Clinical experiences with systemically administered siRNA-based therapeutics in cancer.

Nature Reviews Drug Discovery | AOP, published online 16 November 2015; doi:10.1038/nrd4685

REVIEWS Clinical experiences with systemically administered siRNA-based therapeutics in cancer Jonathan E. Zuckerman1 and Mark E. Davis2

Abstract | Small interfering RNA (siRNA)-based therapies are emerging as a promising new anticancer approach, and a small number of Phase I clinical trials involving patients with solid tumours have now been completed. Encouraging results from these pioneering clinical studies show that these new therapeutics can successfully and safely inhibit targeted gene products in patients with cancer, and have taught us important lessons regarding appropriate dosages and schedules. In this Review, we critically assess these Phase I studies and discuss their implications for future clinical trial design. Key challenges and future directions in the development of siRNA-containing anticancer therapeutics are also considered. RNA interference (RNAi). An intrinsic pathway in eukaryotic cells by which short pieces of RNA are able to induce the degradation of mRNA transcripts containing a complementary sequence.

Small interfering RNA (siRNA). Synthetic double-stranded RNA molecules approximately 21–23 nucleotides long that are capable of engaging the intrinsic RNAi pathway when delivered to the cellular cytoplasm.

Department of Pathology and Laboratory Medicine, University of California Los Angeles, Los Angeles, California 90095, USA. 2 Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA. Correspondence to M.E.D. e-mail: [email protected] doi:10.1038/nrd4685 Published online 16 November 2015 1

The use of RNA interference (RNAi) has significantly altered the way biology is investigated, particularly in the study of functional genomics in mammals. The ability to selectively silence gene expression in vivo with small interfering RNA (siRNA) has been particularly helpful in unravelling mechanistic features of human diseases. However, it remains to be determined whether the use of siRNA will become a practiced therapeutic modality for the treatment of human diseases. If so, will RNAi be as revolutionary to therapy as it has been to the study of biology? Andrew Z. Fire considered this issue in his Nobel Lecture when receiving the 2006 Nobel Prize in Physiology or Medicine1, asking the following questions: “Will direct administration of interfering RNA be a useful clinical tool?” and “If a person has a tumor, why not take a gene that’s essential for that tumor and administer double stranded RNA corresponding to that gene to shut down growth of that tumor?” (REF. 1). Over the past decade, substantial progress has been made to answer these questions. Indeed, siRNA is now recognized as a possible game-changing way to treat cancer, with the potential to inhibit multiple gene targets simultaneously to maximize antitumour efficacy without increased toxicity. Although such anticancer effects have been demonstrated in animal models of cancer 2–6, it is now important to observe whether siRNA will have similar effects in the treatment of human diseases. Multiple ways to engage the RNAi pathway have been proposed and investigated2. The use of naked nucleic acids continues to be explored2–4, and various delivery systems to administer siRNA are also under investigation,

especially to enable targeting of genes that are located outside the liver and that cannot be reached by local administration2–9. Numerous reviews on RNAi therapeutics2–9 and their delivery systems10–13 are available. Additionally, there have been significant advances in optimizing the design of siRNAs to increase potency and limit off-target effects, including chemical modifications to minimize immunostimulatory effects14,15. The systemic administration of siRNA is important in anticancer treatment as this delivery route enables the therapeutic to reach both localized and metastatic cancers. However, specific challenges associated with using systemic administration of siRNA-based therapeutics for cancer, compared with those encountered when treating liver and kidney diseases, must be addressed (see below). Several Phase I studies of experimental siRNA-based anticancer therapeutics have recently been completed. These therapeutics all utilized delivery systems that encapsulate the siRNAs and are systemically administered to patients. In this Review, we assess these clinically tested siRNA-containing therapeutics and discuss key lessons learnt for the future development of such therapies and the design of new clinical studies. All of these Phase I studies involved patients with solid tumours that had not responded to standard therapy. Thus, many types of cancers have been treated. The siRNA targets selected for these studies are not limited to a specific cancer type but can be applicable to a broad spectrum of cancers; the advantages and limitations of this approach are also discussed.

NATURE REVIEWS | DRUG DISCOVERY

ADVANCE ONLINE PUBLICATION | 1 © 2015 Macmillan Publishers Limited. All rights reserved

REVIEWS

Systemic administration A route of administration of a drug into the circulatory system so that the entire body is affected. In the nanoparticle therapeutic field, this usually refers to intravenous administration.

RNA-induced silencing complex (RISC). The protein complex responsible for the identification and destruction of the target mRNA when engaged by siRNA.

Nanoparticles Generally refers to particles between 1 and 100 nm in size.

Liposomes A broad category of drug delivery vehicles based on a backbone of lipid bilayers.

siRNA to treat cancer Increasing numbers of cancer-related gene targets are being elucidated, providing mechanistic bases for a variety of cancer types. The potential use of siRNA as a therapeutic modality to attack cancer at these gene targets has several attractive features. First, essentially any gene can be targeted. Second, well-designed siRNAs can have potencies for mRNA inhibition that are on the order of single-digit, picomolar IC50 values (the concentration required for 50% inhibition)3. Third, chemical modifications and specific sequence designs can be utilized to minimize off-target and immunostimulatory effects without compromising potency and target specificity 4,14,15. Fourth, the RNAi mechanism of action is catalytic, resulting in extended siRNA inhibition of mRNA target expression even after a single dose. That is, siRNA-loaded RNA-induced silencing complex (RISC) is able to engage mRNA for sequence-specific cutting, after which the mRNA fragments are released to allow further cycles of mRNA binding and cleavage1,2. Bartlett and Davis16 showed that the duration of gene silencing is a function of the cell doubling time, which is presumably due to dilution. Gene silencing in nondividing cells can be on the order of a month or more both in vitro and in vivo, but as the time between cell division decreases (as is the case with cancer cells) the silencing time decreases16. These in vitro and in vivo results16,17 have been observed in many other studies, including clinical investigations, and the implications of these effects will be discussed further below as they are relevant to clinical dosing schedules. Fifth, multiple targets can be inhibited simultaneously without changing the fundamental physical composition of the therapeutic. This point is particularly important for the treatment of cancers. Recently, Yuan et al.18 showed that nanoparticles carrying three different siRNAs could be delivered to tumour xenografts. The simultaneous delivery of KRAS-, PIK3CA- and PIK3CB‑targeting siRNAs resulted in improved therapeutic efficacy with no increased toxicity 18. This study demonstrates how siRNA-based therapeutics can be used to simultaneously inhibit multiple gene targets to produce greater antitumour efficacy, without increasing toxicity. Systemic administration of siRNA The initial use of siRNA in clinical studies involved the local administration of naked siRNA molecules, for example, in the eye2–9. Although local administration continues to be explored, the investigation of systemic administration of siRNA has opened new avenues for clinical use. In 2007, an article was published reporting the results of a single patient with chronic myeloid leukaemia treated systemically with siRNA using liposomes as the delivery vehicle19. In the same year, the first clinical trial that systemically administered siRNA was conducted by Quark Pharmaceuticals, and involved the intravenous infusion of a naked siRNA (QPI‑1002, also known as I5NP) designed to inhibit the expression of p53 in the kidney 20 (Phase I and II trials, ClinicalTrials.gov identifier: NCT00802347). Because naked siRNA clears through the kidney, systemic administration of naked

siRNA is an appropriate way to target siRNA to tubule cells in the kidney. Now, numerous other clinical trials use systemic administration, involving either some type of synthetic component (for example, lipids or polymers) in the formulation to facilitate delivery or, more recently, direct conjugation of molecules that assist in the targeting of the diseased cells of interest. Several of these siRNA-based formulations are what would now be called nanoparticles10–13,21. For example, Alnylam Pharmaceuticals has published results from several clinical studies that utilized lipid nanoparticles22,23. The lipid nanoparticles were administered intravenously with the aim of delivering the siRNA to the liver to inhibit gene expression in hepatocytes. The investigational drug patisiran (also known as ALN‑TTR02) is a lipid nanoparticle that contains an siRNA that inhibits hepatocyte-derived transthyretin22. Patisiran is currently in a Phase III trial (NCT01960348), and is the most advanced siRNA-based investigational therapeutic so far. The conjugation of galactose-based agents that bind to the asialoglycoprotein receptor on hepatocytes enables systemic administration via either intravenous infusions or subcutaneous injections24. For example, revusiran (also known as ALN-TTRsc) is delivered via subcutaneous injections (NCT01814839 and NCT02292186). When contemplating the design of a platform therapeutic modality for the treatment of cancer, systemic administration is the preferred route of delivery as it enables the therapeutic to reach the broadest types of cancers (whether they are localized in specific tissues or metastatic). To date, siRNA administration to patients with cancer is primarily via intravenous infusions for systemic delivery (TABLE 1), although there is an example of localized delivery (siG12D LODER)25. Notably, in contrast to the use of antisense molecules for anticancer treatment 26, all the siRNA-based investigational anticancer therapeutics have formulations that include delivery systems. Four siRNA-containing experimental therapeutics have now completed Phase I trials: CALAA‑01 (REFS 27,28) , ALN-VSP 29 , Atu027 (REFS 30,31) and TKM‑PLK1 (REF. 32) (FIG. 1; TABLES 1,2). All four of these experimental therapeutics utilize delivery systems and are administered intravenously. Additionally, PNT2258, which uses single-stranded DNA (ssDNA) rather than siRNA, has also completed Phase I testing 33. FIGURE 2 (REFS 34,35) schematically illustrates the processes involved in the systemic administration of functional siRNA to cancer cells in a patient. If the gene target of interest is in cell populations other than the cancer cells, for example, tumour endothelial cells of the vasculature, then some of the steps may not be needed. Systemic administration of formulated siRNA has the potential to simultaneously reach multiple tumours within a patient (as seen with CALAA‑01 (REF. 27)). In most first‑in‑human trials involving patients with cancer, the disease has progressed with treatment with approved therapies, and many patients have metastatic disease. Therefore, unless specifically excluded (for example, with localized treatments), systemic administration will be necessary. As cancer cells will be growing and dividing, the siRNA dosing schedule for anticancer

