantly increased in
siG12-LODER treated mice. The treatment also showed ben-
efits in orthotopic pancreatic tumors in mice. Overall survival
was prolonged while KRAS expression was reduced.
The last preclinical studies for safety, toxicity, and
toxicokinetics were performed in Sprague-Dawley rats.
This anti-KRAS RNAi system was implanted in subcu-
taneous tissue of the scapular region (111). Animals
(males and females) received one or three siG12D-
LODERs with 0.33 mg siRNA in each implanted de-
vice, administered on days 1, 14, and 28. Regarding the
safety and toxicity, animals were evaluated for clinical
signs, weight, ophthalmology, hematology, biochemistry,
and pathology. For toxicokinetic analysis, blood samples
were collected after each administration (days 1, 14, and
28) at seven temporal points, from 0.25 h to 24 h.
SiG12D-LODER showed safety and tolerability, as no
adverse effect, death, or unscheduled euthanasia oc-
curred. A preclinical finding was a capsule of a thin
fibrotic tissue developed locally in the region of
LODER implantation. Macrophages and multinucleated
giant cells formed a single layer between this capsule
and the cavity. This reaction did not involve adjacent
tissues and showed no evidence of chronic active inflam-
mation or necrosis. Finally, only small amounts of
siG12D siRNA were detected in plasma samples until
30 min after subcutaneous administration of 3 doses.
A first human phase 1/2a clinical trial evaluated siG12D-
LODER in association with chemotherapy for patients with
non-operable Locally Advanced Pancreatic Cancer (LAPC)
(NCT01188785) (112). The study examined safety, tolerabil-
ity, and antitumor effects. Escalating doses of siG12D-
LODER in test were 0.025 mg, 0.75 mg (0.375 mg x 2
siG12D-LODERs) or 3.0 mg (0.375 mg x 8 siG12D-
LODERs) (See Fig. 6b). Some patients received siG12D-
LODER with a concomitant chemotherapy, like gemcitabine
or FOLFIRINOX (n = 4). The route of administration is an
outstanding advantage for this RNAi-based drug. By a stan-
dard endoscope ultrasound (EUS) biopsy, SiG12D-LODER’s
miniature devices were administered into pancreatic tumor
tissue (Fig. 6c). No dose-limiting toxicity occurred, and most
adverse events were grade 1–2. Grade 3–4 adverse reactions
like neutropenia and cholangitis occurred in ten patients
(66.7%). Despite these adverse effects, the drug was consid-
ered safe and well tolerated. SiG12D-LODER showed an
anticancer effect as no patient presented tumor progression.
Seven patients also exhibit a decrease in CA19–9 tumor mark-
er, and the median overall survival was ≈ 15 months. A phase
2 clinical trial (NCT01676259) is now testing the effects of this
siRNA in combination with chemotherapy. Patients with
unresectable LAPC will receive a single dose of 2.8 mg (i.e.
eight 0.35 mg siGD-LODERs) by EUS, in combination with
chemotherapy (gemcitabine + nab-paclitaxel).
Critical Analysis
At first, siG12D-LODER made KRAS a druggable antican-
cer target. New drugs also targeting this oncogene are now in
test an may represent future competitors of siG12D-LODER
(113–115). siG12D-LODER is the only RNAi-based drug
that provides a sustained release of siRNA into the tumor
region thanks to a well-known biodegradable PLGA polymer,
approved by FDA for drug delivery (116–118). This siRNA
represents a hopeful strategy against advanced pancreatic can-
cer. It competes with Atu027 (with a different target) and
much lower administered dose (≈ 3 mg) due to the cited
PLGA devices that deliver RNA duplexes with no chemical
modifications. The formulation into the LODER matrix of
PLGA, however, will increase the drug costs. Indeed,
intratumoral administration may has pros and cons.
Biophase bioavailability is relatively high compared to other
routes of administration. Also, it is free of infusion-related
adverse events experienced by patients treated with I.V. for-
mulated siRNAs. However, metastasis of pancreatic cells
will not be controlled by siG12D-LODER unless it was
administered in association with other antineoplastic
drugs. The procedure for administration the Bpills^ into
pancreatic tumors will require a specialized medical ser-
vice which implies in costs and may reduce the market.
