First-in-Humans Trial of an RNA Interference with …...Akshay K. Vaishnaw 6, Dinah WY Sah . . 6,...

RESEARCH ARTICLE

First-in-Humans Trial of an RNA Interference Therapeutic Targeting VEGF and KSP in Cancer Patients with Liver Involvement

ABSTRACT RNA interference (RNAi) is a potent and specifi c mechanism for regulating gene expression. Harnessing RNAi to silence genes involved in disease holds promise

for the development of a new class of therapeutics. Delivery is key to realizing the potential of RNAi, and lipid nanoparticles (LNP) have proved effective in delivery of siRNAs to the liver and to tumors in animals. To examine the activity and safety of LNP-formulated siRNAs in humans, we initiated a trial of ALN-VSP, an LNP formulation of siRNAs targeting VEGF and kinesin spindle protein (KSP), in patients with cancer. Here, we show detection of drug in tumor biopsies, siRNA-mediated mRNA cleavage in the liver, pharmacodynamics suggestive of target downregulation, and antitumor activity, including com-plete regression of liver metastases in endometrial cancer. In addition, we show that biweekly intrave-nous administration of ALN-VSP was safe and well tolerated. These data provide proof-of-concept for RNAi therapeutics in humans and form the basis for further development in cancer.

SIGNIFICANCE: The fi ndings in this report show safety, pharmacokinetics, RNAi mechanism of action, and clinical activity with a novel fi rst-in-class LNP-formulated RNAi therapeutic in patients with cancer. The ability to harness RNAi to facilitate specifi c multitargeting, as well as increase the number of drug-gable targets, has important implications for future drug development in oncology. Cancer Discov; 3(4); 406–17. ©2012 AACR.

Josep Tabernero 1 , Geoffrey I. Shapiro 4 , Patricia M. LoRusso 7 , Andres Cervantes 2 , Gary K. Schwartz 8 , Glen J. Weiss 9 , Luis Paz-Ares 3 , Daniel C. Cho 5 , Jeffrey R. Infante 10 , Maria Alsina 1 , Mrinal M. Gounder 8 , Rick Falzone 6 , Jamie Harrop 6 , Amy C. Seila White 6 , Iva Toudjarska 6 , David Bumcrot 6 , Rachel E. Meyers 6 , Gregory Hinkle 6 , Nenad Svrzikapa 6 , Renta M. Hutabarat 6 , Valerie A. Clausen 6 , Jeffrey Cehelsky 6 , Saraswathy V. Nochur 6 , Christina Gamba-Vitalo 6 , Akshay K. Vaishnaw 6 , Dinah W.Y. Sah 6 , Jared A. Gollob 6 , and Howard A. Burris III 10

406 | CANCER DISCOVERY�APRIL 2013 www.aacrjournals.org

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Published OnlineFirst January 28, 2013; DOI: 10.1158/2159-8290.CD-12-0429

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APRIL 2013�CANCER DISCOVERY | 407

Authors’ Affi liations: 1 Medical Oncology Department, Vall d’Hebron Uni-versity Hospital, Universitat Autònoma de Barcelona, Barcelona; 2 Institute of Health Research INCLIVA, University of Valencia, Valencia; 3 Hospital Virgen del Rocio, Seville, Spain; 4 Dana-Farber Cancer Institute; 5 Beth Israel Deaconess Medical Center, Boston; 6 Alnylam Pharmaceuticals, Inc., Cam-bridge, Massachusetts; 7 Karmanos Cancer Institute, Detroit, Michigan; 8 Memorial Sloan-Kettering Cancer Center, New York, New York; 9 Virginia G. Piper Cancer Center Clinical Trials at Scottsdale Healthcare, Scottsdale, Arizona; and 10 Sarah Cannon Research Institute, Nashville, Tennessee Note: Supplementary data for this article are available at Cancer Discovery Online (http://cancerdiscovery.aacrjournals.org/). Corresponding Author: Josep Tabernero, Medical Oncology Department, Vall d’Hebron University Hospital, Universitat Autònoma de Barcelona, P. Vall d’Hebron 119-129, 08035 Barcelona, Spain. Phone: 34-93-489-4301; Fax 34-93-274-6059; E-mail: [email protected] doi: 10.1158/2159-8290.CD-12-0429 ©2012 American Association for Cancer Research.