2 | ADVANCE ONLINE PUBLICATION

www.nature.com/reviews/drugdisc © 2015 Macmillan Publishers Limited. All rights reserved

REVIEWS Table 1 | siRNA-based investigational therapeutics in clinical trials for cancer* Company

Investigational therapeutic

siRNA target

Delivery vehicle

Infusion time

Dosing schedule

ClinicalTrials.gov identifier (start date; current status)

Calando CALAA‑01 Pharmaceuticals

RRM2

Polymer (targeted)

30 min

Two 21-day cycles, two doses per week for the first two weeks

• NCT00689065 (Spring 2008; Phase I terminated)

Alnylam ALN-VSP (also Pharmaceuticals known as ALN‑VSP02)

VEGFA and KSP

Lipid (non-targeted)

15 min

Dosing every 14 days

• NCT00882180 (Spring 2009; Phase I completed) • NCT01158079 (Summer 2010; Phase I completed)

Silence Therapeutics

PKN3


4 h

Daily dosing for three weeks, then twice-weekly dosing for four weeks

• NCT00938574 (Summer 2009; Phase I completed) • NCT01808638 (Spring 2013; Phase I/II active, not recruiting)

Tekmira TKM‑PLK1 Pharmaceuticals (also known as TKM‑080301)

PLK1


30 min

Once-weekly dosing for three weeks

• NCT01262235 (Winter 2010; Phase I/II completed) • NCT01437007 (Summer 2011; Phase I completed) • NCT02191878 (Summer 2014; Phase I/II recruiting)

ProNAi Therapeutics

PNT2258 (single-stranded DNA)*

BCL2


2 h

21-day cycles, daily dosing for days 1–5 of each cycle

• NCT01191775 (Summer 2010; Phase I completed) • NCT01733238 (Winter 2012; Phase II active, not recruiting) • NCT02226965 (Winter 2014; Phase II recruiting)

Silenseed

siG12D LODER

KRAS

Biodegradable polymer matrix (local administration)

NA

NA

• NCT01188785 (Winter 2011; Phase I completed) • NCT01676259 (Winter 2015; Phase II not yet recruiting)

Senesco Technologies

SNS01‑T

eIF5AK50R plasmid eIF5A siRNA

Polyethylenimine (non-targeted)

Not reported

Twice-weekly dosing for six weeks

• NCT01435720 (Autumn 2011; Phase II active, not recruiting)

Mirna Therapeutics

MRX34 (microRNA mimic)*

miR‑34


Not reported

21 day cycle, daily • NCT01829971 dosing for five (Spring 2013; Phase I recruiting) days followed by a two-week break

Dicerna DCR-MYC Pharmaceuticals

MYC


1 h

Once-weekly dosing for two weeks followed by a one-week break

• NCT02110563 (Spring 2014; Phase I recruiting) • NCT02314052 (Winter 2014; Phase Ib/II recruiting)

M.D. Anderson Cancer Center

EPHA2


Not reported

Twice-weekly dosing for three weeks

• NCT01591356 (Summer 2015; Phase I recruiting)

Atu027

siRNA‑EPHA2‑ DOPC

DOPC, 1,2‑dioleoyl-sn-glycero‑3‑phosphatidylcholine; eIF5A, eukaryotic translation initiation factor 5A; EPHA2, ephrin type-A receptor 2; KSP, kinesin spindle protein; NA, not applicable; PKN3, protein kinase N3; PLK1, polo-like kinas 1; RRM2, ribonucleotide reductase subunit M2; siRNA, small interfering RNA; VEGFA, vascular endothelial growth factor A. *PNT2258 and MRX34 do not contain siRNA.

treatment will be significantly different to that for liver diseases, whereby hepatocytes are not undergoing rapid cell division. Therefore, as patients with cancer will be dosed at a frequency that is appropriate to treat a disease involving rapidly growing and dividing cells, one must be mindful of the localization and off-target effects of the investigational therapeutic in tissues that are not experiencing cell cycling. For example, nanoparticle delivery

systems tend to localize in the liver, spleen and kidney. However, such passive targeting is beneficial for other diseases; for example, lipid delivery systems localize in the liver, which is why they are used to deliver siRNA for the treatment of liver diseases. To date, no siRNA dosedependent accumulation effects have been recorded that saturate the RNAi pathway in the livers of animals or patients when dosing on schedules to treat cancers



REVIEWS (discussed below); such effects have been observed with expressed short hairpin RNAs (shRNAs)36. Additionally, there have not been any reports of tachyphylaxis-like responses to siRNA treatments in animals or humans.

Cytokines A large group of proteins, peptides or glycoproteins that are secreted by specific cells of the immune system. Cytokines are a category of signalling molecules that mediate and regulate immunity, inflammation and haematopoiesis.

Complement An innate part of the mammalian immune system that complements the ability of antibodies and phagocytic cells to clear pathogens from an organism.

Phase I cancer trials with siRNA Results from Phase I trials evaluating siRNAs (and also PNT2258 (see below)) in the treatment of cancer are now available. The commonalities between the trials are: the experimental therapeutics involve nanoparticle delivery; each is a first‑in‑human trial for patients with solid tumours; and the dosing is via intravenous, systemic administration.

of CALAA‑01 and evidence of siRNA-induced gene knockdown. Fluorescence-based microscopy using a CALAA‑01‑specific stain revealed a dose-dependent accumulation of CALAA‑01 within tumours but not adjacent tissue27. RRM2 protein and mRNA were inhibited in these patients’ tumours post-treatment. Finally, the RNAi mechanism of action of CALAA‑01 was demonstrated via rapid amplification of cDNA ends (RACE; a 5ʹ‑RNA-ligand-mediated PCR technique) analysis, which showed siRNA-induced RRM2 mRNA cleavage fragments in post-treatment tumour samples (at both 24 mg siRNA per m2 (results obtained after the publication of REF. 27) and 30 mg siRNA per m2)27,28.

CALAA‑01. CALAA‑01 (FIG. 1a) is a four-component, polymer-based nanoparticle (~75 nm in diameter) siRNA delivery system. The system is composed of a linear, cationic cyclodextrin-based polymer (adamantane polyethylene glycol (PEG)) that is used as a surface modifier on the nanoparticle to provide steric stabilization, a human transferrin protein-targeting agent and an siRNA designed to reduce the expression of the M2 subunit of ribonucleotide reductase (RRM2)37. RRM2 is a key enzyme in nucleic acid metabolism and is upregulated in many tumour types27,28,34,37. RRM2 suppression is proposed to result in cell cycle arrest and cell death37. The CALAA‑01 Phase I trial (TABLES 1,2) included a Phase Ia open-label, multi-centre, dose-escalation study (3–30 mg per m2) and a Phase Ib extension study (18–27 mg per m2) of patients with advanced solid tumours27,28. CALAA‑01 treatment was continued until disease progressed or treatment was no longer tolerated. A single patient with metastatic melanoma achieved stable disease for 4 months with 30 mg siRNA per m2; however, no other objective tumour responses were observed. Overall, 21% of the patients discontinued the study due to an adverse event. The majority of severe adverse events occurred following a year-long enrolment gap in the study. It was proposed that drug instability (specifically the transferrin-targeting ligand) may have been responsible for the adverse effects observed after the enrolment time gap, because the severity and types of adverse events significantly changed between treatments before and after the recruitment gap28. No maximal tolerable dose was determined in this study, and no clear association between siRNA plasma concentration and adverse events was observed. Furthermore, there was no correlation between elevated plasma levels of cytokines and the severity of adverse effects. No significant changes in serum complement activity were observed following CALAA‑01 administration28. Mild, dose-dependent elevations in some serum cytokines were measured; all elevations were transient in nature, peaking at 4–6 hours post-infusion and returning to baseline by 24 hours. No clinically significant alterations to liver or kidney function were observed28. Pre- and post-treatment biopsies from patients with melanoma (at three increasing doses: 18, 24 and 30 mg siRNA per m2) were obtained during the study. These samples were evaluated for the presence

ALN-VSP. ALN-VSP (FIG. 1b) is a nearly neutral lipid nanoparticle formulation (80‑100 nm in diameter) containing two different chemically modified siRNAs in a 1:1 molar ratio29,38. The siRNAs in ALN-VSP target vascular endothelial growth factor A (VEGFA) and kinesin spindle protein (KSP). Targeting of VEGF is proposed to modulate tumour angiogenesis29, whereas KSP is essential for mitotic spindle formation in proliferative cells. Both proteins show increased expression in various tumour types29. The ALN-VSP Phase I study (TABLES 1,2) was an openlabel, 3 + 3 design, dose-escalation study (0.1–1.5 mg siRNA per kg)29. Treatments were carried out over 4 months. Patients without progressive disease at that time were eligible to continue treatment in an extension study. Four patients who received >0.7 mg siRNA per kg ALN-VSP achieved disease control (stable disease or better at 6 months). A complete response occurred in one patient who remained in remission at the time of study publication. Seven patients receiving 0.4–1 mg siRNA per kg ALN-VSP continued to the extension study, with an average treatment time of 11.3 months. Dynamic contrast-enhanced MRI was used to determine whether ALN-VSP treatment was associated with reduced tumour blood flow, which is proposed to be indicative of an anti-VEGF effect. Twenty-eight patients were evaluable for this study, of which 46% had a ≥40% decrease in transfer constant (KTrans) and a parallel decrease in initial area under the gadolinium concentration time curve. However, there was no evidence of dose-dependent reductions in tumour blood flow indicators and no overall correlation between the change in KTrans and tumour response29. Cytokine induction and symptoms related to cytokine activation (chills and rigor) were observed predominantly in the 1.25 mg siRNA per kg dosing cohort at 4–8 hours after infusion. No significant changes in liver function tests were observed in 40 out of 41 patients. One patient with >70% liver involvement by metastatic primitive neuroectodermal tumour and prior partial hepatectomy and splenectomy developed hepatic failure after the second dose of ALN-VSP (0.7 mg siRNA per kg) and subsequently died. The liver failure was proposed to be due to massive tumour necrosis, progression of extensive disease or ALN-VSP toxicity to the scant remaining live parenchyma.