I.V. route is a comparatively more accessible hospital pro-
cedure. This limitation would be more important whether
siG12D-LODER implantation must have to be repeated,
but it is not the case. Taking the aggressive behavior of
pancreatic tumor cells, siG12D-LODER is a promising
anticancer therapy. Results of a phase 1/2a was positive
in a relatively small number of patients, so we must wait
for an ongoing phase 2 study that is testing siG12DLoder
(2.8 mg) plus standard chemotherapy for patients with
unresectable advanced pancreatic cancer.
ANTIMIR IN CLINICAL STUDIES REGISTERED
IN NIH
Miravirsen
Miravirsen (SPC3649) is a microRNA inhibitor designed for
treatment of hepatitis C infection (119, 120). Curiously, a
microRNA produced by hepatic cells – microRNA 122
(miR-122), is essential in Hepatitis C Virus (HCV) infection
(Fig. 7a) (121). A new antiviral drug acting as a miR-122
inhibitor (AntimiR-122) thus has therapeutic potential for this
liver disease (122). Miravirsen is a 15-base oligonucleotide,
 Titze-de-Almeida, David and Titze-de-Almeida

Fig. 7 The role of miR-122 in HCV infection and antiviral action of miravirsen. (a) miR-122 expressed by hepatic cells is an essential host factor for HCV infection,
as it guides AGO2 protein to 5'-UTR region of the virus genome. The binding of Ago2-miR-122 complex to 5'-UTR region protects viral RNA from nucleolytic
degradation, allowing virus propagation in patients with hepatitis C. (b) Miravirsen is an antisense oligo (AntimiR) that binds to and inhibits miR-122 function,
thereby counteracting HCV infection. Chemical changes in miravirsen structure are LNA (red uppercase) and the inclusion of DNA nucleotides (lowercase).
Reprinted with permission from Reference (119).

locked nucleic acid (LNA) modified, phosphorothioated, and
complementary to 5′ region of mature miR-122. The newly
formed heteroduplexes miravirsen–miR-122 will prevent the
binding of miR-122 to viral RNA, compromising HCV rep-
lication (Fig. 7b) (119).
African green - and cynomolgus monkeys were includ-
ed in preclinical testing on miravirsen activity and safety,
showing positive results (123, 124). Additionally, chronic
HCV-infected chimpanzees treated with miravirsen dem-
onstrated a long-term suppression of viremia and im-
provement in liver pathology, without viral resistance or
side effects (125). This AntimiR-122 was allowed to enter
in phase 1 human clinical trials.
Safety of miravirsen was determined in two phase 1
c l in i c a l t r ia l s ( NC T 00 68 8 01 2, N C T 0 0 97 99 27 ) .
Miravirsen caused a dose-dependent effect, was well-
tolerated and showed no dose-limiting toxicities (119).
The drug entered in phase 2 clinical trial that examined
safety, tolerability and antiviral effects in subjects with
chronic HCV genotype 1 infection (NCT01200420).
Patients with plasma HCV RNA level above
75,000 IU per milliliter had not received prior antiviral
treatment for HCV genotype 1. Individuals (n = 36)
received five weekly subcutaneous injections of
miravirsen at doses of 3 mg, 5 mg, or 7 mg per kilo-
gram of body weight (n = 9 patient per dose) or
placebo over a 29-day period (126). They were followed
over 18 weeks for clinical examinations, biochemistry,
and hematologic tests. Miravirsen caused a significant
dose-dependent and sustained reduction in HCV RNA
levels, as measured by a Real-Time HCV assay. The
mean maximum reduction (log10 IU per milliliter) from
baseline reached 1.2, 2.9, and 3.0 for patients receiving
3 mg, 5 mg, or 7 mg of miravirsen per kilogram, re-
spectively, compared to 0.4 value found in the placebo
group. No dose-limiting adverse events or escape muta-
tions were found in the miR-122 binding sites of HCV
sequence. Most adverse events were grade 1. One se-
vere event occurred, with the loss of consciousness, at
nine weeks after the last dose of 7 mg/Kg. Two pa-
tients treated with this dose presented injection-site re-
actions characteristic of oligonucleotide drugs (i.e. ery-
thema, pruritus, burning). Changes in laboratory param-
eters were not clinically significant to stop miravirsen
treatment. In summary, this AntimiR-122 oligonucleo-
tide was well-tolerated and showed antiviral activity in
patients with chronic HCV infection (126).
