ArticlePDF AvailableLiterature Review

siRNA therapeutics: a clinical reality

April 2019
Science China. Life sciences 63(4)

April 2019
63(4)

Authors:

Sun Yat-Sen University

Since the revolutionary discovery of RNA interference (RNAi), a remarkable progress has been achieved in understanding and harnessing gene silencing mechanism; especially in small interfering RNA (siRNA) therapeutics. Despite its tremendous potential benefits, major challenges in most siRNA therapeutics remains unchanged—safe, efficient and target oriented delivery of siRNA. Twenty years after the discovery of RNAi, siRNA therapeutics finally charts its way into clinics. As we journey through the decades, we reminisce the history of siRNA discovery and its application in a myriad of disease treatments. Herein, we highlight the breakthroughs in siRNA therapeutics, with special feature on the first FDA approved RNAi therapeutics Onpattro (Patisiran) and the consideration of effective siRNA delivery system focusing on current siRNA nanocarrier in clinical trials. Lastly, we present some challenges and multiple barriers that are yet to be fully overcome in siRNA therapeutics.

Timeline of RNAi discovery and the progress of siRNA in clinical application.

…

Schematic representation of siRNA silencing mechanism.

…

The proper folding of TTR tetramer into protein and the misfolding of the tetramer which then translates into a misfolded protein, resulting in the formation of amyloid fibril. Patisiran (Onpattro) is a 100 nm multi-lipid component nanoparticle (SNALP) encapsulating siRNA to silence the mutant TTR gene responsible for tetramer misfolding in the liver of hATTR patients.

…

The pros and cons of siRNA delivery routes

…

Figures - uploaded by Phei Er Saw

Content may be subject to copyright.

Content uploaded by Phei Er Saw

Content may be subject to copyright.

SPECIAL TOPIC: Noncoding RNA: from dark matter to bright star April 2020 Vol.63 No.4: 485–500

•REVIEW•https://doi.org/10.1007/s11427-018-9438-y

siRNA therapeutics: a clinical reality

Phei Er Saw1& Er-Wei Song1,2,3*

1Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen

University, Guangzhou 510120, China;

2Breast Tumor Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510120, China;

3Zhongshan School of Medicine, Breast Surgery, Guangzhou 510080, China

Received September 20, 2018; accepted October 14, 2018; published online April 30, 2019

Since the revolutionary discovery of RNA interference (RNAi), a remarkable progress has been achieved in understanding and

harnessing gene silencing mechanism; especially in small interfering RNA (siRNA) therapeutics. Despite its tremendous

potential benefits, major challenges in most siRNA therapeutics remains unchanged—safe, efficient and target oriented delivery

of siRNA. Twenty years after the discovery of RNAi, siRNA therapeutics finally charts its way into clinics. As we journey

through the decades, we reminisce the history of siRNA discovery and its application in a myriad of disease treatments. Herein,

we highlight the breakthroughs in siRNA therapeutics, with special feature on the first FDA approved RNAi therapeutics

Onpattro (Patisiran) and the consideration of effective siRNA delivery system focusing on current siRNA nanocarrier in clinical

trials. Lastly, we present some challenges and multiple barriers that are yet to be fully overcome in siRNA therapeutics.

siRNA therapeutic, disease treatment, systemic siRNA delivery, clinical application

Citation: Saw, P.E., and Song, E.W. (2020). siRNA therapeutics: a clinical reality. Sci China Life Sci 63, 485–500. https://doi.org/10.1007/s11427-018-9438-y

In 1998, Fire et al. (1998) uncovered the mechanism of

RNAi and have since revolutionized the understanding of

gene regulation when they discovered that the silencing ef-

fectors in Caenorhabditis elegans were double stranded

RNAs. By using an RNA sequence that could interfere with

muscular functions, they treated C. elegans with either sin-

gle-stranded RNA (ssRNA) or double-stranded RNA

(dsRNA) and consistently found that ssRNAs were 10- to

100-fold less effective than dsRNA in silencing the same

mRNA. They observed that the occurrence of effective

knockdown led to phenotypical muscle twitching of C. ele-

gans that was observable. Importantly, the only way to in-

crease the effectiveness of ssRNA was to co-inject the

ssRNAs with the antisense strand, therefore suggesting that

ssRNA hybridization into dsRNA is a prerequisite for gene

silencing (Figure 1) (Sen and Blau, 2006).

siRNA: the “FIRST” of interventions

In mammalian cells. In 2001, Elbashir et al. (2001) first

demonstrated the RNAi mechanism in mammalian cell lines.

They used siRNA to specifically silence the expression of

different genes: siRNA against reporter genes coding for sea

pansy (Renilla reniformis, RL) and two sequence variants of

firefly (Photinus pyralis, GL2 and GL3) luciferases. After

transfection with the siRNAs, there was a significant re-

duction in gene expression level of all the silenced genes, and

the silencing effects were directly proportional to the level of

their endogenous gene expression. In animal models. In

2003, we demonstrated that siRNA targeting Fas protected

mice from fulminant hepatitis; and this work became the first

proof-of-principle that siRNAs could be utilized for the

treatment of a disease in an in vivo model. We first injected

Fas siRNAs into mice before sending Fas into hyperdrive to

mimic the liver failure disease model by hepatitis infections.

SCIENCE CHINA

Life Sciences

*Corresponding author (email: songew@mail.sysu.edu.cn)

All mice without siRNA intervention died of acute liver

failure within a few days, compared to an 82% survival rate

in siRNA-treated mice. Among these mice that survived,

their liver cells incorporated 80%–90% of the injected siR-

NAs (Song et al., 2003). The New England Journal of

Medicine (NEJM) highlighted this work and stated that “the

ability of Fas-specific siRNA to temper the destructive role

of Fas could find widespread clinical application in acute and

chronic liver diseases” (Davidson, 2003). Subsequently,

Hamar et al. (2004) continued to demonstrate the feasibility

of delivering Fas-specific siRNA in protecting mice against

renal ischemic reperfusion injury, verifying the effectiveness

of siRNA therapeutics in the treatment of a myriad of human

diseases (Lieberman et al., 2003). In non-human primates. In

2006, by encapsulating ApoB siRNAs in SNALP for-

mulation and intravenously delivered into non-human

primates, Zimmerman and colleagues showed that apoli-

poprotein B (ApoB) could be specifically silenced with

>90% silencing of ApoB mRNA in the liver even after 48-

h post-administration. Herein, they also proved the spe-

cificity of ApoB siRNA as the cleavage of apoB mRNA

occurred precisely on the site predicted for the RNAi

mechanism. Moreover, within 24 h after treatment, the

level of ApoB protein, serum cholesterol and low-density

lipoprotein levels were significantly reduced. Most im-

portantly, this effect is long-lasting with the highest dose

of siRNA treatment lasted for 11 d; demonstrating a potent

and lasting biological effect of siRNA treatment modality

(Zimmermann et al., 2006). In human. The first in-human

evidence of siRNA silencing was published by Davis and

colleagues in 2011. This data was obtained in conjunction

with the Phase 1 CALAA-1clinical trial (NCT00689065).

CALAA-1 is a nanoparticle delivery system carrying M2

subunit of ribonucleotide reductase (RRM2) siRNA and

was injected systematically into patients of solid cancers

via intravenous injection. Tumor biopsies from patients

obtained after the completion of treatment revealed two

major observations: (i) the delivery carrier was found

abundantly localized in the cytoplasm of cancer cells, and

these amounts correlate directly with the dose levels of

administered nanoparticles and (ii) significant reduction in

both RRM2 mRNA and protein level were detected in

patients. Similar with Zimmermann, they also detected

that the specific mRNA cleavage site corresponded to the

site of siRNA-specific mediated mRNA cleavage (Davis,

2009). In clinics. In August 2018, the Food and Drug

Administration (FDA) has approved the first ever RNA

interference drug–Alnylam’s Onpattro (Patisiran) for the

nerve damage caused by a rare disease hereditary trans-

thyretin-mediated amyloidosis (hATTR) in adults after the

encouraging result in the Phase 3 clinical trial, “Apollo”

(NCT01960348). In the largest cohort study of hATTR

amyloidosis, treatment with Onpattro improved poly-

neuropathy in a majority of patients by reversal of neu-

ropathy impairment of neuropathy impairment, showing a

significant improvement in the Quality of Life (QOL) of

patients as well (Adams et al., 2018).

siRNA: the REAL breakthrough

To circumvent the barriers in systemic siRNA delivery, non-

invasive delivery strategies for directing siRNAs specifically

to target cells needed to be devised (Dykxhoorn and Lie-

berman, 2005;Shankar et al., 2005). Therefore, we designed

a protamine-antibody fusion protein that could deliver siR-

NA specifically to HIV-infected or envelope-transfected

cells by conveniently mixing siRNA with the fusion protein

via electrostatic charge interactions (Song et al., 2005).

NEJM gave high commendations for this study as it pos-

sessed high targeted gene silencing efficacy in vivo despite

its beauty in simplicity (Williams, 2005) and pronounced this

as the magic bullet that fully demonstrated the potential for

successful systemic siRNA delivery. Subsequently, Morris-

sey et al. (2005a) developed a stable nucleic acid-lipid par-

ticle (famously known as SNALPs). As a strategy for siRNA

encapsulation and a delivery vehicle, they demonstrated that

SNALPs could protect and enhance the half-life of siRNAs

in circulation, while effectively reducing hepatitis B re-

plicons specifically in mice liver. Similar results were ob-

served when Zimmermann et al. (2006) demonstrated the

first gene silencing mechanism in non-human primates by

using ApoB-specific siRNA encapsulated in SNALPs to

downregulate ApoB protein, resulting in significant reduc-

tion of ApoB protein, serum cholesterol and low-density li-

poprotein levels.

Taken together, we and many others have proven that

Figure 1 Single stranded (sense or antisense) RNA or double-stranded

RNA encoding for a muscle protein was injected into C. elegans. Both

single-stranded RNA had no effect when injected separately, but started to

twitch when treated with double-stranded RNA, showing similar pheno-

types to C. elegans with defective muscle proteins. This indication denoted

that the gene coding for normal muscle movement had been silenced by the

dsRNA.