REVIEWS a

c

e

Transferrin

siRNA targeting RRM2

~75 nm

~100 nm

b

Single-stranded DNA targeting BCL2

siRNA targeting PKN3

~130 nm

d siRNA-I targeting VEGF siRNA targeting PLK1

siRNA-II targeting KSP 80–100 nm

~80 nm

Figure 1 | Schematic illustrations of the siRNA-based therapeutics used in Phase I trials to treat patients with Nature | Drug solid cancers. a | CALAA‑01 is a polymer-based nanoparticle containing a targeting ligand onReviews its surface (theDiscovery human protein transferrin) and a small interfering RNA (siRNA) that targets the M2 subunit of ribonucleotide reductase (RRM2). b | ALN-VSP is a lipid-based nanoparticle that contains two different siRNAs that target vascular endothelial growth factor A (VEGFA) and kinesin spindle protein (KSP). c | Atu27 is a lipid-based nanoparticles that contains an siRNA that targets protein kinase N3 (PKN3). d | TKM‑PLK1 is a lipid-based nanoparticle that contains an siRNA that targets polo-like kinase 1 (PLK1). e | PNT2258 is a lipid-based nanoparticle that contains single-stranded DNA (rather than siRNA) that targets BCL2.

Eastern Cooperative Oncology Group performance status A quantification of the general well-being, daily activity and physical ability of a patient with cancer. In the setting of a clinical trial, set performance status cut-off levels are one criterion used to determine eligibility for trial enrolment and treatment.

Pharmacodynamics The study of the biochemical and physiological effects of a drug on the body, including mechanistic action, duration of action, therapeutic effects and toxicities.

The level of tumour-specific delivery of the VEGFAand KSP-targeted siRNAs was measured in 12 patients who volunteered to have pre- and post-treatment biopsies. The average siRNA concentration of both siRNAs in tissue was similar (VEGFA: 21.3 ± 39.1 ng per g tissue; KSP: 13.6 ± 20.8 ng per g tissue). Two patients in this study population had post-treatment biopsies with >95% viable tumour, suggesting that both siRNAs could be successfully delivered to tumour tissue. However, the proportion of siRNA in tumour versus other tissue types could not be determined in this study. VEGFA and KSP mRNA levels were also measured in these samples. Three patients had significant VEGF mRNA knockdown (range of 20–75%). Of these patients, only one was observed to also have a concomitant significant KSP mRNA knockdown. No patients were observed to have solely KSP mRNA knockdown. siRNA mechanisms of action were confirmed via RACE analysis (VEGFA siRNA cleavage product only) in two patients; however, the majority of the biopsy tissue for these studies was normal liver with little viable tumour. Atu027. Atu027 (FIG. 1c) is a cationic lipoplex-based siRNA delivery system composed of a blunt ended, chemically modified 23‑mer RNA oligonucleotide (siRNA payload) and three cationic lipids30,31. The siRNA payload of Atu027 targets protein kinase N3 (PKN3) gene expression in tumour and secondary organ vascular endothelium. PKN3 inhibition is proposed to stabilize vessel integrity and attenuate inflammatory responses in the vasculature of tumours and secondary organ sites via modulation of actin and adherens junction dynamics31. These effects are thought to reduce the vascular permeability of both tumour and host vasculature, thereby inhibiting mobilization and engraftment of metastatic tumour cells.

The Atu027 Phase I study (TABLES 1,2) was a 7‑week, openlabel, single-centre, monotherapeutic, dose-escalation study in patients with advanced solid tumours. Patients with stable disease after this period could continue on treatment until disease progressed. Disease stabilization was observed in 52% of the study participants, including 8 patients who had stable disease at the end of the study (6 of these patients continued on compassionate use). Stabilization or partial regression of metastatic lesions was observed 1 week after the last treatment dose in 50% of the patients assessed. Eleven patients achieved treatment response at the end of the study. One patient showed complete regression of pulmonary lesions. There was a 10% improvement in the Eastern Cooperative Oncology Group performance status in treated patients, which was associated with high doses. Transient elevations in complement activation products (C3a, Bb and SC5b‑9) were observed in patients at all dosing levels. However, no allergic reactions were observed. Additionally, no antibodies against the siRNA molecule of Atu027 were detected. Regarding the pharmacodynamics of Atu027, the soluble variant of vascular endothelial growth factor receptor 1 (sVEGFR1 (sFLT)) was significantly decreased in 12 out of the 20 patients after repeated Atu027 treatments. Only a single dose-limiting toxic event (serum lipase elevation) was observed. No maximal tolerable dose was reached in this study. Atu027 is currently being evaluated in a Phase II trial in combination with gemcitabine for patients with locally advanced or metastatic pancreatic adenocarcinoma (NCT01808638). As of the summer of 2014, the Phase IIa portion of this study had completed enrolment.



REVIEWS Table 2 | Results from clinical trials for cancer that used siRNA-based investigational therapeutics* Thera peutic

Trial size

Treatment regimen

Pretreatment medications

Common adverse events

CALAA‑01 24

0.60 mg siRNA per kg dose levels exhibited clinical benefit (three stable disease, one partial response). Transient cytokine activation occurred — peaking and normalizing at 6 hours and 25 hours post-dose, respectively — which was associated with peak TKM‑080301 levels. The maximal tolerable dose was estimated to be 0.75 mg siRNA per kg. Pre- and post-dose liver lesion biopsy specimens from one patient

were collected. RACE analysis of the post-dose specimen confirmed the presence of the PLK1 mRNA cleavage product. TKM‑080301 is currently in Phase II trials as monotherapy for hepatocellular carcinoma (NCT02191878), and gastrointestinal neuroendocrine tumours and adreno cortical carcinoma (NCT01262235). Enrolment in these clinical studies was completed at the end of 2014. PNT2258. PNT2258 (FIG. 1e) is a liposomal formulation (~130 nm in diameter) consisting of a 24‑base chemically unmodified DNA oligonucleotide (designated PNT100) and four lipid molecules33. The therapeutic nucleic acid payload, PNT100, binds to the 5ʹ‑untranscribed regulatory regions of the BCL2 gene and blocks transcription of BCL2 via DNA interference33. Blocking BCL2 expression promotes cancer cell apoptosis. Although PNT2258 does not induce RNAi, the results of the trial are discussed here because the study also involved a systemically administered formulation of a nucleic acid for anticancer treatment33.



REVIEWS The Phase I study of PNT2258 (TABLES 1,2) was an openlabel, single-centre, monotherapeutic, dose-escalation study in patients with advanced solid tumours33. Patients received PNT2258 doses ranging from 1 to 150 mg DNA per m2 over a median of 2 completed cycles. Twentyseven per cent of patients achieved stable disease at the end of cycle 2, as confirmed by CT scans. Four patients (two with non-small cell lung cancer, two with sarcoma) remained on the study for >4 cycles. PNT2258 exhibited a dose-dependent increase in AUC. A less than dose proportional increase in the AUC was observed in higher dosing cohorts, which was suggested to be due to achieving the maximum PNT2258 exposure. Additionally, extension of the half-life of PNT2258 was observed with multiple doses, suggesting prolonged clearance kinetics due to saturation of the mononuclear phagocyte system (MPS). PNT2258 administration resulted in decreased lymphocyte and platelet counts (surrogate serum markers for BCL2 inhibition). Lymphocyte and platelet counts reached a nadir (≤ grade 1) within hours and at 9 days post-infusion, respectively. PNT2258 is currently in a Phase II trial for relapsed refractory non-Hodgkin lymphoma (NCT01733238) and diffuse large B‑cell lymphoma (NCT02226965).

Critical evaluation of Phase I trials with siRNA Critical evaluations of and lessons learnt from the pioneering clinical trials of the siRNA-based therapeutics CALAA‑01 (REFS 27,28), ALN-VSP29,38, Atu027 (REFS 30,31) and TKM‑PLK1 (REF. 32) , as well as of PNT2258 (which contains single-stranded DNA) are discussed below. siRNA design, delivery and dosing. The siRNAs used in these first‑in‑human trials all engage the RNAi pathway at RISC and targeted different genes. The potencies of the siRNA varied between nanomolar (CALAA‑01 (REF. 37)) and picomolar (ALN-VSP3) IC50 values for in vitro mRNA inhibition. As picomolar IC50 values can now be routinely obtained for most gene targets, it is anticipated that these values of siRNA potencies will become commonplace in future clinical studies. Thus, issues other than potency will probably drive the design of siRNAs that are used in future investigational therapeutics. All of the siRNAs with the exception of CALAA‑01 were chemically modified. As reviewed elsewhere, chemical modifications are made for multiple reasons, for example, to enhance nuclease stability, modulate immune stimulation and/or minimize offtarget effects14,15. There has been a substantial amount of work reported on unravelling the immunostimulatory properties of siRNAs and how to mitigate these effects through chemical modifications39. Additionally, there was early concern about the purity of clinical-grade siRNA because single-stranded, small RNAs can be more immunostimulatory than duplex siRNAs 40. Control over the manufacturing and formulation processes have eliminated this potential problem, and the first‑in‑human trials described here were conducted