A retrospective analysis of the same patients enrolled in
that study (126), evaluated safety and efficacy of miravirsen
after an extended period (127). Patients with chronic hepatitis
C had received five weekly subcutaneous injections of
miravirsen or placebo over a period of 29 days. About half
RNAi-based drugs in phase II – III clinical trials
of miravirsen-treated patients underwent treatment with
peginterferon and ribavirin for 3 or 6 weeks and were followed
up for 35 months. This study highlighted the long-term safety
and efficacy of miravirsen.
An in vitro study was aimed at evaluating the ability of
miravirsen to act in association with antiviral drugs and po-
tential mutations in viral sequences (128). Miravirsen im-
proved the action of antiviral drugs interferon α2b, ribavirin,
VX-222, 2′-methylcytidine, and telaprevir. However, muta-
tions in the HCV 5′ untranslated region (5′-UTR) occurred
after serial passages (day 72) in vitro. Moreover, mutations were
also found in samples from subjects that completed a phase 2
clinical trial (NCT01200420) (128). Association between
miravirsen and telaprevir was further evaluated in phase 1
clinical trial with healthy volunteers (NCT01646489).
Finally, miravirsen may represent a therapeutic option for
patients with chronic hepatitis C genotype 1 that are null
responders to PEGylated interferon alpha plus ribavirin, as
will be revealed by ongoing clinical trials (NCT02031133,
NCT01727934, NCT02508090, and NCT01872936).
Critical Analysis
Miravirsen is the only drug of this review that targets a
microRNA sequence. Most RNAi-based drugs in clinical
testing are siRNAs (not targeting miRNAs) because they
represent the first molecules used for gene silencing and
experimental therapeutics, soon after the Fire & Melo’s
striking finding (1, 79). MicroRNAs compose the second
- but not the less important - Bwave of discovery^ on
RNAi, playing a role in many diseases including host-
pathogen viral interaction (129). Miravirsen is an anti-
HCV miR-122 inhibitor. Curiously, miravirsen is ad-
ministered via subcutaneous injection. This route is suit-
able for chronic diseases, as used for insulin injections
for example. The rate of absorption is constant and
slow, providing a sustained effect for the weekly injec-
tions of miravirsen (126, 130). Regarding structure,
miravirsen is a single stranded antisense DNA oligo,
containing chemical modifications for stability and spec-
ificity. It is administered naked, reducing the costs of
formulation in nanoparticles. That is of capital impor-
tance, as patients will receive high quantities of
miravirsen of ≈ 2400 mg (7 mg/Kg X 70 Kg X 5
weekly injections) or even higher dosing. Indeed, other
antiviral drugs against HCV are available (131).
Miravirsen showed a dose-dependent reduction in
HCV levels. Antiviral effects of this AntimiR-122 are
now under evaluation in ongoing phase II studies, to
PEGylated Interferon and antivirus drugs resistant
HCV. Miravirsen is one more option in HCV Roche
treatments.
CHALLENGES AND PROGRESS OF RNAI
TECHNOLOGY
RNAi is the major discovery of the last two decades in life
sciences. The molecular mechanisms of gene regulation by
noncoding RNAs is still a matter of capital importance (7,
132). Indeed, RNAi has been very helpful for scientists in a
more general way, offering a straightforward method to si-
lence genes of interest. Much information on cell biology
and even the mechanisms of disease are now available thanks
to RNAi techniques (133, 134). As soon as RNAi conquered
its place in research laboratories at the beginning of twenty-
first century, an exciting question emerged: would this method
be transposed from bench to clinics? (135–138). Fortunately, a
decade after the Nobel prize was awarded to Fire and Mello
for their discovery of RNAi, ten synthetic RNAi-based drugs
entered in phases 2–3 clinical trials. These represent good
candidates to reach the pharmaceutical market in the coming
years (79, 106, 134, 139).