486 Saw, P.E., et al. Sci China Life Sci April (2020) Vol.63 No.4

siRNA therapeutics is indeed potent and effective against

various human diseases. Most importantly, our publications

also resulted in the intellectual property licensing of a potent

in vivo siRNA delivery system to Alnylam- the company that

owns Onpattro and several other siRNA therapeutics pipe-

line in clinical trials, therefore serving as a bridge between

academic and translational research. With on-going colla-

borations between our institution and Harvard Medical

School, we are creating a global avenue for international

clinical siRNA research that could accelerate bench-to-bed-

side transition and thus benefiting more patients in gene si-

lencing therapeutics. The timeline of RNAi discovery and

the progress of siRNA in clinical application is summarized

in Figure 2.

siRNA: the mechanism

siRNAs are short synthetic RNA duplexes commonly con-

sisted of two 21-mer oligonucleotides strand with 19 nu-

cleotides (nt) of complementary bases and a 2-nt overhang at

each 3′-end (de Fougerolles et al., 2005). Once in cell cy-

toplasm, this siRNA molecule will be incorporated into a

RNA-induced silencing complex (RISC), a nuclease-con-

taining multi-protein complex (Hammond et al., 2000). The

siRNA duplexes will be separated within the RISC complex.

The strand with the more stable 5′-end is integrated to the

active RISC complex. This siRNA then guides the RISC

complex to seek and cleave through the action of catalytic

RISC protein, a member of the argonaute family (Ago2).

(Figure 3) (Nair et al., 2014;Ryther et al., 2005).

siRNA: when silence is golden

RNAi therapeutics represent a paradigm shift in treating

human disease. The ability of siRNA to target and silence

virtually any gene of interest provides powerful new tools for

biomedical research and drug discovery (de Fougerolles et

al., 2007). In the development of small molecule drugs, many

oncogenic targets (i.e., RAS and MYC) fall into “un-

druggable” category since there is no “active binding site”

amenable by conventional small drug-like molecules (Sou-

cek and Evan, 2010). Therefore, the development of small

molecule inhibitors targeting these intractable proteins posed

major challenges (Dang et al., 2017;Soucek and Evan,

2010). The use of siRNA therapeutics could turn the tables

around for many devastating diseases, as siRNA could easily

be designed and the identification of a potent siRNA could

be done within weeks as compared to years for a drug de-

velopment (Petrocca and Lieberman, 2011). Besides, potent

siRNA sequences are usually active at extremely low (pi-

comolar) concentrations; and this could be designed for any

gene of interest with appropriate tools (Petrocca and Lie-

berman, 2011). Notably in cancer treatment, siRNA ther-

apeutics promise negligible side effects as compared to

conventional chemotherapeutics. Many have tried to silence

k-RAS or c-MYC siRNA in mouse models, hoping to de-

velop RNAi-based therapeutics by targeting these “un-

druggable” oncogenes (Bäumer et al., 2015;Wang et al.,

2005). By addressing targets that are otherwise untreatable

with existing medicines, RNAi not only promises an efficient

therapeutic modality, but also diminish the possibility of

drug resistance, a side effect so often crippled a che-

Figure 2 Timeline of RNAi discovery and the progress of siRNA in clinical application.

487

Saw, P.E., et al. Sci China Life Sci April (2020) Vol.63 No.4

motherapeutic regime. Table 1 below summarized the de-

tailed comparison between siRNA therapeutics and small

molecule drugs.

siRNA success story: Patisiran (Onpattro)

On August 10, 2018, the world is euphoric at the ground-

breaking announcement of the FDA approval of Patisiran

(Onpattro) or known as ALN-TTR02 for the treatment of

Hereditary Transthyretin Amyloidosis (hATTR). hATTR,

also referred to as transthyretin familial amyloid poly-

neuropathy (TTR-FAP) or familial amyloid cardiomyopathy

(TTR-FAC), is a rare, progressive, and fatal disease (Ando et

al., 2013;Conceição et al., 2016;Gertz, 2017). Unlike non-

hereditary, wild-type ATTR amyloidosis which primarily

affects only the heart, hATTR amyloidosis affects multiple

organs, such as the heart, nervous system, gastrointestinal

tract, and kidney, leading to various complications (Ando et

al., 2013;Coelho et al., 2013b;Hawkins et al., 2015). hATTR

amyloidosis is characterized by the extracellular deposition

of misfolded transthyretin (TTR) protein. In a normal folding

condition, TTR tetramer is made up of 4 single-chain

monomers. A mutation in TTR gene would destabilize the

tetramer and causes its dissociation into monomers, which

would then aggregate into amyloid fibrils and accumulate in

multiple organs throughout the body causing fatality (Figure

4) (Ando et al., 2013;Gertz, 2017;Johnson et al., 2012).

Since this TTR gene mutation occurs in the liver, silencing

the TTR gene promises to reverse the mutation effect of TTR

gene, therefore resolving the disease.

Earlier this year, Alnylam first presented the APOLLO

Phase 3 study of Onpattro (NCT01960348) at the 16th In-

ternational Symposium on Amyloidosis. These results were

later published in NEJM in July 2018, a month shy of its

FDA approval (Adams et al., 2018). In a series of clinical

assessment, Alnylam presented 87.8% mean serum TTR

knockdown for over 18 months, improvement in all primary

and secondary endpoints, met all exploratory endpoints in

cardiac subpopulations, 50% reduction in all-cause hospita-

lization and mortality, and 45% reduction in cardiac hospi-

talization and all-cause mortality. With these promising

results, Alnylam concluded that Patisiran is capable of sig-

nificantly improving multiple clinical manifestations of

hereditary transthyretin amyloidosis (Adams et al., 2018).

While the approval was historic, the fact remains that the

treatment is expensive as the drug is currently priced at

$450,000. Considering that siRNA treatment is transient and

that repetitive treatment might be needed, this could turn out

Figure 3 Schematic representation of siRNA silencing mechanism.

Table 1 Comparison between siRNA therapeutics and small molecule drug. Adapted from Bumcrot et al., 2006;Lam et al., 2015;Vaishnaw et al., 2010

siRNA Small molecule drug

Specificity High, sequence-driven Low-medium, conformation driven

Potency Typically pM Varies

Number of accessible targets >> 1000 500–1000

Nature of action Inhibition of target Inhibition/activation of target

Lead optimization Rapid, 4 to 8 weeks Slow, 2 to 4 years

Selectivity High Variable

Manufacturing Common, rapid, scalable Varies, can be complex

Serum stability Low (half-life of minutes) High (half-life of hours)

Toxicity Low High

Delivery Difficult Easy

Serum stability Stable Unstable

488 Saw, P.E., et al. Sci China Life Sci April (2020) Vol.63 No.4

to be a massive burden to patients. According to Alnylam’s

chief executive officer John Maraganore, this price-tag is no

surprise as the drug approval is the culmination of 16 years

of research and the investment of almost $2.5 billion. On top

of that, currently there are only about 3000 diagnosed

hATTR patients in US, thus the drug will not likely bring

much profit margin to Alnylam. Despite being the first RNAi

therapeutic approved, Patisiran will not be the only drug

available for hATTR. In July 2018, Tegsedi (Inotersen) a 2′-

O-methoxyethyl-modified antisense oligonucleotide devel-

oped by Akcea. Therapeutics was approved in Europe and is

currently being reviewed by the FDA while Vyndaqel (Ta-

famidis), also a small molecule drug owned by Pfizer re-

ceived Breakthrough Therapy designation from the FDA for

the treatment of patients with transthyretin cardiomyopathy.

With only an estimated 50,000 cases of hATTR worldwide,

some would argue that the approval of Onpattro is only a

drop in the ocean as compared to the drugs targeting major

diseases such as cancer. According to a statistic given by the

National Cancer Institute, in 2018 alone, an estimated

1,735,350 new cases of cancer will be diagnosed and

609,640 people will die from the disease in the United States.

Nevertheless, this is definitely a milestone in RNAi research

and we now see a glimpse of hope that could pave the way

for more RNAi therapeutics to be approved in the near fu-

ture. In an excerpt from FDA Commissioner Scott Gottlieb,

M.D., on the approval of Onpattro: “This approval is part of a

broader wave of advances that allow us to treat disease by

actually targeting the root cause, enabling us to arrest or

reverse a condition, rather than only being able to slow its

progression or treat its symptoms. In this case, the effects of

the disease cause a degeneration of the nerves, which can

manifest in pain, weakness and loss of mobility.”

siRNA: route of administration

The biggest hurdle in achieving potent siRNA therapeutics

did not lie on the design of potent siRNA, but rather on how

to administer biologically active siRNAs to the target tissue.

Various siRNA delivery strategies are being developed and

examined; and had been reviewed elsewhere (de Fougerolles

et al., 2007;de Fougerolles, 2008). Although there are var-

ious routes to administer siRNA, they are generally divided

into two major pathways: local and systemic delivery

(Whitehead et al., 2009). The choice of administration is

dependent on the accessibility of target site and desired ef-

fects, which would in turn determine the total dose of siRNA

required, the amount of siRNA successfully arriving at target

tissue, potential side effects and to consider the need of an

agent that facilitates delivery (Wittrup and Lieberman, 2015;

Zuckerman and Davis, 2015). One should always be in

check-and-balance and weigh their pros and cons when de-

ciding on the choice of siRNA administration route.

For local siRNA delivery, high concentration of siRNA

can be delivered to the target tissue via local injection, in-

travitreal, intranasal, topical absorption and many other

methods as described in Table 2. This proved to be useful in

the treatment of “locally-treatable” diseases such as age-re-

lated macular degeneration (AMD) affecting only the eyes

and therefore therapeutics should be given through in-

travitreal administration. Apart from higher bioavailability

of siRNA, local siRNA delivery method also promises re-

duction in adverse effects, simple formulation and ease of

administration among others (Akhtar and Benter, 2007).

Herein, we highlight some siRNA drug candidates currently

in clinical trials; targeting different diseases via local deliv-

ery (Table 3).

Nevertheless, systemic delivery of siRNA is vital for

Figure 4 The proper folding of TTR tetramer into protein and the misfolding of the tetramer which then translates into a misfolded protein, resulting in the

formation of amyloid fibril. Patisiran (Onpattro) is a 100 nm multi-lipid component nanoparticle (SNALP) encapsulating siRNA to silence the mutant TTR

gene responsible for tetramer misfolding in the liver of hATTR patients.