with siRNAs for which single strands of RNA are not an issue. Interestingly, polymer-based delivery systems such as that used with CALAA‑01 (REF. 41) and atelocollagen42 do not appear to produce the immunostimulatory effects observed with lipid-based delivery systems. This lack of immunostimulation may be due to the highly hydrophilic nature of polymer-based nanoparticles43. Indeed, the lack of immunostimulation with CALAA‑01 was observed in animal studies41 and in humans28. As the siRNAs were all delivered in nanoparticle systems, the siRNAs are not exposed to nucleases until they are released from the nanoparticles (naked non-chemically modified siRNAs have low stability in biological fluids44). Bartlett and Davis45 showed that the prolonged mRNA knockdown times of nuclease-stabilized siRNAs originate primarily from effects before and during cell internalization, before the siRNAs can interact with the intracellular RNAi machinery. Thus, because dosing will be frequent, chemical modifications for intracellular stability are not as necessary for cancer as for delivering siRNA to liver, for which dosing occurs every month or two (hepatocyte cell division occurs infrequently)16. The maximum frequency of systemic administration of the therapies varied: Atu027 and CALAA‑01 were administered twice per week; TKM‑PLK1 once per week; and ALN-VSP once every other week28,29,31,32. CALAA‑01, TKM‑PLK1 and ALN-VSP were dosed up to 0.75–1.0 mg siRNA per kg 28,29,32, whereas Atu027 dosing never exceeded 0.336 mg siRNA per kg 31. Atu027 was the only investigational therapeutic that did not utilize pre-medications, and was administered at a slower infusion rate (4 hours versus 30 minutes with CALAA‑01 and TKM‑PLK1 and 15 minutes with ALN-VSP). Although pre-medications could be an issue for patients with chronic illnesses, for example, those with various liver diseases, their use is common with cancer therapeutics and does not impose a significant problem for patients with cancer. Thus, infusions lasting 30 minutes or more with the use of standard pre-medications are likely to continue with investigational therapeutics containing siRNAs. Although the duration of gene inhibition with delivered siRNA is a function of the cell doubling time16,17, the cancer cell doubling times and the various metastases are not known in an individual patient. Moreover, although there are general trends in the growth rates of certain types of cancers, a priori data from patients before their treatment are not known. This issue is important only if the action of the siRNA does not induce cell death. Some treatments with siRNA can cause growth arrest rather than cell death (this is the case for RRM2 inhibition by siRNA in melanoma independent of the activating oncogenic mutations46). If this is the case, then the cancer cells will not be cycling and siRNA inhibition will be longer in duration (this scenario may have been observed with CALAA‑01 in a patient with melanoma27). Also, because the delivery of the siRNA from a single dose will not reach some of the cancer cells in the tumour, frequent dosing, if it can be accomplished within safety limitations, provides the best opportunity to reach a greater number of the cancer cells in the tumour. Given these variables, dosing schedules tend to be arbitrarily



REVIEWS 1 + CDP

Tumour cell

+ siRNA

9

AD-PEG AD-PEG-Tf

Nucleus RISC 8

RISC 7

AAA

AAA

siRNA 6 5 2

4

Intracellular drug release Endocytosis

Tumour cells

3 Blood ﬂow Leaky vasculature

Nanoparticle

Tumour boundary

Figure 2 | Schematic illustration of systemic delivery of siRNA via nanoparticles. There are many functions that must be performed at the right place and at the right time to produce an antitumour effect with small interfering RNA (siRNA) Nature Reviews | Drug Discovery that is systemically administered to a patient with cancer. First, the nanoparticle formulation is administered intravenously to the patient (steps 1 and 2). The nanoparticles must circulate, reach the tumours and move through the tumour to contact the cancer cells (step 3). The nanoparticles must engage the surface of the cancer cells (step 4) and be internalized (step 5). As the nanoparticles normally enter cells via endocytosis, they must then actively escape the endocytic pathway and release the siRNA into the cytoplasm (step 6). The siRNA then must engage the RNAi pathway (step 7) at some point (most of the time the siRNA engages the RNA-induced silencing complex (RISC) but there are also those that engage Dicer). Loaded RISC then cuts mRNA to yield new RNA fragments (step 8) and the loss of mRNA leads to the loss in protein (step 9). AD-PEG, adamantane polyethylene glycol; CDP, cyclodextrin-based polymer; Tf, transferrin. Figure is adapted from Davis, M. E., Fighting cancer with nanoparticle medicines — the nanoscale matters. MRS Bulletin 37, 828–835, reproduced with permission.

selected. Based on the data from these initial clinical trials, it appears that twice per week to weekly systemic administrations can be accomplished with dosages less than ~1 mg siRNA per kg. Dosing frequencies higher than these values are probably not necessary because of the catalytic nature of the mRNA knockdown with RNAi. Thus, the use of metronomic dosing that is common with chemotherapeutics is not likely to be used with therapeutics that harness the RNAi mechanism of action. Nanoparticle delivery systems. CALAA‑01 is a polymerbased nanoparticle, whereas each of the other investigational siRNA-containing therapeutics discussed in this article are lipid-based nanoparticles. It is anticipated that future clinical studies will involve both types of delivery systems, and each has its own advantages and limitations. Lipid-based nanoparticles are highly efficient at

delivering siRNA to the liver and are therefore the delivery system of choice for diseases involving the liver. However, as the dosing schedule is frequent with cancer, liver accumulation with lipid-based systems needs to be carefully investigated. In the clinical study with ALN-VSP, the most positive RACE data were obtained from biopsies that were primarily normal liver tissue29. These results indicate that lipid-based nanoparticles can deliver functional siRNA to normal liver tissue, even when attempting to deliver the siRNA to tumours (evidence for RNAi in tumours (RACE) from lipid-based nanoparticle delivery has been reported for TKM‑PLK1 (REF. 32)). Fortunately, there do not appear to be any dose-limiting adverse events that have occurred in the clinical trials with ALN-VSP, TKM‑PLK1 and Atu027 that can be attributed to this phenomenon. CALAA‑01 does not deliver functional siRNA to hepatocytes; therefore, this is not an issue with



REVIEWS this investigational therapeutic and no significant liver toxicities were observed in animal or human studies28. Nanoparticles can also accumulate in tissues other than the liver (although in lower amounts), for example, kidney and spleen; therefore, the issues highlighted above for the liver must also be considered for these other organs. For example, CALAA‑01 does localize to the kidney, and dose-limiting toxicities in non-human primates were observed in the kidney (at very high doses of CALAA‑01 that would not be reached in patients, >20 mg siRNA per kg)41. A pre-dosing hydration protocol was used in the clinical trial with CALAA‑01, and no clinically significant alterations in kidney function were observed28. CALAA‑01 is the only siRNA-based investigational therapeutic to contain a so-called targeting agent 34. The human protein transferrin is used as a targeting agent to engage transferrin receptors, which are typically upregulated on cancer cells. Thus, the function of transferrin on these nanoparticles is to enhance the amount and rate of uptake into the cancer cells. Pre- and post-dosing biopsies had significant amounts of transferrin receptors in tissues that also showed loss of RRM2 protein after treatment with CALAA‑01 (REF. 27). The inclusion of a targeting agent adds complexity to nanoparticle delivery systems, and the costs versus the benefits of such agents have been discussed47. With CALAA‑01, there are clear benefits in delivery using the transferrin-targeting agent (the targeted nanoparticles outperformed the non-targeted versions in numerous animal studies48,49), but it appears that transferrin may also have been the cause of long-term drug instability 28. Targeting agents can have functions other than facilitating uptake into cancer cells50; for example, if the targeting agents also have antitumour activity, antibodies or their fragments51 can also block signalling from their binding receptors52. However, transferrin exhibits no antitumour efficacy of its own, and was utilized in CALAA‑01 to enhance uptake into cancer cells only 34.

Pharmacokinetics The movement of a drug into, through and out of the body; that is, its absorption, bioavailability, distribution, metabolism and excretion.

Pharmacokinetics. Regarding the pharmacokinetics of these siRNA-containing experimental therapeutics, all showed dose-dependent increases in the maximal serum siRNA concentration (Cmax) and area under the curve (AUC). These delivery systems did not demonstrate any dose accumulations over time, except for PNT2258, which showed increased AUC with multiple dosing over most of the dosing cohorts. These data suggest that PNT2258 administration might saturate the MPS. The other lipid nanoparticle systems primarily clear through the liver (but also clear through the spleen) and therefore are likely to have increased hepatocyte uptake and consequently a much larger reservoir for drug clearance compared to PNT2258 (which may be limited to primarily Kupffer cell uptake). CALAA‑01 primarily clears through the kidney and is therefore not subject to MPS saturation53,54. Similar increases in AUC have also been observed with PEGylated liposomal doxorubicin and have been correlated with decreasing monocyte counts55. A major concern with nanoparticle delivery systems is the development of a humoral immune response and production of antibodies leading to rapid plasma clearance of the delivery systems after repeated doses. None