Development of RNAi drugs faces a crucial obstacle: how
to deliver such polar / hydrophilic oligonucleotides across
lipid barriers of the organism? In fact, delivery of nucleic acids
into cells and tissues is still a matter of concern, but not a
technical barrier for drug development. Three ophthalmic
drugs of this review are naked siRNAs, which means they
could transfect cells without the help of any carrier. PF-
04523655 and QPI-1007 are injected by IVT route, while
SYL040012 is an eye drops. Moreover, naked siRNAs can
be administered systemically. QPI-1002, a drug used to pre-
vent kidney injury, is given by I.V. route and rapidly accumu-
lates in proximal tubule cells. Kidney and liver are organs that
naturally receive the largest amount of oligonucleotides given
by this route (30, 31). The anti-HCV miravirsen is another
example of naked oligonucleotide administered by I.V. route
properly internalized by liver cells (119). This AntimiR-122 is
a short single-stranded antisense DNA nucleotide with two
key advantageous changes in chemical structure: LNA which
increases stability to hybridize with miR-122 and a
phosphorotioated backbone for nuclease resistance (123,
140). In fact, five weekly injections of miravirsen caused a
long-term and dose-dependent suppression of miR-122 in pa-
tients with hepatitis C (126). Gymnosis is a term coined for
delivery of short single-stranded oligonucleotides into cells
without assistance of transfection agents (141, 142).
TKM-080301, Atu027, and patisiran are not naked
siRNAs. Instead, these drugs were complexed with particles
for delivery of RNAi molecules which are revised elsewhere
(56, 57, 143, 144). Cationic liposomes (i.e. lipofectamine) are
lipid vesicles commonly used for transfection of siRNAs and
non-viral vectors in different cell types and experimental as-
says (145–148). All three drugs are administered by I.V. route,
formulated in lipid particles with technological improvements
over the conventional liposomes. TKM-080301 uses stable-

nucleic-acid-lipid particles (SNALPs). The major improve-
ment is a lipid bilayer composed by cationic and fusogenic
lipid and coated with polyethylene glycol (PEG). SNALPs
protect siRNAs from serum nucleases attack while allowing
cellular endosomal uptake for cytoplasmatic release of the
siRNAs (149, 150). PEGylation minimizes phagocytosis and
avoid renal excretion of siRNAs (134). Arbutus Biopharma
developed its own proprietary delivery system – lipid nano-
particle (LNP) platform, which also includes amino lipids and
cholesterol (151). TKM-080301 reduced tumors located in
two different organs, liver and adrenal cortex (59, 62).
Regarding Atu027, it was formulated in a lipid particle con-
taining three distinct lipids, AtuFect01 (positively charged),
fusogenic DPhyPE helperlipid (neutral) and MPEG-2000-
DSPE (PEGylated). This siRNA-lipoplex technology allowed
a marked uptake of 19-mer siRNA duplexes by the vascular
endothelium in the liver, heart and lung (65, 68, 69). Atu027
showed efficacy against tumors developing in prostate and
pancreas and prevented lymph node lung metastasis in ro-
dents (65, 66). This siRNA was also effective in patients with
advanced refractory cancer of different organs (i.e. breast,
pancreas, colon and others) (67).
Tissue-targeted delivery of RNAi therapeutics was a re-
markable challenge surpassed by patisiran and siG12D-
LODER. Patisiran was designed to knockdown a mutated
and nonmutated TTR gene that causes amyloidosis (99).
Beyond a treatment for a genetic disease, this medication
brought a clear example of a tissue-targeted RNAi delivery
after I.V. injection. The lipid nanoparticle that carries the
siRNA is opsonized by apolipoprotein E, allowing its binding
to receptors on hepatocytes. After endocytosis and fusion with
endosomal membranes, the siRNAs are finally released in cell
cytoplasm to trigger RNAi on TTR mRNA. Previous at-
tempts were also made to deliver nucleic acids into spe-
cific organs. Immunoliposomes coated with monoclonal
antibodies for binding to receptors of insulin and transfer-
rin present in blood-brain barrier and neuronal cell mem-
brane, were able to deliver nonviral vectors for RNAi into
cerebral neurons (152).
SiG12D-LODER showed how regional administra-
tion could be used as an innovative way for site-
specific siRNA delivery. Miniature cylindrical Bpills^
made with the biocompatible polymer PLGA were im-
planted regionally into pancreatic tumors by endoscope
ultrasound (EUS) biopsy for a long term delivery of
KRAS-targeted siRNAs, showing anticancer effects
(109, 112). Regional application of RNAi-based thera-
peutics is a viable route to treat diseases in organs of
difficult access. Devices like catheters or stents for local
injections might circumvent the delivery of nucleic acids
in such tissues (120). Some systemic therapeutics for
brain diseases may also fail because of the blood-brain
barrier (153). In this sense, the method of convection-
Titze-de-Almeida, David and Titze-de-Almeida
enhanced delivery (CED) is a viable strategy to circum-
vent this barrier and administrate RNAi drugs in brain
areas of interest (154–156).