489

Saw, P.E., et al. Sci China Life Sci April (2020) Vol.63 No.4

siRNA to reach a specific target, or multiple organs, for

example diseases like cancer (Dunning et al., 2016; Kol-

dehoff and Elmaagacli, 2009; Koldehoff et al., 2010), hy-

percholesterolemia (Kosmas et al., 2018;Tadin-Strapps et

al., 2011), amyloidosis (Adams et al., 2018;Coelho et al.,

2013a), viral infections (Morrissey et al., 2005b), and others.

Also, siRNA should retain its biological activity upon

reaching target site. Therefore, it is vital for siRNAs to be

formulated into different carrier systems so that they may be

protected from nuclease digestion and have improved phar-

macokinetics and bioactivity (Morrissey et al., 2005a). Im-

proved nuclease-stability is especially important for siRNA

duplexes that are exposed to nuclease-rich environments

during systemic circulation as naked siRNAs will degrade in

serum within minutes (Layzer et al., 2004). Designing sys-

temic delivery carrier for siRNA do possess significantly

larger hurdles than local delivery and therefore causing some

drug candidates to be withdrawn or terminated at various

stages of trials (Kanasty et al., 2013). siRNA drug candidates

targeting different diseases via systemic delivery are listed

below in Table 4.

siRNA delivery: the hurdles

Theoretically, siRNA could be designed to target and silence

virtually all mRNA. However, the major obstacle in clinical

application of siRNA lies in developing safe and effective

method of delivering siRNA to target cells, and not in de-

signing potent siRNAs (Burnett and Rossi, 2012;Whitehead

et al., 2009). siRNA is a large (~13 kD) polyanionic mac-

romolecule and could not cross the cell membrane unaided

due to negative charge repulsion of cell membranes (de

Fougerolles et al., 2007;Robbins et al., 2008). siRNA re-

quires specific vehicles that could facilitate its intracellular

uptake and cytosolic delivery for bioactivity. Apart from that,

these delivery systems should circumvent all barriers faced

by siRNA; these include reducing its susceptibility to serum

nucleases, renal filtration, and uptake by the mononuclear

phagocyte system (depicted in Figure 5)(Jain and Styliano-

poulos, 2010;Kumari et al., 2011;Whitehead et al., 2009;

Wittrup and Lieberman, 2015). Consequently, much attempts

have been made to develop an efficient siRNA carrier which

would mitigate the barriers and toxicity issues. Generally,

delivery system is divided into viral or non-viral techniques.

Although virus-like nanoparticles proved to be highly effi-

cient in delivering siRNA, safety concerns surrounding the

long-term infusion of viral vectors rendered it less attractive

as compared to non-viral vectors, namely lipid-based nano-

particles (Saw et al., 2017;Zatsepin et al., 2016;Zimmer-

mann et al., 2006) and polymeric nanoparticles (Xu et al.,

2017;Xu et al., 2016). Besides encapsulation, siRNA could

also be adsorbed onto nanoparticle surface (Elbakry et al.,

2009;Steinbacher and Landry, 2014), complexed with po-

sitive-charged condensation agent (Beloor et al., 2015;Ta-

galakis et al., 2014), conjugation with other

biomacromolecules (Jeong et al., 2009;Springer and Dowdy,

2018), or being modified to increase siRNA stability

(Choung et al., 2006;Kenski et al., 2012;Terrazas and Kool,

2009). Multiple approaches to overcome siRNA delivery

hurdles are summarized in Table 5.

siRNA delivery systems in current clinical trials

There are innumerable publications on siRNA delivery

system every year, yet interestingly only few made it to the

clinical trial stage. Currently only three siRNA delivery

systems are in active clinical trials; namely lipid-based na-

noparticles (LNPs), N-acetylgalactosamine conjugated siR-

Table 2 The pros and cons of siRNA delivery routes

Route of administration Purpose Advantages Disadvantages

Intravenous injection Systemic delivery Broad distribution of siRNA, high localization

in liver

Non-specific, higher dose needed,

clearance by RES and renal

Subcutaneous injection Systemic delivery Broad distribution of siRNA, high localization

in liver, avoid RES and renal clearance Non-specific, skin toxicity

Local injection Localized delivery High localized concentration of siRNA, lower

dose needed, reduce systemic side effect

Not applicable to all organs

and tissues

Topical application Transepithelial absorption (oral,

rectal, vaginal mucosa)

Higher patient compliance, non-invasive, high

local concentration of siRNA, lower dose

needed, reduce systemic side effect

The need to bypass thick

mucosal layer

Intravitreal injection Localized delivery High local concentration, bypass systemic

barriers

Lower patient compliance, eye

irritation

Intrathecal/intraventricular

injection

Delivery to central nervous

system (CNS)

Bypass the dense blood-brain barrier, high

local concentration, reduce systemic side

effects

Low patient compliance, direct

toxicity to CNS

Inhalation/intranasal/

intratracheal administration Pulmonary delivery High local concentration, reduce systemic

side effects

Higher loss of drug in aerosol,

low patience compliance, especially

for intratracheal administration

490 Saw, P.E., et al. Sci China Life Sci April (2020) Vol.63 No.4

Table 3 Local delivery of siRNA-based therapeutics and their current status in clinical trialsa)

Drug

Method of

delivery/

Delivery vehicle

Disease Clinical Trial No. Phase Status Year

completed Ref.

Bevasiranib

Opko Health

Target: VEGF

Intravitreal/NC Age-related macular degeneration

NCT 00722384 Phase I Completed 2007 (Dejneka et al., 2008;

Garba and Mousa,

2010;Landa et al.,

2009;Singerman,

2009;Stepien et al.,

2009)

NCT 00259753 Phase II Completed 2007

NCT 00499590 Phase III Terminated 2009

NCT 00557791 Phase III Withdrawn N/A

Diabetic macular edema NCT 00306904 Phase II Completed 2007

AGN-745

Allergan

Target: VEGFR

Intravitreal/NC Age-related macular degeneration,

Choroidal neovascularization

NCT 00363714 Phase I/II Completed 2007 (Cho et al., 2009;

Kleinman et al., 2008)

NCT 00395057 Phase II Terminated 2009

ALN-RSV01

Alnylam

Target: RSV-N

Intranasal/NC Respiratory Syncytial Virus Infection

NCT 00496821 Phase II Completed 2007 (Alvarez et al., 2009;

DeVincenzo et al.,

2010;Zamora et al.,

2011)

NCT 00658086 Phase II Completed 2009

NCT 01065935 Phase IIb Completed 2012

TD101

Transderm

Target: k6a

Transdermal/NC Pachyonychia Congenita NCT 00716014 Phase I Completed 2008 (Leachman et al., 2010)

Excellair

Zabecor

Target: Syk

Intranasal/NC Asthma

– Phase I Completed 2009

(Burnett et al., 2011)

– Phase II On-going N/A

PF-655

Quark/Pfizer

Target: RTP801

Intravitreal/NC

Age-related macular degeneration NCT 00725686 Phase I Completed 2010

Choroidal neovascularization NCT 00713518 Phase II Completed 2011

Diabetic macular edema NCT01445899 Phase II Completed 2013

Diabetic retinopathy, diabetes

complications NCT 00701181 Phase II Terminated 2011

SYL1001

Sylentis

Target: TRPV1

Intravitreal/NC Ocular pain, dry eye syndrome

NCT 01438281 Phase I Completed 2012

NCT 01776658 Phase I/II Completed 2015

NCT 02455999 Phase II Completed 2016

NCT 03108664 Phase III Recruiting Est: 2018

SYL040012

(Bamosiran)

Sylentis

Target: β2-AR

Intravitreal/NC

Ocular hypertension, glaucoma NCT 00990743 Phase I Completed 2010

(Vaishnaw et al., 2010)

NCT 01227291 Phase I/II Completed 2012

Ocular hypertension, open angle

glaucoma NCT 01739244 Phase II Completed 2013

Open angle glaucoma NCT 02250612 Phase II Completed 2016

SIG12D

Silenseed

Target: KRAS

Implant/LODER

polymer

Pancreatic Ductal Adenocarcinoma,

Pancreatic cancer

NCT 01188785 Phase I Completed 2013

(Golan et al., 2015)

NCT 01676259 Phase II Recruiting Est: 2020

QPI-1007

Quark

Target: Caspase-2

Intravitreal/NC

Optic Atrophy, Non-arteritic Anterior

Ischemic Optic Neuropathy NCT 01064505 Phase I Completed 2013

Glaucoma, Angle closure, Primary,

Acute NCT 01965106 Phase II Completed 2015

Non-arteritic Anterior Ischemic Optic

Neuropathy NCT 02341560 Phase II/III Recruiting Est: 2020

RXI-109

Rxi

Target: CTGF

Transdermal/NC

Cicatrix, scar prevention NCT 01640912 Phase I Completed 2014

Hypertrophic scars NCT 02246465 Phase I Completed 2014

NCT 02030275 Phase II Completed 2016

Keloid NCT 02079168 Phase II Completed 2016

AMD, CN, Subretinal scarring,

subretinal fibrosis NCT 02599064 Phase I/II On-going Est: 2018

OLX-10010

OliX

Target: CTGF

Transdermal/NC Cicatrix, hypertrophic NCT 03569267 Phase I Recruiting Est: 2018

STP705

(Cotsiranib®)

Sirnanomics

Target: TGF-β and

Cox-2

Subcutaneous

injection/

polypeptide

nanoparticle

(PNP)

Hypertrophic scars NCT 02950317 Phase I Recruiting Est: 2018

a) VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor; RSV-N, human respiratory syncytial virus neuclo-

protein; k6a, keratin 6a; syk, spleen tyrosine kinase; RTP801 (also known as REDD1/Dig2/DDIT4), DNA damage inducible transcript 4; TRPV1, the

transient receptor potential cation channel subfamily V member 1; β2-AR, Adrenoreceptor Beta-2; KRAS, kirsten rat sarcoma; CTGF (also known as CCN2),

connective tissue growth factor; TGF-β, transforming growth factor-beta; COX2 (also known as PTGS2), prostaglandin-endoperoxide synthase 2.

491

Saw, P.E., et al. Sci China Life Sci April (2020) Vol.63 No.4

Table 4 Systemic delivery of siRNA-based therapeutics and their current status in clinical trials

Drug Method of

delivery Disease Clinical Trial No. Phase Status Year

completed Ref.