of the siRNA delivery systems showed any evidence of pharmacokinetic attenuation over multiple doses, suggesting that significant antibody production is either not occurring or, if it is occurring, not producing antibodies that are altering the pharmacokinetic properties. There is some uncertainty about how the amount of the nanoparticle dose should be scaled when transitioning from preclinical animal studies to humans. Specifically, should the dose be scaled based on body weight or body surface area? Thus, the pharmacokinetic results from Phase I trials are important as they provide the initial data for understanding the translation from animals to humans. The Cmax from CALAA‑01 scales across species based on body weight28 (FIG. 3a). Using data from monkeys and humans only 31, the AUC of Atu027 scales with body weight (FIG. 3b). However, the AUC of another nanoparticle drug delivery system — CRLX101 (which does not deliver siRNA but a small-molecule drug) — scaled with body surface area56. Therefore, the pharmacokinetic scaling of these systems across species will probably depend on their mechanism of clearance. Systems that clear primarily through the kidney may scale with body weight, whereas those that clear through the MPS may scale based with body surface area. Safety. The data from the clinical trials described here indicate that this new class of investigational therapeutics can be safely dosed to humans. Most therapeutics were well tolerated28,29,31–33. The primary toxicities among the trials were infusion-related reactions and cytokine release symptoms that were successfully managed with supportive therapies and, in some cases, by lengthening the infusion time (TABLE 2). Thrombocytopaenia was also observed in many of these trials. Maximum tolerable doses were reached in the TKM‑08030 and PNT2248 trials32,33. No maximal tolerable dose was reached in the Atu027 study owing to the lack of sufficient dose-limiting toxic events in the dose range investigated31. No clear doselimiting toxicity was reached in the CALAA‑01 trial28. The toxicities in the CALAA‑01 trials were thought to be primarily due to instability of the protein component of the delivery system over time rather than true toxicity of the intact therapeutic. Importantly, pharmacodynamic effects (siRNA activity) were observed at the doses investigated in these trials28. So far, limited hepatotoxicity has been observed in humans treated with these siRNA delivery systems. These results are surprising in light of the hepatotoxicity observed in many of the preclinical studies of these siRNA therapies. A single patient with >70% liver involvement by metastatic primitive neuroectodermal tumour developed hepatic failure after the second dose of ALN-VSP (0.7 mg siRNA per kg) and subsequently died29. This liver failure was proposed to be due to massive tumour necrosis, progression of extensive disease or ALN-VSP toxicity to the scant remaining live parenchyma. A single grade 3 aspartate aminotransferase elevation was observed in the PNT2258 study 33. Oligonucleotides are able to specifically induce activation of the alternative pathway of the complement system. ALN-VSP and Atu027 were both associated with mild



REVIEWS b

1,000,000

siRNA AUC (pg hour per ml)

Cmax plasma siRNA (ng per ml)

a

10,000

100

1 0.05

0.5

5

50

10,000 7,500 5,000 2,500 0

Dose (mg per kg) Rat

Monkey

Human

0

0.5

1.0

1.5

2.0

2.5

3.0

Dose (mg per kg) Dog

Human

Monkey

Figure 3 | Pharmacokinetic scaling across different species. a | CALAA‑01 in four species. b | Atu027 in monkeys and humans. AUC, area under the curve; Cmax, maximal serum siRNA concentration; siRNA, small interfering RNA. Figure part a is reprinted with permission from REF. 28; data for part b are from REF. 31. Nature Reviews | Drug Discovery

Enhanced permeability and retention effect A phenomenon whereby macromolecules and nanoparticles passively accumulate within tumour tissue from the bloodstream. The mechanism behind this phenomenon is poorly understood and is thought to be partially due to the increased permeability of tumour vasculature and poor tumour lymphatic drainage.

complement activation in a dose-dependent manner 29,31. These activations were transient and resolved within 24 hours after dosing. No significant adverse events relating to this complement activation were observed. No significant changes in serum complement activity were observed following CALAA‑01 administration28. Complement measurements were not reported for the TKM‑080301 or PNT2258 studies32,33. These findings suggest that oligonucleotide-induced complement activation may occur in humans, but is not clinically significant. Substantial preclinical research has demonstrated that non-chemically modified siRNAs can be immuno stimulatory under certain conditions. CALAA‑01 treatment resulted in mild, transient elevations in some serum cytokines (interleukin‑6 (IL‑6), IL‑10, tumour necrosis factor (TNF) and interferon-γ (IFNγ)) that were not correlated with adverse events28. ALN-VSP treatment also induced cytokine production, including IL‑10, IL‑1 receptor A, IL‑6, granulocyte-colony stimulating factor and TNF. Symptoms related to cytokine activation (chills and rigor) were observed at higher doses 4–8 hours after infusion29. No cytokine activation was observed following dosing with Atu027 (REF. 31). TKM‑080301 treatment transiently induced the production of IL‑6, monocyte chemoattractant protein 1 (MCP1; also known as CCL2) and IL‑8 in 25% of patients. The cytokine release was associated with peak levels of TKM‑080301; for example, IL‑6 concentrations ranged from a maximum of ~30 pg per ml with 0.15 mg siRNA per kg to 2,000 pg per ml with 0.9 mg siRNA per kg 32. In preclinical studies, renal toxicity was the doselimiting toxicity for the CALAA‑01 delivery system. A rigorous pre- and post-hydration protocol in the clinical studies effectively prevented this toxicity in humans. In fact, no significant changes in serum creatinine and blood urea nitrogen were observed in this clinical study 28. Efficacy and pharmacodynamics. The clinical experiences with siRNA therapeutics in solid malignancies are so far restricted to small Phase I trials. Therefore, only limited conclusions about their overall therapeutic efficacy can be drawn. The best response observed to date was a single complete response in the ALN-VSP trial29.

Partial responses were achieved in the ALN-VSP, Atu027 and TKM‑080301 trials. The primary therapeutic effect across all trials was stable disease; stable disease was the best response achieved in the CALAA‑01 and PNT2258 trials28,33. It should be noted that each of these trials enrolled heterogeneous groups of patients with a variety of advanced solid malignancies, the majority of which had failed multiple prior therapies. No pre-selection for patients most likely to benefit from siRNA-specific target knockdown was performed in any trial. Important data about the ability of nanoparticle therapies to deliver siRNAs to human tumours was elicited from these clinical studies. The mechanistic underpinning of nanoparticle-based drug delivery to solid tumours — the enhanced permeability and retention effect — is supported by studies of nanoparticle deposition and siRNA delivery in xenografted tumours in mice. Given the considerable differences between the morphology of xenografted tumours in mice and tumours in humans, there has been uncertainty with regard to the ability of nanoparticles to deliver drugs or siRNA to human tumours as has been observed in animals. The data gathered from these Phase I studies are highly encouraging in this regard. The CALAA‑01 nanoparticle system was shown to accumulate in human metastatic melanoma tumours in a dose-dependent manner 27. Furthermore, RACE cleavage fragments specific for RRM2 siRNA RNAi activity and knockdown of RRM2 mRNA and protein were also detected in these samples27. Both VEGFA and KSP siRNAs were detected in tumour tissue following treatment with the ALN-VSP delivery system29. Significant VEGF knockdown was also observed in these samples. siRNA mechanism of action was confirmed via RACE analysis (VEGF siRNA cleavage product only) in two patients treated with ALN-VSP; however, the majority of the biopsy tissue for these studies was normal liver with little viable tumour 29. Pre- and post-dose liver lesion biopsy specimens from one patient enrolled in the TKM‑080301 study were subjected to RACE analysis, and the results confirmed the presence of the PLK1 mRNA cleavage product 32. These data are suggestive that these nanoparticle systems are effectively reaching tumour tissue and delivering functional siRNA.



REVIEWS Future directions The initial first‑in‑human trials with systemically administered siRNA-based anticancer therapies showed that this new class of investigational therapeutic can be safely dosed to humans. This positive step forward in the development of RNAi-based therapies paves the way for the development of newer formulations and newer clinical trial designs. As there is no substitute for human data the results from these initial trials need to be correlated to animal data to learn when the animal data are capable of predicting results from humans. Although there are good reasons for some to take the ‘glass is half empty’ stance57–59 and state that better delivery is needed, we feel that the ‘glass is half full’ in that the movement from discovery to first clinical trials of this potentially revolutionary new therapeutic modality has occurred in a relatively short period of time, and these trials have generated a wealth of important data that will be useful to the field. In the meantime, there are several important issues to consider for future clinical trials. siRNA design. It is clear that siRNAs can be designed, manufactured at appropriate quality for human use and administered to patients with cancer to elicit an RNAi mechanism of action on a target mRNA. As previously mentioned, the siRNAs used in the clinical trials described here all engaged the RNAi pathway at RISC. siRNAs can also be designed to enter the RNAi pathway at Dicer (for example, with siRNAs developed by Dicerna; see the clinical trial listed in TABLE 1). Current evidence indicates that Dicer expression levels can vary in humans60–62. For example, Dicer expression can be decreased in epithelial ovarian cancers, which was associated with poor clinical outcome in one study61. Additionally, Dicer can be dysregulated in triple negative breast cancer 62. Thus, it will be interesting to find out whether there will be clinical advantages for targeting the RNAi pathway at positions other than RISC. Other types of RNAs. The use of siRNA in the clinic is leading the way to the use of other therapeutic RNAs. Delivery of a microRNA (miRNA) mimic63 has already reached the clinic (MRX34; see TABLE 1). The silencing of miRNAs is being investigated as experimental anticancer therapeutics64 because tumours can be oncomiR addicted (analogous to oncogene addictions). It would not be surprising to see the delivery of RNAs of the CRISPR system (clustered, regularly interspaced, short palindromic repeat system) in the near future65,66.

CRISPR system An RNA-guided gene-editing platform that utilizes the bacterial protein Cas9 and a synthetic guide RNA to introduce a double-stranded DNA break at a specific location within the genome.