DRUGGING RNAI MOLECULES
Inaugurating a new pharmacological drug class is far from an
easy matter, besides consuming efforts and a high amount of
time and money. The road from bench to clinics has been a
difficult way but not an insurmountable barrier (138). In this
scenario, RNAi has shown a quite particular fragmented time-
line. First occurred a disproportional optimism soon after the
Fire and Melo’s Nobel Prize in 2006 - called as Birrational
exuberance^ by Haussecker and Kay (157). Then RNAi bio-
tech suffered a backlash as that huge expectation was not
achieved (2008 to 2011). Fortunately, the companies entered
into a more stable and mature phase of drug development.
Drugging RNAi is an evolving matter. Advances in the
chemical structure of siRNAs and delivery systems have made
the drugs safe and efficient, as revised elsewhere (139, 158,
159). Conjugating siRNAs with specific molecules is a prom-
ising strategy for a targeted-delivery (160). Among them is N-
acetylgalactosamine (GalNAc), a ligand for a receptor found
in hepatocytes. siRNA-GalNac conjugates injected subcuta-
neously can trigger a marked knock-down of target mRNA
in liver. Such technology developed by Alnylam is been used
in various products (161). This delivery strategy was success-
fully employed in a product named Revusiran for the treat-
ment of ATTR amyloidosis (162), the same disease and target
gene also addressed by Patisiran that is formulated in lipid
carrier. Revusiran had reached the phase 3 but was
discontinued by Alnylan in May 2016 due to safety concerns
(163). While reinforcing the importance of Monitoring
Committees during ongoing clinical trials, it highlights the
vulnerability of any new technological platform.
Many other promising strategies for delivery and siRNA
conjugation have been developed, including monoclonal an-
tibodies and aptamers (164). Monoclonal antibodies are at-
tractive molecules for tissue-specific delivery of siRNAs
(165). The rationale of this approach looks perfect, but the
efficacy is still limited (166). Both the cellular uptake and the
following cytoplasm trafficking and siRNA delivery are issues
that may compromise the success of such strategy (134). A
recent work presented the named THIOMAB platform for
antibody-siRNA conjugates delivery. Although offering po-
tential improvements in conjugation, the method revealed
some limitations about cell receptors capable of internalizing
siRNAs, intracellular trafficking and endosomal escape (167).
Regarding aptamers, these are synthetic oligonucleotides se-
lected to bind with high affinity to receptors (168). RNA
aptamers can be conjugated with siRNAs for a targeted
delivery (169, 170). A critical point is the selection of
RNAi-based drugs in phase II – III clinical trials
receptors that efficiently internalize cargo to cell cyto-
plasm, as addressed by a new methodology named Bcell-
internalization SELEX^ (171).
Research on mechanisms of siRNA internalization in tar-
get cells and further trafficking, as well as the development of
ligands and delivery systems are matters of capital importance
in RNAi biotechnology. Any system for oligonucleotide deliv-
ery, either using a nanoparticle or a conjugative molecule, will
present pros and cons regarding cost, specificity, efficiency,
and even toxicity.
SUMMARY
Synthetic RNAi-based drugs have shown ability to silence
different genes, acting in diseases with distinct pathogenesis,
from cell death by apoptosis to those caused by uncontrolled
cell proliferation. The established diseases for RNAi therapeu-
tics are: 1- death of kidney cells after injury by ischemia, cis-
platin or transplantation (QPI-1002); 2- tumor growth in dif-
ferent organs, and lung metastasis (TKM-080301, Atu027,
siG12D-LODER); 3- HCV genotype 1 infection in chronic
hepatitis C (miravirsen); 4- ophthalmic disorders, including
AMD, NAION, glaucoma, and ocular pain (PF-04523655,
QPI-1007, SYL040012, SYL1001); 5- transthyretin-mediated
amyloidosis (patisiran). Clinical trials revealed these products
are safe and well tolerated, as most adverse effects were mild
to moderate, and occurred during drug injection, either local-
ly or systemically. Infusion-related reactions after I.V. injec-
tions are transient and could be prevented by glucocorticoids,
antihistamines and others. In conclusion, RNAi therapeutic
drugs will take place in the pharmaceutical market in the
few coming years.
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