QPI-1002

Quark

Target: p53

Intravenous/NC

Acute renal failure, acute kidney injury NCT 00683553 Phase I Terminated 2010

(Demirjian et al.,

2017)

Delayed graft function, complication of

kidney transplant NCT 00802347 Phase I/II Completed 2014

Acute kidney injury NCT 02610283 Phase II Completed 2018

Delayed graft function NCT 02610296 Phase III On-going Est: 2019

Cardiac surgery NCT 03510897 Phase III On-going Est: 2021

TKM-ApoB

Tekmira

Target: ApoB

Intravenous/

SNALP Hypercholesterolemia NCT 00927459 Phase I Terminated 2010

(Jeffs et al., 2005;

Morrissey et al.,

2005b)

ALN-VSP02

Alnylam

Target: KSP and VEGF

Intravenous/LNP Solid tumors

NCT 00882180 Phase I Completed 2011 (Cervantes et al.,

2011;Tabernero et

al., 2013)

NCT01158079 Phase I Completed 2012

CALAA-01

Calandro

Target: RRM2

Intravenous/

Cyclodextrin

polymer-based

nanoparticles

Solid tumor, cancer NCT 00689065 Phase I Terminated 2012 (Davis, 2009;

Davis et al., 2010)

ALN-TTR01

Alnylam

Target: TTR

Intravenous/first

generation LNP

Transthyretin mediated amyloidosis

(ATTR) NCT 01148953 Phase I Completed 2012 (Coelho et al.,

2013a)

ALN-PCS02

Alnylam

Target: PCSK9

Intravenous/

Second

generation LNP

Elevated LDL-Cholesterol (LDL-C) NCT 01437059 Phase I Completed 2012 (Fitzgerald et al.,

2014)

TKM-PLK1

Tekmira

Target: PLK-1

Intravenous

/LNP

Colorectal cancer with hepatic

metastases, pancreas vancer eith

hepatic metastases, gastric cancer with

hepatic metastases, breast cancer with

hepatic metastases, ovarian cancer with

hepatic metastases

NCT 01437007 Phase I Completed 2012

(Northfelt et al.,

2013;Semple et

al., 2011)

Cancer, neuroendocrine tumors, NET,

adrenocortical carcinoma, ACC NCT 01262235 Phase I Completed 2015

TKM-100201

Tekmira

Target: VP24 and VP35

Intravenous/LNP Ebola virus infection NCT 01518881 Phase I Terminated 2012 (Dunning et al.,

2016)

BCR-ABL

University of

Duisburg-Essen

Target: BCR-ABL gene

Intravenous/

Anionic

liposome

Chronic myeloid leukemia – Phase I – –

(Koldehoff and

Elmaagacli, 2009;

Koldehoff et al.,

2010)

Atu-027

Silence

Target: PKN3

Intravenous/

cationic lipoplex

Advanced solid tumors NCT 00938574 Phase I Completed 2012 (Aleku et al., 2008;

Strumberg et al.,

2012a;Strumberg

et al., 2012b)

Carcinoma, pancreatic ductal NCT 01808638 Phase I/II Completed 2016

ND-L02-S0201

Nitto Biopharma

Target: HSP 47

Intravenous/

Vitamin A

coupled liposome

Fibrosis NCT 01858935 Phase I Completed 2014 (Sato et al., 2008;

Soule et al., 2018)

Moderate to extensive hepatic

fibrosis (METAVIR F3-4) NCT 02227459 Phase I Completed 2017

ARC AAT

Arrowhead

Target: AAT gene

Intravenous/

Dynamic

PolyConjugates

Alpha 1-antitrypsin dDeficiency

NCT 02363946 Phase I Terminated 2016

NCT 02900183 Phase II Withdrawn –

ARC 520

Arrowhead

Pharmaceutical

Target: coagulation factor

VII (F7)

Intravenous/

Dynamic

PolyConjugates

Hepatitis B

NCT 01872065 Phase I Terminated 2014

(Lanford R. E.,

2013;Schluep et

al., 2017;Yuen,

2014)

NCT 02535416 Phase I Completed 2016

NCT 02738008 Phase II Terminated 2016

NCT 02604199 Phase II Terminated 2017

Chronic hepatitis B

NCT 02349126 Phase II Terminated 2016

NCT 20604212 Phase II Terminated 2016

NCT 02452528 Phase II Terminated 2016

NCT 02065336 Phase II Terminated 2017

Hepatitis B, Hepatitis D NCT 02577029 Phase II Terminated 2016

ALN-PCSsc (Inclisiran)

Alnylam Target: PCSK9

Subcutaneous/

GalNAc

conjugate

Hypercholesterolemia NCT 02314442 Phase I Completed 2014

(Kosmas et al.,

2018)

Atherosclerotic cardiovascular disease

(ASCVD), familial

hypercholesterolemia (FH), diabetes

NCT 02597127 Phase II Completed 2017

Renal impairment NCT 03159416 Phase I Active, not

recruiting Est: 2018

(To be continued on the next page)

492 Saw, P.E., et al. Sci China Life Sci April (2020) Vol.63 No.4

(Continued)

Drug Method of

delivery Disease Clinical Trial No. Phase Status Year

completed Ref.

ALN-PCSsc (Inclisiran)

Alnylam Target: PCSK9

Subcutaneous/

GalNAc

conjugate

Homozygous FH NCT 02963311 Phase II Active, not

recruiting Est:2018

(Kosmas et al.,

2018)

Heterozygous FH, elevated cholesterol NCT 03397121 Phase III Active, not

recruiting Est: 2019

ASCVD, elevated cholesterol NCT 03400800 Phase III Active, not

recruiting Est: 2019

NCT 03399370 Phase III

ASCVD, Symptomatic atherosclerosis,

Type 2 Diabetes NCT 03060577 Phase II Active, not

recruiting Est: 2022

ASCVD NCT 03705234 Phase III Not yet

recruiting Est: 2049

ALN-TTRSC (Revusiran)

Alnylam

Target: TTR

Subcutaneous/

GalNAc

conjugate

Transthyretin-mediated amyloidosis

(ATTR)

NCT 01814839 Phase I Completed 2015

(Zimmermann et

al., 2013)

NCT 02797847 Phase I Completed 2018

NCT 01981837 Phase II Completed 2015

NCT 02292186 Phase II Completed 2017

TTR mediated familial amyloidotic

cardiomyopathy (FAC), amyloidosis,

hereditary, amyloid neuropathies,

familial amyloid neuropathies,

transthyretin cardiac amyloidosis

NCT 02319005 Phase III Completed 2017

DCR-MYC

Dicerna

Target: MYC

Intravenous/

LNP-formulated

Dicer substrate

siRNA

(DsiRNA)

Solid tumors, multiple myeloma,

non-hodgkins lymphoma, pancreatic,

neuroendocrine tumors, PNET, NHL

NCT 02110563 Phase I Terminated 2016

(Chipumuro et al.,

2016;Tolcher et

al., 2015)

ALN-AT3SC

(Fitusiran)

Alnylam

Target: AT3

Subcutaneous/

GalNAc

conjugate

Hemophilia A, B

NCT 02035605 Phase I Completed 2017

(Sorensen B et al.,

2015;Sorensen et

al., 2014)

NCT 02554773 Phase II On-going Est: 2021

NCT 03417245 Phase III Recruiting Est: 2019

NCT 03417102 Phase III Recruiting Est: 2019

Hemophilia A, B; inhibitors NCT 03549871 Phase III Recruiting Est: 2021

ALN-AS 1

(Givosiran)

Alnylam

Target: ALAS 1

Subcutaneous/

GalNAc

conjugate

Acute intermittent porphyria NCT 02452372 Phase I Completed 2017

NCT 02949830 Phase I/II On-going Est: 2020

AIP, acute hepatic porphyria (AHP),

acute porphyria (AP) NCT 03505853 Phase I Recruiting Est: 2018

AHP, AIP, AP, hereditary copropor-

phyria (HCP), variegate porphyria

(VP), ALA dehydratas, deficient por-

phyria (ADP)

NCT 03338816 Phase III On-going Est: 2021

ARB-1467

(TKM HBV)

Arbutus

Target: HbsAg

Intravenous/LNP Hepatitis B, Chronic NCT 02631096 Phase II Completed 2018

DCR-PHXC-101

Dicerna

Target: LDHA

Subcutaneous/

GalNAc

conjugate

Primary hyperoxaluria NCT 02956317 Phase I Recruiting Est: 2018

ARO-HBV

Arrowhead

Pharmaceutical

Target: HBV gene product

Subcutaneous/

TRiM platform Hepatitis B NCT 03365947 Phase I/II Recruiting Est: 2019

ARO-AAT

Arrowhead

Pharmaceutical

Target: AAT gene

Subcutaneous/

TRiM platform Alpha 1-antitrypsin deficiency NCT 03362242 Phase I On-going Est: 2019

AMG 890

(formerly ARO-LPA)

Amgen

Target: undisclosed

Subcutaneous/

TRiM platform Cardiovascular diseases NCT 03626662 Phase I Recruiting Est: 2019

siRNA-EphA2

MD Anderson

Target: EphA2

Intravenous/LNP Advanced cancers NCT 01591356 Phase I Recruiting Est: 2021 (Landen et al.,

2005)

a) ApoB, apolipoprotein B; KSP, kinesin-like protein; RRM2, ribonucleoside-diphosphate reductase subunit M2; TTR, transthyretin; PCSK9, Proprotein

convertase subtilisin/kexin type 9; PLK-1, Polo-like Kinase 1; VP24, Membrane-associated protein VP24; VP35, Membrane-associated protein VP35; BCR-

ABL, The ABL gene from chromosome 9 joins to the BCR gene on chromosome 22, to form the BCR-ABL fusion gene; PKN3, Serine/threonine-protein

kinase N3; HSP47, Heat Shock Protein 47; AT3, Antithrombin 3; ALAS1, Delta-aminolevulinate synthase 1; AAT, alpha-1 antitrypsin; EphA2, Ephrin type-

A receptor 2; LDHA, lactate dehydrogenase A.