Nanoparticle delivery systems. Accumulating information on the fundamental behaviour of nanoparticle delivery systems provides hope that this information will be translated into improved anticancer efficacy. Recent studies of the properties of nanoparticles that can improve their biodistribution to tumours in animals have been published50,67. Additionally, information on how nanoparticles behave in humans (data from several clinical trials on multiple clinical batches of a drug-containing nanoparticles are now available56) and how these data compare to results in animals studies is

emerging 56. It is clear that the more homogeneous the nanoparticle formulation the better the performance68,69. Clinical lots of 20–30 nm diameter nanoparticles can show excellent reproducibility in their properties and performance in humans56. It is expected that newer nanoparticle formulations will carry multiple siRNAs and will become safer and more efficacious. At this time, it has been shown that two siRNAs can be used in the clinic (ALN-VSP) and that twice per week dosing can be achieved with several different nanoparticles at amounts approaching 1 mg siRNA per kg. Lipid-based delivery systems have the potential for long-term lipid accumulation because they can incorporate into biological membranes. This effect and others can lead to toxicity issues. Recently, siRNAs have been delivered to animals using biodegradable lipids that display advantages such as more-rapid elimination and greater safety 70,71. Biodegradable polypeptide-based polymer conjugates have also been reported72. Although biodegradability can show functional advantages in animal models, there is the possibility of added regulatory complexity when using these types of delivery systems. That is, the regulatory agencies may require additional safety studies of the fragments formed from the bio degradable molecules. With the delivery systems that have been described in this Review, the components are not biodegradable. However, when the nanoparticles disassemble, the delivery components are all sufficiently small that they can be cleared via the kidney. Although cleared renally, delivery components were not explicitly reported in these studies. However, it has been shown that a non-biodegradable, polymer-based nanoparticle carrying a drug molecule does indeed excrete individual polymer molecules (that in their assembled form create the nanoparticle) of the same molecular weight as that infused into the patient 56. Research continues on understanding the multiple phenomena occurring when a nanoparticle is used in vivo to deliver a therapeutic agent 73. As further understanding is made, safety and delivery can be improved. Although mechanisms of adverse events are typically investigated in vitro and in mice74,75, it is of importance to realize that they are only models for what may occur in patients. Thus, studies that more closely approximate effects in humans76 are of increasing importance. Nanoparticle delivery of siRNAs takes advantage of the leaky vasculature in tumours77. There are numerous reports of how the tumour vasculature affects the delivery of nanoparticles in tumours of animals50,67,77,78; however, there is relatively little known about the tumour vasculature in humans79. PEGylated liposomes containing either 111In or 99mTc have been used to monitor their biodistribution in patients80,81; both studies revealed that the liposomes do accumulate in tumours. Koukourakis et al.80 also showed that the tumour accumulation of the liposomes correlated with the tumour blood vessel density (measured by anti‑CD31 staining). However, tumour regression could not be correlated to the blood vessel density, which may be due to a multitude of factors. With the delivery of siRNA to cancer cells in solid tumours, reaching the tumour is only a



REVIEWS prerequisite (FIG. 2) to tumour regression. The intracellular trafficking and release of the siRNA can have critical roles in efficacy 82. Importantly, CALAA‑01 was shown to reach cancer cells in the tumours of patients with melanoma in a dose-dependent manner, and the siRNA was delivered to provide target mRNA knockdown by an RNAi mechanism (as confirmed by the presence of the correct RACE RNA fragments)27. There is no doubt that further research in humans to address tumour localization and intracellular trafficking of the siRNA would be of tremendous importance to the field. Although the use of combinations of siRNAs may have great potential to provide efficacious anticancer therapeutics, there remains concern about the effectiveness of the delivery pathway for the siRNA (discussed above). Additionally, initial clinical studies show that the nanoparticles are heterogeneously distributed in animal and human tumours (CALAA‑01 (REF. 27) and CRLX101 (REFS 56,83)). Thus, it is likely that there must be some type of bystander effect with siRNA to achieve significant tumour regression. It is possible that some form of paracrine or exocrine secretion of mRNA, miRNA or protein is occurring (for example, via exosomes and microvesicles). For example, exosomes can perform cell‑to‑cell communication within a given tumour 84 as well as between other tissues in the body 84–89. In the future, the nanoparticles carrying siRNA will probably become more multifunctional. For example, nanoparticles carrying siRNAs could utilize targeting agents to enable simultaneous attack of numerous pathways. Recent efforts with antibody-conjugated siRNAs show that intracellular trafficking and siRNA can be difficult with these types of constructs82. By contrast, antibody-containing nanoparticles do not have such difficulties as the nanoparticles can contain functionalities to enhance endocytic escape and siRNA release. Additionally, and in contrast to antibodies conjugated to siRNA, the antibody-containing nanoparticle can carry a large number of siRNA molecules that permit multiple gene target silencing. Clinical trials and target selection. The clinical results discussed here all involve targets that are not cancer-type specific. Additionally, it appears that these initial trials did not screen patients to ascertain their gene status before dosing. For example, the regulatory agencies both at the local and federal levels explicitly ruled out a provision for mandatory biopsies in all patients in the CALAA‑01 trial27. On the positive side of the argument for using these types of Phase I trials, they are typically completed faster than those that require more specific patient characteristics. It is also possible that such trials may reveal compound efficacy in patients who were previously thought to have a low likelihood of treatment success. On the negative side of the argument for these types of trials, the chances of determining pharmacodynamics and activity are greatly diminished. Although Phase I trials are primarily about measuring safety, it is now important to also obtain pharmacodynamic information and, if possible, some measure of activity. When patients are stratified into the trial based on their genetic

signature, the chances of obtaining successful pharmaco dynamics and activity are greatly increased, especially with selective therapeutics such as siRNAs. In future clinical trials, it would be extremely helpful to pre-screen patients so that only those who have tumour-driving gene mutations are treated; that is, tumour-driving oncogenes can then be inhibited by the siRNAs being delivered. A full discussion on target selection is beyond the scope of this Review; however, we offer a few thoughts based on our experiences using siRNA-based therapeutics. First, it is our opinion that gene targets that are appropriate for clinical application in cancer are those that induce cancer cell death, not just growth arrest, when inhibited by siRNA. Second, patients should be screened for the target, and even Phase I trials should be designed with patients stratified into the trials based on pre-screening characteristics of the tumours. Although this criterion will probably lengthen the time of the trial by making it more difficult to recruit patients, it should greatly increase the opportunities to measure the ability of the siRNA therapeutic to modulate the target and have pharmacodynamic and activity readouts. If these types of results can be obtained in Phase I trials, they should allow for the better design and accelerated completion of later stage trials. Two examples of potential siRNA targets for future clinical trials are epidermal growth factor receptor (EGFR) and NRAS. Although there are numerous drugs that target EGFR, there is still a need for therapeutics that can inhibit EGFR while enabling high patient quality of life; for example, without inducing skin rash. New therapeutics against EGFR continue to be tested in the clinic for colorectal cancer 90 and lung cancer 91. For both of these cancer types, patient pre-screening protocols are already in place. One potential problem with developing an siRNA-based therapeutic against EGFR is that patient enrolment in a clinical trial may be challenging because patients have an increasing number of options to participate in clinical trials with EGFR inhibitors. Another interesting target for an siRNA-based therapeutic is NRAS in melanoma92. Currently, patients with melanoma are pre-screened, and those with BRAFmutant melanoma now have important new therapeutic options. Although there are several options that are being investigated for patients with NRAS-mutant melanoma, none of them directly attack NRAS (because there are no drugs available to do so)92. Given the lack of existing treatment options ror patients with NRASmutant melanoma, clinical trial recruitment may not be an issue with this target. These two examples illustrate just a few of the issues that need to be considered when selecting the appropriate siRNA target for clinical trials.

Summary Several Phase I trials evaluating the use of siRNA for the treatment of solid cancers have now been completed. All trials to date have used nanoparticle-based delivery systems to deliver therapeutic siRNA to tumour tissue following systemic administration. Despite concerns about possible overwhelming immunostimulation following systemic siRNA administration in humans, the siRNA therapeutics



REVIEWS data reported to date have shown that these therapeutics are well tolerated, with only modest and treatable immuno stimulatory reactions. Results from multiple clinical trials have demonstrated the successful delivery of functional siRNA to human tumours, providing proof‑of‑principle of RNAi-based therapeutics for cancers in humans. Clinical trials being conducted that deliver siRNA to the liver for non-cancer indications are at later stages of development than those used for treating cancer. Some of these trials are in Phase III, and the

1.

Fire, A. Z. Gene silencing by double-stranded RNA (Nobel Lecture). Angew. Chem. Int. Ed. 46, 6966–6984 (2007). This paper provides a brief history and background on RNAi. 2. Burnett, J. C. & Rossi, J. J. RNA-based therapeutics: current progress and future prospects. Chem. Biol. 19, 60–71 (2012). 3. Vaishnaw, A. K. et al. A status report on RNAi therapeutics. Silence 1, 14 (2010). 4. de Fougerolles, A., Vornlocher, H.‑P., Maraganore, J. & Lieberman, J. Interfering with disease: a progress report on siRNA-based therapeutics. Nat. Rev. Drug Discov. 6, 443–453 (2007). 5. Sepp-Lorenzino, L. & Ruddy, M. K. Challenges and opportunities for local and systemic delivery of siRNA and antisense oligonucleotides. Clin. Pharmacol. Ther. 84, 628–632 (2008). 6. Whitehead, K. A., Langer, R. & Anderson, D. G. Knocking down barriers: advances in siRNA delivery. Nat. Rev. Drug Discov. 8, 129–138 (2009). 7. Chen, S.‑H. & Zhaori, G. Potential clinical applications of siRNA technique: benefits and limitations. Eur. J. Clin. Invest. 41, 221–232 (2011). 8. Pecot, C. V., Calin, G. A., Coleman, R. L., Lopez-Berestein, G. & Sood, A. K. RNA interference in the clinic: challenges and future directions. Nat. Rev. Cancer 11, 59–67 (2011). 9. Wu, S. Y., Lopez-Berestein, G., Calin, G. A. & Sood, A. K. RNAi therapies: drugging the undruggable. Sci. Transl. Med. 6, 240ps7 (2014). 10. Davis, M. E., Chen, Z. G. & Shin, D. M. Nanoparticle therapeutics: an emerging treatment modality for cancer. Nat. Rev. Drug Discov. 7, 771–782 (2008). This paper reviews the area of nanoparticle-based anticancer therapeutics. 11. Egusquiaguirre, S. P., Igartua, M. Hernández, R. M. & Pedraz, J. L. Nanoparticle delivery systems for cancer therapy: advances in clinical and preclinical research. Clin. Transl. Oncol. 14, 83–93 (2012). This paper reviews the different types of delivery of siRNA and the materials used to facilitate the delivery of siRNA. 12. Kanasty, R., Dorkin, J. R., Vegas, A. & Anderson, D. Delivery materials for siRNA therapeutics. Nat. Mater. 12, 967–977 (2013). 13. Gomes da Silva, L. C., Simões, S. & Moreira, J. N. Challenging the future of siRNA therapeutics against cancer: the crucial role of nanotechnology. Cell. Mol. Life Sci. 21, 1417–1438 (2014). 14. Behlke, M. A. Chemical modification of siRNAs for in vivo use. Oligonucleotides 18, 305–320 (2008). 15. Rettig, G. R. & Behlke, M. A. Progress toward in vivo use of siRNAs-II. Mol. Ther. 20, 483–512 (2012). This paper describes advances in siRNA biochemistry for in vivo use. 16. Bartlett, D. W. & Davis, M. E. Insights into the kinetics of siRNA-mediated gene silencing from live-cell and live-animal bioluminescent imaging. Nucl. Acids Res. 34, 322–333 (2006). 17. Bartlett, D. W. & Davis, M. E. Impact of tumor-specific targeting and dosing schedule on tumor growth inhibition after intravenous administration of siRNA-containing nanoparticles. Biotech. Bioeng. 99, 975–985 (2008). 18. Yuan, T. L. et al. Development of siRNA payloads to target KRAS-mutant cancer. Cancer Discov. 4, 1182–1197 (2014). This paper nicely demonstrates the in vivo use of multiple different siRNAs within the same nanoparticle and the enhanced efficacy that can be obtained from such an approach.