493

Saw, P.E., et al. Sci China Life Sci April (2020) Vol.63 No.4

NA system (GalNAc-siRNA), and Targeted RNAi Molecule

(TRiM) platform. LNPs have several advantages compared

to other nanocarriers such as high in vivo compatibility, re-

latively low toxicity, ability to encapsulate large amount of

siRNA, biodegradable, stable in storage, straightforward

synthesis and scale-up procedures. Stable nucleic acid lipid

particles (SNALPs, 70–120 nm) are the second generation of

LNP formulation used in the development of Patisiran

(Onpattro) (Figure 6A). At present, many delivery systems in

clinical trials from Tekmira Pharmaceuticals and Alnylam

Pharmaceuticals are formulated using SNALPs and they are

currently in different stages of clinical trial for various dis-

Figure 5 The mechanism of siRNA and the barriers of siRNA delivery. Naked siRNA faces more biological barriers as compared to siRNA encapsulated,

adsorbed or attached to nanoparticles. Biological barriers include low serum stability, failure to extravasate due to poor tissue penetration, poor diffusion

through extra-cellular matrix, ineffective release from endosomes and failure to dissociate and release the siRNA from the carrier.

Table 5 Multiple approaches in circumventing siRNA delivery hurdles. Adapted from de Fougerolles et al., 2007;Dykxhoorn and Lieberman, 2006a,b;

Lieberman and Sharp, 2015;Petrocca and Lieberman, 2011;Wittrup and Lieberman, 2015

Avoiding excretion and improve

circulation half-life

PEGylation-increases molecular weight of siRNA to avoid renal clearance, simultaneously decreasing

anti-biofouling effect and opsonization by RES.

Nanoparticle formulation–usually around 10–100 nm, above the molecular weight cut-off for renal clearance.

Cholesterol conjugation of siRNA-cholesterol bounds to endogenous apolipoprotein resulting in longer

circulation time.

Avoiding nuclease degradation

Chemically modified siRNA-phosphothioate linkage to siRNA backbones confers nuclease resistance.

Nanoparticle formulation-protecting siRNA inside the core of delivery vehicle ensures that siRNA is biologically

active upon reaching target site.

Extravasation

Targets tissues with leaky vessels (i.e., liver, spleen or tumor tissues) that allow nanoparticles carrying siRNA to

extravasate out of blood vessels into target tissues.

Endothelial transcytosis-applicable to all tissues if carriers can be made to bind endothelial specific receptors to

induce transcytosis.

Specifically targets endothelial cells (i.e., endothelial cells in tumor tissues overexpressed VEGF and EDB) for

entry.

Binding to target cells and

intracellular transport

Targeting ligand-conjugates siRNA or delivery vehicle to cell specific receptor targeting moiety (i.e., peptide,

aptamer, antibody) for specific binding and subsequent endocytosis.

Hijacking endogenous ligand-cholesterol-conjugated siRNA and nanocarriers bind to apolipoproteins in

circulation therefore enhancing their liver homing properties.

Endosomal escape

Increases endosomal accumulation-even if siRNA or carriers do not possess endosomal escape mechanism,

sufficient amount of siRNA present could compensate for poor endosomal release.

Endosomal membrane destabilizing peptide/polymer/lipid-masked charge reversal peptide/polymer could be

activated in acidic pH resulting in unmasking positive charged particle therefore destabilizing the endosomal

membrane. Lipid nanoparticles are also activated by the low pH in endosome, resulting in reversed hexagonal

membrane formation, therefore destabilizing the membrane.

494 Saw, P.E., et al. Sci China Life Sci April (2020) Vol.63 No.4

eases (Singh and Peer, 2016). MD Anderson Cancer Center

developed a neutral-charged DOPC-based LNPs system to

encapsulate EphA2 siRNA (Landen et al., 2005) for the

treatment of advanced cancers, and have just started re-

cruitment for a Phase I clinical trial (NCT01591356).

GalNAc-conjugated siRNA system chemically links the

siRNA to a trimer of N-acetylgalactosamine (GalNAc)

(Foster et al., 2018;Nair et al., 2017;Springer and Dowdy,

2018). In preclinical rodent and non-human primate, it has

been proven that GalNAc binds with high avidity to the

Asialoglycoprotein receptor (ASGPR) which is pre-

dominantly expressed on liver hepatocytes (Morell et al.,

1971;Nair et al., 2014). Through subcutaneous administra-

tion, the GalNAc-conjugated siRNA then enters the blood

stream and localizes in the liver leading to the binding of

GalNAc ligand towards hepatocyte-restricted asialoglyo-

protein receptor (ASGPR). This specific binding causes he-

patocyte specific uptake of GalNAc-siRNA conjugate,

leading to durable gene silencing in the whole liver with

minimal toxicity. Interestingly, the GalNAc-siRNA con-

jugate system was predicted to eventually replace most

therapeutic LNPs for liver targets as the conjugates are

simpler in structure, cheaper to manufacture, better tolerated

and can be subcutaneously administered (Wittrup and Lie-

berman, 2015). Alnylam is currently running four clinical

trials based on GalNAc-siRNA conjugate system (with three

of them in Phase 3 clinical trial) (Figure 6B).

Recently, Arrowhead Pharmaceutics unveiled their new

targeted RNAi molecule (TRiM) platform which uses li-

gand-mediated tissue-specific targeted delivery of siRNA.

The uniqueness of this platform lies in its ability to screen for

potent siRNA, high affinity targeting ligands and linkers to

optimize the pharmacokinetics of each drug candidate,

leading to a more personalized siRNA delivery platform

(Figure 6C). This simple yet robust siRNA delivery system

could pave the way for next generation siRNA delivery as

simple manufacturing promises a reduction in production

cost combined with the selection of multiple administration

routes (i.e., subcutaneous injection and inhalation). The ad-

vantage of being small in size reduces potential safety and

possibility of inflammatory responses. Arrowhead Pharma-

ceutics currently have three TRiM mediated siRNA delivery

in clinical trials and others still under development.

siRNA: concurrent challenges

To date, almost 100,000 siRNA-related publications can be

found in PubMed. The first decade after siRNA discovery

(1998–2008) saw a steady exponential increase in siRNA-

related publications (Figure 7A). During this period of time,

RNAi became the “go-to” techniques for all research, and

this hype has led major pharmaceutic companies to invest

billions in RNAi therapeutics in hopes to treat major diseases

without speculating the hurdles and technical obstacles,

leading to the initiation of 14 clinical trials. Not too sur-

prisingly, most clinical trials in the early times failed to meet

expectations. In the most striking case, OPKO Health an-

nounced the shutdown of its Phase III trial of an RNAi

treatment for wet macular degeneration even after successful

Phase I and II trials. Other RNAi-based drugs provoked

strong innate immune reactions, failed to produce an im-

provement on the disease, or both (see full references in

Tables 4 and 5). In the latter decade (2008–2018), although

publications were maintained at about 7,000 publications per

year, there is an increase of clinical trials to 44 studies in-

itiated (Figure 7B), indicating that there is an improvement

in the clinical development of RNAi therapeutics. Never-

theless, this is roughly 0.06% successful translation rate from

laboratory to clinics. This seemingly unparallel translation

Figure 6 siRNA delivery system currently in clinical trials. A, Stable nucleic acid lipid particle (SNALP) developed by Alnylam, also the delivery vehicle

of Patisiran (Onpattro). B, siRNA conjugated to triantennary GalNac to form siRNA-GalNAc conjugate for specific hepatocyte targeting. C, Targeted RNAi

Molecule (TRIM platform) consisted of targeting ligand, linker and chemically stabilized siRNA for simple yet robust siRNA delivery.

495

Saw, P.E., et al. Sci China Life Sci April (2020) Vol.63 No.4

success of siRNA might suggest a direct evidence of diffi-

culties in developing clinically-translatable siRNA ther-

apeutics. Herein, we highlight some of the challenges that

still need to be addressed in siRNA delivery.

Long-term safety of siRNA. RNAi has just emerged in

clinical tests since early 2000. There is no long-term and

comprehensive patient safety data to prove that RNAi ther-

apeutic is absolutely safe and tolerable in human (Dana et al.,

2017). Since siRNA is a potent gene silencing machinery,

there is a high probability of off-target effects in which an

unrelated gene similar to the target gene can also be silenced

(Jackson and Linsley, 2010). Although this could be cir-

cumvented by lowering siRNA administration dose or in-

troducing modifications on the siRNA (i.e., circular siRNA,

Dicer-siRNA complex) (Zhang et al., 2018), such potential

can cause adverse effects on the patients’ health.

Off-target effect of siRNA. Although siRNA is generally

known to be specific with high probability of achieving the

desired on-target silencing, they could generate several types

of off-target effects. When an imperfect pairing of siRNA

and mRNA occurs (usually with sequence motifs in the

3′URT regions of mRNAs), it sets off microRNA-like off-

target silencing effect (Birmingham et al., 2006;Jackson et

al., 2003;Jackson et al., 2006). Interestingly, the delivery

vehicles used to deliver siRNA could be non-specifically

taken up by myeloid or dendritic cells, activating the Toll-

like receptors (TLR) and thus triggering an innate immune

response in these cells (Hornung et al., 2005;Judge et al.,

2005;Kariko et al., 2004;Robbins et al., 2009;Sioud, 2005;

Sledz et al., 2003)

Immunogenicity of siRNA. Based on the few published

data confirming siRNA’s non-immunogenicity, researchers

took it for granted that siRNA is generally non-immunogenic

and had not repeated the immunogenetic assay with all

siRNA sequences. Since all siRNA sequence is unique and

specific, they should all be tested individually to rule out the

possibility of becoming immunogenic especially after few

cycles of administration. Few published studies actually

measured serum cytokine levels after siRNA administration.

Most would simply employ control siRNAs to ensure that

immune stimulation or other unintended events did not oc-

cur.

Long-term safety of nanocarrier. Although numerous for-

mulations are being devised for siRNA delivery, focus

should remain on biological stability, specificity and safety

of these nanocarriers, which would ease bench-to-bedside

transition. Complex formulations involving multiple com-

ponents are not only difficult to scale-up but could also be

costly and challenging from a production regulatory stand-

point.

Immunogenicity of nanocarrier. Naked RNA may be poor

immunogen by itself, but siRNA conjugated to peptides,

polymers (i.e., PEGylation), antibodies could easily become

immunogenic. PEGylation is one of the most common way

to increase systemic stability of siRNA. However, the Phase

III trial of PEGylated RNA aptamer against factor IX (Re-

gado Biosciences) had to be discontinued due to severe

anaphylactic reaction to the PEG part of the conjugate

leading to death in some severe cases, raising significant

concerns in nanocarrier’s immunogenicity (Wittrup and

Lieberman, 2015).

Non-specific liver uptake in systemic siRNA delivery. As

the liver is the main blood-filtering organ, it is undoubtedly

easier to deliver RNAi-based therapeutics to liver cells than

to any other organs. Accordingly, development of siRNA

drugs has mostly focused on targets in the liver (Lieberman

and Sharp, 2015). However, long-term non-specific uptake

could lead to long-term liver toxicity. One should be focus-

ing on strategies that will bring about high target tissue (i.e.,

solid tumor) to liver ratio or increase siRNA bioavailability

at the target tissue, especially for non-liver targeted delivery,

thereby increasing therapeutic efficacy. If a suitable method

of siRNA delivery could be developed to minimize non-

specific liver uptake, the potential applications of RNAi-

based therapies can be greatly expanded.

Heterogeneity of disease (cancer). In a solid tumor, mul-

tiple mutations could emerge from different pathways at

various time-points. In other words, a single siRNA silencing

Figure 7 siRNA related publications and number of clinical trials in the first decade of RNAi discovery (A) and the latter decade (B).

496 Saw, P.E., et al. Sci China Life Sci April (2020) Vol.63 No.4

one specific oncogene might not be sufficient to effectively

cause long-term tumor inhibition. Several siRNAs can be

used in combination for targeting different survival pathways

to provide a synergistic silencing effect (also known as

cocktail siRNA). In addition, delivering siRNA in combi-

nation with currently FDA-approved anti-cancer drugs may

sensitize the treatment of a tumor (especially for drug re-

sistance tumors) and may become novel therapeutic mod-

alities (Behlke, 2006).

Long process of bench to bedside transition. The process

of a new drug development is excruciatingly long. With the

transition of siRNA drug in mind, one should consider in-

itiating early discussions with FDA to obtain expert re-

commendations that could speed up the development

process. Currently, it is estimated to take about 10 years from

target discovery to IND approval. Since RNAi therapeutics

are considered new as compared to conventional drug, the

hope is to obtain expedited IND approval for siRNA-based

drug to move these promising technologies towards clinical

application in a shorter period of time (Ozcan et al., 2015).

Conclusion

The FDA approval of Patisiran is timely and is seen as an

acknowledgement of the immense potential that siRNA

possess for treating a myriad of diseases. With many other

promising candidates currently at Phase 3 clinical trials, we

can predict more FDA approval of siRNA-based therapeutics

in the years to come. Combining meticulous design of the

target gene and siRNA delivery system, we envision that one

day we would be able to achieve a “knock-out” of all disease-

causing genes to eliminate incurable diseases such as pro-

geria, systemic lupus, Alzheimer, malignant brain tumors

and many more. siRNA therapeutics is now a clinical reality.

Acknowledgements This work was supported by the National Key Re-

search and Development Program of China (2016YFC1302300), the Na-

tional Natural Science Foundation of China (81720108029, 81621004, and

81490750), Guangdong Science and Technology Department

(2016B030229004), Guangzhou Science Technology and Innovation Com-

mission (201803040015), and the Fountain-Valley Life Sciences Fund of

University of Chinese Academy of Sciences Education Foundation.

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Recent Updates on Epigenetic-Based Pharmacotherapy for Atherosclerosis

Article

Full-text available

Apr 2024

Atherosclerosis is one of the most dominant pathological processes responsible in cardiovascular diseases (CVD) caused by cholesterol accumulation accompanied by inflammation in the arteries which will subsequently lead to further complications, including myocardial infarction and stroke. Although the incidence of atherosclerosis is decreasing in some countries, it is still considered the leading cause of death worldwide. Atherosclerosis is a vascular pathological process that is chronically inflammatory and is characterized by the invasion of inflammatory cells and cytokines. Many reports have unraveled the pivotal roles of epigenetics such as DNA methylation, post-translational histone modifications, and non-coding RNAs (ncRNAs) in atherogenesis, which regulate the expression of numerous genes related to various responsible pathways. Many studies have been conducted to develop new therapeutical approaches based on epigenetic changes for combating atherosclerosis. This review elaborates on recent updates on the development of new atherosclerosis drugs whose mechanism of action is associated with the modulation of DNA methylation, posttranslational histone modifications, and ncRNA-based gene regulation.

Identification of hub genes and therapeutic siRNAs to develop novel adjunctive therapy for Duchenne muscular dystrophy

Article

Full-text available

May 2024
BMC MUSCULOSKEL DIS

Objective Duchenne muscular dystrophy (DMD) is a devastating X-linked neuromuscular disorder caused by various defects in the dystrophin gene and still no universal therapy. This study aims to identify the hub genes unrelated to excessive immune response but responsible for DMD progression and explore therapeutic siRNAs, thereby providing a novel treatment. Methods Top ten hub genes for DMD were identified from GSE38417 dataset by using GEO2R and PPI networks based on Cytoscape analysis. The hub genes unrelated to excessive immune response were identified by GeneCards, and their expression was further verified in mdx and C57 mice at 2 and 4 months (M) by (RT-q) PCR and western blotting. Therapeutic siRNAs were deemed as those that could normalize the expression of the validated hub genes in transfected C2C12 cells. Results 855 up-regulated and 324 down-regulated DEGs were screened from GSE38417 dataset. Five of the top 10 hub genes were considered as the candidate genes unrelated to excessive immune response, and three of these candidates were consistently and significantly up-regulated in mdx mice at 2 M and 4 M when compared with age-matched C57 mice, including Col1a2, Fbn1 and Fn1. Furthermore, the three validated up-regulated candidate genes can be significantly down-regulated by three rational designed siRNA (p < 0.0001), respectively. Conclusion COL1A2, FBN1 and FN1 may be novel biomarkers for DMD, and the siRNAs designed in our study were help to develop adjunctive therapy for Duchenne muscular dystrophy.

Targeted siRNA Therapy for Psoriasis: Translating Preclinical Potential into Clinical Treatments

Article

Full-text available

May 2024

Psoriasis is a chronic inflammatory skin disease characterized by the excessive proliferation of keratinocytes and heightened immune activation. Targeting pathogenic genes through small interfering RNA (siRNA) therapy represents a promising strategy for the treatment of psoriasis. This mini-review provides a comprehensive summary of siRNA research targeting the pathogenesis of psoriasis, covering aspects such as keratinocyte function, inflammatory cell roles, preclinical animal studies, and siRNA delivery mechanisms. It details recent advancements in RNA interference that modulate key factors including keratinocyte proliferation (Fibroblast Growth Factor Receptor 2, FGFR2), apoptosis (Interferon Alpha Inducible Protein 6, G1P3), differentiation (Grainyhead Like Transcription Factor 2, GRHL2), and angiogenesis (Vascular Endothelial Growth Factor, VEGF); immune cell infiltration and inflammation (Tumor Necrosis Factor-Alpha, TNF-α; Interleukin-17, IL-17); and signaling pathways (JAK-STAT, Nuclear Factor Kappa B, NF-κB) that govern immunopathology. Despite significant advances in siRNA-targeted treatments for psoriasis, several challenges persist. Continued scientific developments promise the creation of more effective and safer siRNA medications, potentially enhancing the quality of life for psoriasis patients and revolutionizing treatments for other diseases. This article focuses on the most recent research advancements in targeting the pathogenesis of psoriasis with siRNA and explores its future therapeutic prospects.

Supramolecular Nanoparticles of Histone and Hyaluronic Acid for Co-Delivery of siRNA and Photosensitizer In Vitro

Article

Full-text available

May 2024
INT J MOL SCI

Small interfering RNA (siRNA) has significant potential as a treatment for cancer by targeting specific genes or molecular pathways involved in cancer development and progression. The addition of siRNA to other therapeutic strategies, like photodynamic therapy (PDT), can enhance the anticancer effects, providing synergistic benefits. Nevertheless, the effective delivery of siRNA into target cells remains an obstacle in cancer therapy. Herein, supramolecular nanoparticles were fabricated via the co-assembly of natural histone and hyaluronic acid for the co-delivery of HMGB1-siRNA and the photosensitizer chlorin e6 (Ce6) into the MCF-7 cell. The produced siRNA-Ce6 nanoparticles (siRNA-Ce6 NPs) have a spherical morphology and exhibit uniform distribution. In vitro experiments demonstrate that the siRNA-Ce6 NPs display good biocompatibility, enhanced cellular uptake, and improved cytotoxicity. These outcomes indicate that the nanoparticles constructed by the co-assembly of histone and hyaluronic acid hold enormous promise as a means of siRNA and photosensitizer co-delivery towards synergetic therapy.

Advancements in clinical RNA therapeutics: Present developments and prospective outlooks

Article

Full-text available

May 2024

RNA molecules have emerged as promising clinical therapeutics due to their ability to target “undruggable” proteins or molecules with high precision and minimal side effects. Nevertheless, the primary challenge in RNA therapeutics lies in rapid degradation and clearance from systemic circulation, the inability to traverse cell membranes, and the efficient intracellular delivery of bioactive RNA molecules. In this review, we explore the implications of RNAs in diseases and provide a chronological overview of the development of RNA therapeutics. Additionally, we summarize the technological advances in RNA-screening design, encompassing various RNA databases and design platforms. The paper then presents an update on FDA-approved RNA therapeutics and those currently undergoing clinical trials for various diseases, with a specific emphasis on RNA medicine and RNA vaccines.

Target Reprogramming the 3′UTR of Tumor‐Suppressor Genes by a siRNA Composite Nanoparticle for Glioblastoma Therapy

Article

Full-text available

Jun 2024
ADV FUNCT MATER

Glioblastoma multiforme (GBM) is the most aggressive and common adult brain tumor. Current therapies primarily involve surgical resection, followed by chemotherapy and radiotherapy. However, the low survival rate due to high toxicity, side effects, and poor targeting necessitates novel treatments. Post‐transcriptional regulation pathways, particularly the regulation of alternative polyadenylation based on the mRNA 3′UTR, play a critical role in cell growth and development and the progression of tumors. Targeting tumor cells and reprogramming the 3′UTR of tumor suppressor genes to achieve post‐transcriptional regulation is expected to be a new channel for GBM therapy. Herein, a novel siRNA delivery system is developed based on mesoporous silica nanoparticles: siRNA composite nanoparticles (siRNA CNP). Encapsulation in MSNs overcomes siRNA degradation and cellular entry issues, while lipid bilayer coating improves biocompatibility and stability. Surface‐modified nucleic acid aptamer SL1 (Apt‐SL1) significantly enhances GBM targeting, offering therapeutic potential. The findings show that siRNA CNP effectively silences the promoter CFIm25 of distal poly(A) in tumor cells, inducing gene reprogramming, inhibiting tumor growth, and promoting apoptosis in nude mice. The siRNA CNP demonstrates a significant effect in reprogramming tumor suppressor genes and treating GBM. It offers a novel and promising therapeutic avenue for GBM.