results from these trials will hopefully lead to regulatory approvals. These exciting results are paving the way for the commercialization of approved ‘drugs in vials’ using RNAi. Our hope is that the use of siRNA can also be exploited to reach extra-hepatic targets in the near future. Although more difficult than delivery to the liver (for reasons discussed in this Review), the regulatory approval of therapeutics that deliver siRNA to extrahepatic tumours could provide game-changing therapy for patients with cancer.

19. Koldehoff, M., Steckel, N. K., Beelen, D. W. & Elmaagacli, A. H. Therapeutic application of small interfering RNA directed against BCR–ABL transcripts to a patient with imatinib-resistant chronic myeloid leukaemia. Clin. Exp. Med. 7, 47–55 (2007). 20. Molitoris, B. A. et al. siRNA targeted to p53 attenuates ischemic and cisplatin-induced acute kidney injury. J. Am. Soc. Nephrol. 20, 1754–1764 (2009). 21. Petros, R. A. & DeSimone, J. M. Strategies in the design of nanoparticles for therapeutic applications. Nat. Rev. Drug Discov. 9, 615–627 (2010). 22. Coelho, T. et al. Safety and efficacy of RNAi therapy for transthyretin amyloidosis. N. Engl. J. Med. 369, 819–829 (2013). This study reports clinical results for lipid-based siRNA delivery to the liver. 23. Fitzgerald, K. et al. Effect of an RNA interference drug on the synthesis of proprotein convertase subtilisin/ kexin type 9 (PCSK9) and the concentration of serum LDL cholesterol in healthy volunteers: a randomised, single-blinded placebo-controlled, Phase 1 trial. Lancet 383, 60–68 (2014). This study reports results from a Phase I trial that utilized a lipid-based formulation of siRNA for delivery to the liver. 24. Nair, J. K. et al. Multivalent N-acetylgalactosamineconjugated siRNA localizes in hepatocytes and elicits robust RNAi-mediated gene silencing. J. Am. Chem. Soc. 136, 16958–16961 (2014). 25. Golan, T. et al. RNAi therapy targeting KRAS in combination with chemotherapy for locally advanced pancreatic cancer patients. Oncotarget [online], http://www.impactjournals.com/oncotarget/index.php? journal=oncotarget&page=article&op=view&path% 5B%5D=4183&path%5B%5D=9307 (2015). 26. Farooqi, A. A., Rehman, Z. & Muntane, J. Antisense therapeutics in oncology: current status. Onco. Targets Ther. 7, 2035–2042 (2014). 27. Davis, M. E. et al. Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles. Nature 464, 1067–1070 (2010). This paper reported the first example of the RNAi mechanism of action in a human from the delivery of siRNA via a polymer-based nanoparticle, that localized in human tumours in amounts that correlated with the dose levels systemically administered to the patients. 28. Zuckerman, J. E. et al. Correlating animal and human Phase Ia/Ib clinical data with CALAA 01, a targeted, polymer-based nanoparticle containing siRNA. Proc. Natl Acad. Sci. USA 111, 11449–11454 (2014). This paper summarizes all the clinical and preclinical results with CALAA 01. 29. Tabernero, J. et al. First in humans trial of an RNA interference therapeutic targeting VEGF and KSP in cancer patients with liver involvement. Cancer Discov. 3, 406–417 (2013). This study reports the first human results with ALN-VSP, a lipid-based formulation of two siRNAs. It is also the first example of two siRNAs used in the clinic. 30. Strumberg, D. et al. Phase I clinical development of Atu027, a siRNA formulation targeting PKN3 in patients with advanced solid tumors. Int. J. Clin. Pharm. Ther. 50, 76–78 (2012). 31. Schultheis, B. et al. First in human Phase I study of the liposomal RNA interference therapeutic Atu027 in patients with advanced solid tumors. J. Clin. Oncol. 32, 4141–4148 (2014). This paper presents the first human data with Atu027, a lipid-based formulation of siRNA.


32. Ramanathan, R. K. et al. A Phase 1 dose escalation study of TKM 080301, a RNAi therapetuic directed against PLK1, in patients with advanced solid tumors. J. Clin. Oncol. 31 (Suppl), TPS2621 (2013). 33. Tolcher, A. W. et al. A Phase 1 study of the BCL2 targeted deoxyribonucleic acid inhibitor (DNAi) PNT2258 in patients with advanced solid tumors. Cancer Chemoth. Pharm. 73, 363–371 (2014). 34. Davis, M. E. The first targeted delivery of siRNA in humans via a self-assembling cyclodextrin polymer-based nanoparticle: from concept to clinic. Mol. Pharm. 6, 659–668 (2009). 35. Davis, M. E. Fighting cancer with nanoparticle medicines — the nanoscale matters. MRS Bull. 37, 828–835 (2012). 36. Grimm, D. et al. Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways. Nature 441, 537–541 (2006). 37. Heidel, J. D. et al. Potent siRNA inhibitors of ribonucleotide reductase subunit RRM2 reduce cell proliferation in vitro and in vivo. Clin. Cancer Res. 13, 2207–2215 (2007). 38. Barros, S. A. & Gollob, J. A. Safety profile of RNAi nanomedicines. Adv. Drug Deliv. Rev. 64, 1730–1737 (2012) 39. Judge, A. D. et al. Confirming the RNAi-mediated mechanism of action of siRNA-based cancer therapeutics in mice. J. Clin. Invest. 119, 661–673 (2009).  40. Sioud, M. Single-stranded small interfering RNA are more immunostimulatory than their double-stranded counterparts: a central role for 2ʹ‑hydroxyl uredines in immune responses. Eur. J. Immunol. 36, 1222–1230 (2006). 41. Heidel, J. D. et al. Administration in non-human primates of escalating intravenous doses of targeted nanoparticles containing ribonucleotide reductase submit M2 siRNA. Proc. Natl Acad. Sci. USA 104, 5715–5721 (2007). 42. Inaba, S. et al. Atelocollagen-mediated systemic delivery prevents immunostimulatory adverse effects of siRNA in mammals. Mol. Ther. 20, 356–366 (2012). 43. Moyano, D. F. et al. Nanoparticle hydrophobicity dictates immune response. J. Am. Chem. Soc. 134, 3965–3967 (2012). 44. Hickerson, R. P. et al. Stability study of unmodified siRNA and relevance to clinical use. Oligonucleotides 18, 1–10 (2008). 45. Bartlett, D. W. & Davis, M. E. Effect of siRNA nuclease stability on the in vitro and in vivo kinetics of siRNAmediated gene silencing. Biotechnol. Bioeng. 97, 909–921 (2007). 46. Zuckerman, J. E., Hsueh, T., Koya, R. C., Davis, M. E. & Ribas, A. siRNA knockdown of ribonucleotide reductase inhibits melanoma cell line proliferation alone or synergistically with temozolomide. J. Invest. Derm. 131, 453–460 (2011). 47. Cheng, Z. et al. Multifunctional nanoparticles: cost versus benefit of adding targeting and imaging capabilities. Science 338, 903–910 (2012). 48. Hu Lieskovan, S., Heidel, J. D., Bartlett, D. W., Davis, M. E. & Triche, T. J. Sequence-specific knockdown of EWS‑FL1 by targeted, nonviral delivery of small interfering, RNA inhibits tumor growth in a murine model of metastatic Ewing’s sarcoma. Cancer Res. 65, 8984–8992 (2005). 49. Bartlett, D. W., Su, H., Hildebrandt, I. J., Weber, W. A. & Davis, M. E. Impact of tumor-specific targeting on the biodistribution and efficacy of siRNA nanoparticles measured by multimodality in vivo imaging. Proc. Natl Acad. Sci. USA 104, 15549–15554 (2007).