Effect of a novel siRNA delivery system, siRNA ternary complex, on melanoma lung metastasis

Article

May 2024
J DRUG TARGET

Emerging Biomarkers of Acute Myocardial Infarction, An Overview of the Newest MicroRNAs

Article

May 2023

Drugs targeting CTGF in the treatment of pulmonary fibrosis

Article

Full-text available

May 2024
J CELL MOL MED

Pulmonary fibrosis represents the final alteration seen in a wide variety of lung disorders characterized by increased fibroblast activity and the accumulation of substantial amounts of extracellular matrix, along with inflammatory damage and the breakdown of tissue architecture. This condition is marked by a significant mortality rate and a lack of effective treatments. The depositing of an excessive quantity of extracellular matrix protein follows the damage to lung capillaries and alveolar epithelial cells, leading to pulmonary fibrosis and irreversible damage to lung function. It has been proposed that the connective tissue growth factor (CTGF) plays a critical role in the advancement of pulmonary fibrosis by enhancing the accumulation of the extracellular matrix and exacerbating fibrosis. In this context, the significance of CTGF in pulmonary fibrosis is examined, and a summary of the development of drugs targeting CTGF for the treatment of pulmonary fibrosis is provided.

Layer‐by‐Layer Assembly of Renal‐Targeted Polymeric Nanoparticles for Robust Arginase‐2 Knockdown and Contrast‐Induced Acute Kidney Injury Prevention

Article

Apr 2024

The mitochondrial enzyme arginase‐2 (Arg‐2) is implicated in the pathophysiology of contrast‐induced acute kidney injury (CI‐AKI). Therefore, Arg‐2 represents a candid target for CI‐AKI prevention. Here, we developed layer‐by‐layer (LbL) assembled renal‐targeting polymeric nanoparticles to efficiently deliver small interfering RNA (siRNA), knockdown Arg‐2 expression in renal tubules, and evaluated prevention of CI‐AKI. Firstly, near‐infrared dye‐loaded poly(lactic‐co‐glycolic acid) (PLGA) anionic cores were electrostatically‐coated with cationic chitosan (CS) to facilitate the adsorption and stabilization of Arg‐2 siRNA. Next, nanoparticles were coated with anionic hyaluronan (HA) to provide protection against siRNA leakage and shielding against early clearance. Sequential layering of electrostatically adsorbed CS and HA improved loading capacity of Arg‐2 siRNA and yielded LbL‐assembled nanoparticles. Renal targeting and accumulation was enhanced by modifying the outermost layer of HA with a kidney targeting peptide (HA‐KTP). The resultant PLGA/CS/HA‐KTP siRNA nanoparticles exhibited proprietary accumulation in kidneys and proximal tubular cells at 24 hours post‐tail vein injection. In iohexol‐induced in vitro and in vivo CI‐AKI models, PLGA/CS/HA‐KTP siRNA nanoparticles alleviated oxidative and nitrification stress, and rescued mitochondrial dysfunction while reducing apoptosis, thereby demonstrating a robust and satisfactory therapeutic effect. Thus, PLGA/CS/HA‐KTP siRNA nanoparticles offer a promising candidate therapy to protect against CI‐AKI. This article is protected by copyright. All rights reserved

Inclisiran: A New Promising Agent in the Management of Hypercholesterolemia

Article

Full-text available

Jul 2018

The discovery of proprotein convertase subtilisin-kexin type 9 (PCSK9), a serine protease which binds to the low-density lipoprotein (LDL) receptors and targets the receptors for lysosomal degradation, offered an additional route through which plasma LDL-cholesterol (LDL-C) levels can be controlled. Initially, the therapeutic approaches to reduce circulating levels of PCSK9 were focused on the use of monoclonal antibodies. To that effect, evolocumab and alirocumab, two human monoclonal antibodies directed against PCSK9, given on a background of statin therapy, have been shown to markedly decrease LDL-C levels and significantly reduce cardiovascular risk. The small interfering RNA (siRNA) molecules have been used recently to target the hepatic production of PCSK9. siRNA interferes with the expression of specific genes with complementary nucleotide sequences by affecting the degradation of mRNA post-transcription, thus preventing translation. Inclisiran is a long-acting, synthetic siRNA directed against PCSK9 and it has been shown to significantly decrease hepatic production of PCSK9 and cause a marked reduction in LDL-C levels. This review aims to present and discuss the current clinical and scientific evidence pertaining to inclisiran, which is a new promising agent in the management of hypercholesterolemia.

Patisiran, an RNAi Therapeutic, for Hereditary Transthyretin Amyloidosis

Article

Full-text available

Jul 2018

BACKGROUND Patisiran, an investigational RNA interference therapeutic agent, specifically inhibits hepatic synthesis of transthyretin. METHODS In this phase 3 trial, we randomly assigned patients with hereditary transthyretin amyloidosis with polyneuropathy, in a 2:1 ratio, to receive intravenous patisiran (0.3 mg per kilogram of body weight) or placebo once every 3 weeks. The primary end point was the change from baseline in the modified Neuropathy Impairment Score+7 (mNIS+7; range, 0 to 304, with higher scores indicating more impairment) at 18 months. Other assessments included the Norfolk Quality of Life–Diabetic Neuropathy (Norfolk QOL-DN) questionnaire (range, −4 to 136, with higher scores indicating worse quality of life), 10-m walk test (with gait speed measured in meters per second), and modified body-mass index (modified BMI, defined as [weight in kilograms divided by square of height in meters] × albumin level in grams per liter; lower values indicated worse nutritional status). RESULTS A total of 225 patients underwent randomization (148 to the patisiran group and 77 to the placebo group). The mean (±SD) mNIS+7 at baseline was 80.9±41.5 in the patisiran group and 74.6±37.0 in the placebo group; the least-squares mean (±SE) change from baseline was −6.0±1.7 versus 28.0±2.6 (difference, −34.0 points; P<0.001) at 18 months. The mean (±SD) baseline Norfolk QOL-DN score was 59.6±28.2 in the patisiran group and 55.5±24.3 in the placebo group; the least-squares mean (±SE) change from baseline was −6.7±1.8 versus 14.4±2.7 (difference, −21.1 points; P<0.001) at 18 months. Patisiran also showed an effect on gait speed and modified BMI. At 18 months, the least-squares mean change from baseline in gait speed was 0.08±0.02 m per second with patisiran versus −0.24±0.04 m per second with placebo (difference, 0.31 m per second; P<0.001), and the least-squares mean change from baseline in the modified BMI was −3.7±9.6 versus −119.4±14.5 (difference, 115.7; P<0.001). Approximately 20% of the patients who received patisiran and 10% of those who received placebo had mild or moderate infusion-related reactions; the overall incidence and types of adverse events were similar in the two groups. CONCLUSIONS In this trial, patisiran improved multiple clinical manifestations of hereditary transthyretin amyloidosis. (Funded by Alnylam Pharmaceuticals; APOLLO ClinicalTrials .gov number, NCT01960348.)

Antimetastatic activity of Atu027, a liposomal small interfering RNA formulation, targeting protein kinase N3 (PKN3): Final results of a phase I study in patients with advanced solid tumors.

Article

May 2012

e13597 Background: Atu027 contains siRNA-lipoplexes, which elicits RNAi mediated suppression on PKN3 in vascular endothelial cells. In various xenograft mouse models, silencing of PKN3 expression and significant inhibition of invasive growth, lymph node and pulmonary metastasis formation was shown. Methods: Atu027 was applied to patients (pts) as a single 4h-infusion with subsequent follow-up for 3 wks. Thereafter pts were treated twice weekly for 4 weeks. In case of SD, pts were treated until PD. Dose escalation was associated with assessment of toxicity, pharmacokinetics (PK), and multiplex biomarker analyses in plasma from treated pts. Results: A total of 33 pts have received Atu027 of 11 dose levels (DL) up to 0.336 mg/kg. No pre-medication was required. No cytokine activation (TNF-α, IL-1β, IFN-γ, IL-6) was observed. In some subjects transient activation of the complement system (C3a, Bb, sC5b-9) was found, but without any clinical relevance. PK-data showed dose-dependent increase in plasma siRNA as well as lipid levels. Among various biomarkers tested, sVEGFR-1 plasma levels decreased significantly upon treatment. Across all dose levels, Atu027 was well-tolerated. Adverse events possibly related to Atu027 were fatigue grade G1 (6pts), hair loss G1 (2pts), sweating G1 (1pt), and abdominal pain G2 (1pt). G3 AEs not considered as DLTs were elevated lipase (2 pts, DL2+DL10) and diarrhea (1 pt, DL5). So far, no DLTs were seen in the last DL. Stable disease after 3 and 6 months was observed in 10 and 3 pts, respectively. Two pts with neuroendocrine cancer had disease stabilization for 9 and 12 months, respectively, including partial regression of pulmonary metastases in 1 pt. Another patient with breast cancer had regression of liver metastases. Conclusions: Atu027 is well-tolerated and anti-metastatic activity has been observed. Soluble VEGFR-1 might serve as a biomarker. So far, 0,336 mg/kg is the recommended dose for further phase II trials.

A phase I dose-escalation study of TKM-080301, a RNAi therapeutic directed against polo-like kinase 1 (PLK1), in patients with advanced solid tumors: Expansion cohort evaluation of biopsy samples for evidence of pharmacodynamic effects of PLK1 inhibition.