REVIEWS 50. Sykes, E. A. et al. Investigating the impact of nanoparticle size on active and passive tumor targeting efficiency. ACS Nano. 8, 5696–5706 (2014). 51. Senzer, N. et al. Phase I study of a systemically delivered p53 nanoparticle in advanced solid tumors. Mol. Ther. 21, 1096–1103 (2013). 52. Bertrand, N. et al. Cancer nanotechnology: the impact of passive and active targeting in the era of modern cancer biology. Adv. Drug Del. Rev. 66, 2–25 (2014). 53. Zuckerman, J. E., Choi, C. H. J., Han, H. & Davis, M. E. Polycation-siRNA nanoparticles can disassemble at the kidney glomerular basement membrane. Proc. Natl Acad. Sci. USA 109, 3137–3142 (2012). 54. Naeye, B. et al. In vivo disassembly of IV administered siRNA matrix nanoparticles at the renal filtration barrier. Biomaterials 34, 2350–2358 (2013). 55. Song, G., Wu, H., Yoshino, K. & Zamboni, W. C. Factors affecting the pharmacokinetics and pharmacodynamics of liposomal drugs. J. Liposome Res. 22, 177–192 (2012). 56. Eliasof, S. et al. Correlating preclinical animal studies and human clinical trials of a multifunctional, polymeric nanoparticle. Proc. Natl Acad. Sci. USA 110, 15127–15132 (2013). This paper reports correlations between preclinical and clinical data for the polymer-based, drug-containing nanoparticle denoted CRLX101. 57. Venditto, V. J. & Szoka, F. C. Jr. Cancer nanomedicines: so many papers and so few drugs! Adv. Drug Del. Rev. 65, 80–88 (2013). 58. Stein, C. A. & Goel, S. Therapeutic oligonucleotides: the road not taken. Clin. Cancer Res. 17, 6369–6372 (2011). 59. [No authors listed.] Time to deliver. Nat. Biotech. 32, 961 (2014). 60. Merritt, W. M., Bar-Eli, M. & Sood, A. K. The dicey role of Dicer: implications for RNAi therapy. Cancer Res. 70, 2571–2574 (2010). 61. Merritt, W. M. et al. Dicer, drosha, and outcomes in patients with ovarian cancer. N. Engl. J. Med. 359, 2641–2650 (2008). 62. Caffrey, E. et al. Prognostic significance of deregulated dicer expression in breast cancer. PLoS ONE 8, e83724 (2013). 63. Daige, C. L. et al. Systemic delivery of a miR34a mimic as a potential therapeutic for liver cancer. Mol. Cancer Ther. 13, 2352–2360 (2014). 64. Cheng, C. J. et al. MicroRNA silencing for cancer therapy targeted to the tumour microenvironment. Nature 518, 107–110 (2015). 65. Pennisi, E. The CRISPR craze. Science 341, 833–836 (2013). 66. Dounda, J. A. & Charpenter, E. The new frontier of genome engineering with CRISPER Cas9. Science 346, 1258096 (2014).

67. Tang, L. et al. Investigating the optimal size of anticancer nanomedicine. Proc. Natl Acad. Sci. USA 111, 15344–15349 (2014). 68. Adjei, I. M. et al. Heterogeneity in nanoparticles influences biodistribution and targeting. Nanomedicine 9, 267–278 (2014). 69. Zhang, J. et al. Assessing the heterogeneity level in lipid nanoparticles for siRNA delivery: size-based separation, compositional heterogeneity, and impact on bioperformance. Mol. Pharm. 10, 397–405 (2013). 70. Maier, M. A. et al. Biodegradable lipids enabling rapidly eliminated lipid nanoparticle for systemic delivery of RNAi therapeutics. Mol. Ther. 21, 1570–1578 (2013). 71. Whitehead, K. A. et al. Degradable lipid nanoparticles with predictable in vivo siRNA delivery activity. Nat. Commun. 5, 4277 (2014). 72. Barrett, S. E. et al. Development of a liver-targeted siRNA delivery platform with a broad therapeutic window utilizing biodegradable polypeptide-based polymer conjugates. J. Control. Release 183, 124–137 (2014). 73. Lane, L. A., Qian, X., Smith, A. M. & Nie, S. Physical chemistry of nanomedicine: understanding the complex behavior of nanoparticles in vivo. Annu. Rev. Phys. Chem. 66, 521–547 (2015). 74. Landesman-Milo, D. & Peer, D. Toxicity profiling of several common RNAi-based nanomedicines: a comparative study. Drug Deliv. Transl. Res. 4, 96–103 (2014). 75. Hwang, T., Aljuffali, I. A., Lin, C., Chang, Y. & Fang, J. Cationic additives in nanosystems activate cytotoxicity and imflammatory response of human neutrophils: lipid nanoparticles versus polymeric nanoparticles. Int. J. Nanomed. 10, 371–385 (2015). 76. Robbins, G. R. et al. Analysis of human innate immune responses to PRINT fabricated nanoparticles with cross validation using a humanized mouse model. Nanomedicine 11, 589–599 (2015). 77. Li, L. et al. Tumor vasculature is a key determinant of the efficiency of nanoparticle-mediated siRNA delivery. Gene Ther. 19, 775–780 (2012). 78. Cabral, H. et al. Accumulation of sub 100nm polymeric micelles in poorly permeable tumours depends on size. Nat. Nanotech. 6, 815–823 (2011). 79. Prabhaker, U. et al. Challenges and key considerations of the enhanced permeability and retention effect for nanomedicine drug delivery in oncology. Cancer Res. 73, 2412–2417 (2013). 80. Koukourakis, M. I. et al. Liposomal doxorubicin and conventionally fractionated radiotherapy in the treatment of locally advanced non-small-cell lung cancer and head and neck cancer. J. Clin. Oncol. 17, 3512–3521 (1999).


81. Karrington, K. J. et al. Effective targeting of solid tumors in patients with locally advanced cancer by radiolabeled pegylated liposomes. Clin. Cancer Res. 7, 243–254 (2001). 82. Cuellar, T. L. et al. Systemic evaluation of antibodymediated siRNA delivery using an industrial platform of THIOMAB–siRNA conjugates. Nucleic Acids Res. 43, 1189–1203 (2015). 83. Weiss, G. J. et al. First in human Phase 1/2a trial of CRLX101, a cyclodextrin-containing polymercamptothecin nanopharmaceutical in patients with advanced solid tumor malignancies. Invest. New Drugs 31, 986–1000 (2013). 84. Suetsugu, A. et al. Imaging exosome transfer from breast cancer cells to stroma at metastatic sites in orthotopic nude-mouse models. Adv. Drug Del. Rev. 65, 383–390 (2013). 85. Sehgal, A. Chen, Q. Gibbings, D., Sah, D. W. Y. & Bumcrot, D. Tissue-specific gene silencing monitored in circulating RNA. RNA 20, 1–7 (2014). 86. Antanaviciute, I. et al. Long-distance communication between laryngeal carcinoma cells. PLoS ONE 9, e99196 (2014). 87. Melo, S. A. et al. Cancer exosomes perform cellindependent microRNA biogenesis and promote tumorigenesis. Cancer Cell 26, 707–721 (2014). 88. Hannafon, B. N. & Ding, W. Intercellular comminucaiton by exosome-derived microRNAs in cancer. Int. J. Mol. Sci. 14, 14240–14269 (2013). 89. Roberts, C. T. Jr & Kurre, P. Vesicle trafficking and RNA transfer add complexity and connectivity to cell–cell communication. Cancer Res. 73, 3200–3205 (2015). 90. Dienstmann, R. et al. Safety and activity of the first in class Sym004 anti-EGFR antibody mixture in patients with refractory colorectal cancer. Cancer Discov. 5, 598–609 (2015). 91. Sharma, S. V., Bell, D. W., Settleman, J. & Haber, D. A. Epidermal growth factor receptor mutations in lung cancer. Nat. Rev. Cancer 7, 169–181 (2007). 92. Fedornko, I. V., Gibney, G. T., Sondak, V. K. & Smalley, K. S. M. Beyond, BRAF: where next for melanoma therapy? Br. J. Cancer 112, 217–226 (2015).

Competing interests statement

The authors declare competing interests: see Web version for details.

FURTHER INFORMATION ClinicalTrials.gov: https://clinicaltrials.gov ALL LINKS ARE ACTIVE IN THE ONLINE PDF


Persistent neurotoxicity of systemically administered interferon-alpha.

Systemically administered antifungal agents. A review of their clinical pharmacology and therapeutic applications.

Corrigendum: Effects of Systemically Administered Hydrocortisone on the Human Immunome.

Systemically Administered, Target Organ-Specific Therapies for Regenerative Medicine.

Fate of systemically administered cocaine in nonhuman primates treated with the dAd5GNE anticocaine vaccine.

Therapeutic interaction of systemically-administered mesenchymal stem cells with peri-implant mucosa.

Potent antinociceptive effects of clonidine systemically administered in an experimental model of clinical pain, the arthritic rat.

Clinical experiences with platinum and etoposide therapy in lung cancer.

Cutaneous immediate hypersensitivity in man: effects of systemically administered adrenergic drugs.

Spinal transection reduces both spinal antinociception and CNS concentration of systemically administered morphine in rats.

Beneficial Effects of Systemically Administered Human Muse Cells in Adriamycin Nephropathy.

Macrophage-dependent clearance of systemically administered B16BL6-derived exosomes from the blood circulation in mice.

Systemically administered AAV9-sTRAIL combats invasive glioblastoma in a patient-derived orthotopic xenograft model.

Clinical experiences with radiation induced thyroid cancer after chernobyl.

Systemically administered IgG anti-toxin antibodies protect the colonic mucosa during infection with Clostridium difficile in the piglet model.

PTH Induces Systemically Administered Mesenchymal Stem Cells to Migrate to and Regenerate Spine Injuries.

Oligonucleotide therapeutics in cancer.

PTH Induces Systemically Administered Mesenchymal Stem Cells to Migrate to and Regenerate Spine Injuries.

Corrigendum to "PTH Induces Systemically Administered Mesenchymal Stem Cells to Migrate to and Regenerate Spine Injuries".

Distribution of Systemically Administered Nanoparticles Reveals a Size-Dependent Effect Immediately following Cardiac Ischaemia-Reperfusion Injury.

Clinical therapeutics for phenylketonuria.

Clinical pharmacology and therapeutics.

Targeted Transthoracic Acoustic Activation of Systemically Administered Nanodroplets to Detect Myocardial Perfusion Abnormalities.

Systemically administered anti-TNF therapy ameliorates functional outcomes after focal cerebral ischemia.