Article

May 2013

TPS2621 Background: TKM-080301 is a lipid nanoparticle formulation of a small interfering RNA (siRNA) directed against PLK1, a serine/threonine kinase that regulates multiple critical aspects of cell cycle progression and mitosis. Anti-tumor activity, RNA interference and pharmacodynamic effects of PLK1 inhibition have been conclusively demonstrated in preclinical models. Demonstration of pharmacodynamic effects of PLK1 inhibition in patient biopsy samples is an exploratory objective of this first-in-human study. Methods: TKME080301 is being evaluated in an open-label, non-randomized, dose-escalation study in patients with advanced solid tumors or lymphoma. Sequential cohorts of 3 to 6 patients receive TKME080301 as a 30-minute intravenous infusion on Days 1, 8 and 15 of a 28-day cycle. Treatment can continue until disease progression, based on overall clinical benefit. Tumor response is determined according to RECIST criteria. Primary study objectives include determination of safety, maximum tolerated dose and dose limiting toxicities. Secondary objectives include characterization of pharmacokinetics and the preliminary assessment of anti-tumor activity. Five cohorts have been enrolled and a tentative Phase 2 dose has been identified. An expansion cohort of 10 patients began enrolling in February, 2013. The focus of the expansion cohort will be to collect additional safety and pharmacokinetic data at the tentative Phase 2 dose, as well as pharmacodynamic data from mandatory biopsy samples. Pre- and post-dose biopsy samples will be evaluated for potential evidence of PLK1 inhibition using 5’ RACE (rapid amplification of cDNA ends) polymerase chain reaction (to identify the predicted PLK1 mRNA cleavage product), histology (to assess for the presence of aberrant mitotic figures) and immunohistochemistry. An update on enrollment and pharmacodynamic evaluations will be presented. Clinical trial information: NCT01262235.

A Subcutaneously Administered Investigational RNAi Therapeutic (ALN-AT3) Targeting Antithrombin for Treatment of Hemophilia: Interim Weekly and Monthly Dosing Results in Patients with Hemophilia A or B

Article

Dec 2015

Background: Hemophilia A and B are bleeding disorders characterized by a profound defect in thrombin generation (TG). Furthermore, in the presence of normal levels of endogenous anticoagulants a deficiency in factor VIII and IX results in major hemostatic imbalance and a bleeding phenotype. ALN-AT3 is a subcutaneously administered investigational RNAi therapeutic targeting the endogenous anticoagulant antithrombin (AT) that aims to restore the hemostatic balance by increasing TG. Methods: We are conducting a phase 1 multi-center study (NCT02035605) in healthy volunteers and patients with moderate to severe hemophilia A or B. Part A of this study has been completed and assessed a single ascending dose study in healthy volunteers. Parts B and C are multiple ascending dose studies in patients with hemophilia who are receiving weekly or monthly dosing, respectively. Primary endpoints are safety and tolerability. Secondary endpoints include PK, AT knockdown; change in thrombin generation and whole blood clot formation as measured by Calibrated Automated Thrombin generation and ROTEM thromboelastometry. Exploratory endpoints include evaluations of bleed pattern and control. Results: Part A enrolled 4 healthy volunteers, randomized (3:1) to 30 mcg/kg ALN-AT3 or placebo; no serious adverse events (SAE) or injection site reactions were observed. A total of 12 patients with severe hemophilia (10 hemohilia A; 2 hemophilia B) were enrolled in Part B and received 3 weekly subcutaneous doses of ALN-AT3 at 15 (n=3), 45 (n=6), and 75 (n=3) mcg/kg. Similar to part A, weekly administration of ALT-AT3 was generally safe and well tolerated in patients with hemophilia; no SAEs, discontinuations, clinical thromboembolic events or clinically significant D-dimer increases were reported. In the 75 mcg/kg dosing cohort, the mean maximum AT knockdown was 59% (p<0.05, relative to baseline), with nadir levels achieved between days 28 and 42. Maximum plasma AT knockdown of 86% was achieved, resulting in thrombin generation increases that correlated with AT knockdown and a bleed-free period of 114 days in the patient achieving the highest level of AT knockdown. The association between AT KD and TG was assessed in a post hoc exploratory analysis in which AT KD was categorized into tertiles. Part C aims to enroll several cohorts (n=3 per cohort) and will assess a monthly dosing schedule (x3 doses) of ALN-AT3. Patients in cohort 1 and 2 were dosed at 225 and 450 mcg/kg, respectively. Up to 4 additional cohorts may be enrolled within Part C. Updated safety, PK, AT knockdown, TG results as well as bleed patterns from Parts B and C will be presented. Conclusions: Emerging clinical data suggest that targeting AT could be a promising approach for restoring hemostatic balance in hemophilia. The potential for low volume subcutaneous administration, monthly dosing, and applicability to patients with hemophilia A and B with and without inhibitors make ALN-AT3 a potentially encouraging investigational therapy. Disclosures Pasi: Octapharma: Research Funding; Biogen, Octapharma, Genzyme, and Pfizer: Consultancy, Honoraria. Off Label Use: ALN-AT3 is an investigational RNAi therapeutic targeting the endogenous anticoagulant antithrombin.. Mant:Quintiles: Employment, Equity Ownership. Creagh:Bayer Healthcare UK: Honoraria. Austin:SOBI: Other: member of advisory board and received educational support; Pfizer: Other: member of advisory board and received educational support; Novo Nordisk: Other: member of advisory board and received educational support; CSL Behring: Other: member of advisory board and received educational support; Bio Products Laboratory: Other: member of advisory board and received educational support; Bayer: Other: member of advisory board and received educational support; Baxter: Other: member of advisory board and received educational support. Brand:Alnylam: Honoraria. Chowdary:Bayer: Consultancy; Biogen Idec: Consultancy; Baxter: Consultancy; CSL Behring: Consultancy, Research Funding; Novo Nordisk: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; SOBI: Consultancy. Ragni:Tacere Benitec: Membership on an entity's Board of Directors or advisory committees; Alnylam: Research Funding; Bristol Myers Squibb: Research Funding; Biogen: Research Funding; Shire: Membership on an entity's Board of Directors or advisory committees, Research Funding; Dimension: Research Funding; Bayer: Research Funding; SPARK: Research Funding; Genentech Roche: Research Funding; Pfizer: Research Funding; Vascular Medicine Institute: Research Funding; Baxalta: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Biomarin: Research Funding; CSL Behring: Research Funding. Chen:Alnylam Pharmaceuticals: Employment, Equity Ownership. Akinc:Alnylam Pharmaceuticals: Employment, Equity Ownership. Sorensen:Alnylam Pharmaceuticals: Employment, Equity Ownership. Rangarajan:Octapharma: Other: Investigator.

Safety and activity of DCR-MYC, a first-in-class Dicer-substrate small interfering RNA (DsiRNA) targeting MYC, in a phase I study in patients with advanced solid tumors.

Article

May 2015

Hereditary ATTR amyloidosis: burden of illness and diagnostic challenges

Article

Jun 2017
AM J MANAG CARE

Morie A Gertz

Hereditary transthyretin-mediated (hATTR) amyloidosis is a progressive disease characterized by deposition of amyloid fibrils in various organs and tissues of the body. There are a wide variety of clinical presentations for this multisystemic disorder, so it is often misdiagnosed or subject to delayed diagnosis. Although the exact prevalence is difficult to determine, existing estimates suggest a worldwide prevalence of 50,000 individuals, with varying phenotypic presentations of disease. Due to the heterogeneous nature of its presentation, incorrect or delayed diagnosis can severely impact quality of life for these patients. hATTR amyloidosis can lead to significant disability and mortality. After an accurate diagnosis of hATTR amyloidosis is established, new patients should undergo appropriate therapy as soon as possible. Current treatment options for hATTR amyloidosis are limited, but orthotopic liver transplant serves as an established option for patients with early-stage disease. Consequently, there is a need for new, effective, and safe therapies.

GalNAc-siRNA Conjugates: Leading the Way for Delivery of RNAi Therapeutics

Article

May 2018

Short-interfering RNA (siRNA)-induced RNAi responses have great potential to treat a wide variety of human diseases from cancer to pandemic viral outbreaks to Parkinson's Disease. However, before siRNAs can become drugs, they must overcome a billion years of evolutionary defenses designed to keep invading RNAs on the outside cells from getting to the inside of cells. Not surprisingly, significant effort has been placed in developing a wide array of delivery technologies. Foremost of these has been the development of N-acetylgalactosamine (GalNAc) siRNA conjugates for delivery to liver. Tris-GalNAc binds to the Asialoglycoprotein receptor that is highly expressed on hepatocytes resulting in rapid endocytosis. While the exact mechanism of escape across the endosomal lipid bilayer membrane remains unknown, sufficient amounts of siRNAs enter the cytoplasm to induce robust, target selective RNAi responses in vivo. Multiple GalNAc-siRNA conjugate clinical trials, including two phase III trials, are currently underway by three biotech companies to treat a wide variety of diseases. GalNAc-siRNA conjugates are a simple solution to the siRNA delivery problem for liver hepatocytes and have shown the RNAi (and antisense oligonucleotide) field the path forward for targeting other tissue types.

Safety, tolerability, and pharmacokinetics of BMS-986263/ND-L02-s0201, a novel targeted lipid nanoparticle delivering HSP47 siRNA, in healthy participants: A randomised, placebo-controlled, double-blind, phase 1 study

Article

Apr 2018
J HEPATOL

Advanced siRNA Designs Further Improve in vivo Performance of GalNAc-siRNA Conjugates

Article

Jan 2018

Significant progress has been made in the advancement of RNAi therapeutics by combining a synthetic triantennary N-acetylgalactosamine ligand targeting the asialoglycoprotein receptor with chemically modified siRNA designs, including the recently described Enhanced Stabilization Chemistry. This strategy has demonstrated robust RNAi-mediated gene silencing in liver after subcutaneous administration across species, including human. Here we demonstrate that substantial efficacy improvements can be achieved through further refinement of siRNA chemistry, optimizing the positioning of 2′-deoxy-2′-fluoro and 2′-O-methyl ribosugar modifications across both strands of the double stranded siRNA duplex to enhance stability without compromising intrinsic RNAi activity. To achieve this, we employed an iterative screening approach across multiple siRNAs to arrive at advanced designs with low 2′-deoxy-2′-fluoro content that yield significantly improved potency and duration in preclinical species, including non-human primate. Liver exposure data indicate that the improvement in potency is predominantly due to increased metabolic stability of the siRNA conjugates.

siRNA therapeutics: a clinical reality

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