INTRODUCTION

RNA interference (RNAi) is an endogenous cellular mecha-nism for controlling gene expression in which siRNAs bound to RNA-induced silencing complex (RISC) mediate target mRNA cleavage and degradation through a catalytic process involv-ing the Argonaute 2 endonuclease ( 1, 2 ). The use of siRNAs to specifi cally silence genes involved in disease pathogenesis holds promise for the development of a new and far-reaching class of therapeutics ( 3, 4 ). Because the RNAi pathway is present in all mammalian cell types, the primary challenge for effective gene silencing in vivo is delivery of the siRNA to the appropriate organ(s) with productive cellular uptake leading to engage-ment of RISC in the cytosol. Systemic delivery is required for the widest application of RNAi therapeutics, and to that end much effort has been focused on the development of siRNA formulations that confer “drug-like” properties favorable to delivery and uptake following parenteral administration ( 5 ).

Various nanoparticle formulations have been evaluated for their ability to silence targets in vivo ( 6–10 ); among these, lipid nanoparticles (LNP) have been shown to be highly effec-tive in delivering siRNAs to the liver and silencing a number of different hepatocyte gene targets across multiple species, including rodents and nonhuman primates ( 11–14 ). Deliv-ery to tumors has also been shown in murine orthotopic liver tumor and subcutaneous tumor models, in which both mRNA silencing and on-target pharmacology were observed following i.v. dosing ( 15 ). Although results in animal models with LNPs are promising, it remains to be shown whether these fi ndings will translate into an effective way to deliver siRNAs for the treatment of human disease. To examine the activity and safety of LNP-formulated siRNAs in humans, we initiated the fi rst phase I trial using this approach to treat patients with advanced cancer and liver metastases.

RESULTS

ALN-VSP Composition and Preclinical Safety and Activity

ALN-VSP is composed of an LNP containing 2 different siRNAs targeting VEGF-A (hereafter referred to as VEGF) ( 16 )

and kinesin spindle protein (KSP, encoded by the KIF11 gene; ref. 17 ) in a 1:1 molar ratio. Both siRNAs were chemically modifi ed to reduce their immunostimulatory potential (refs. 18–20 ; Supplementary Fig. S1). ALN-VSP has a particle diam-eter of 80 to 100 nm and is essentially uncharged with a zeta potential of less than 6 mV at pH 7.4. Consistent with other liposomes of a similar size ( 21, 22 ), ALN-VSP distributes primarily to liver and spleen following parenteral administra-tion due to the fenestrated endothelium in those organs. Dis-tribution to tumors with leaky microvasculature containing endothelial pores is thought to occur through the enhanced permeability and retention (EPR) mechanism described for liposomes and other nanoparticles ( 23, 24 ).

The organs with demonstrable toxicity in preclinical ani-mal toxicology studies with ALN-VSP were primarily liver in rats and spleen in monkeys. A 50% reduction in spleen weight associated with lymphoid atrophy was observed in monkeys treated with 4 doses of 6 mg/kg of ALN-VSP every 2 weeks but not in control animals treated with the same dose of LNP containing an siRNA-targeting luciferase (Supplementary Table S1). These findings were indicative of an on-target siRNA effect rather than a nonspecific effect of the LNP itself, and the lymphopenia reported in patients treated with a small-molecule KSP inhibitor ( 25, 26 ) suggested that KSP rather than VEGF inhibition was underlying the splenic changes in monkeys occurring with ALN-VSP.

In an orthotopic liver tumor model using severe combined immunodefi cient (SCID)/beige mice implanted with Hep3B human hepatocellular carcinoma cells, i.v. administration of a single dose of ALN-VSP to mice with established tumors resulted in dose-dependent suppression of both VEGF and KSP mRNA, with up to 50% reduction observed 24 hours after a 4 mg/kg dose. This fi nding was accompanied by the genera-tion of specifi c RNAi-mediated VEGF and KSP mRNA cleav-age products, as measured using the 5′ Rapid Amplifi cation of cDNA Ends (5′ RACE) assay (refs. 13, 14 , 27, 28 ; Supplementary Fig. S2A). In addition to these effects on mRNA expression, the pharmacodynamic changes expected with KSP and VEGF inhibition were also observed. Specifi cally, within 48 hours following a single dose of ALN-VSP, tumor cells frozen in mitosis with unipolar mitotic spindles (monoasters) were seen throughout liver tumors in ALN-VSP–treated mice but not in control animals (Supplementary Fig. S2B), consistent with KSP inhibition. In mice given repetitive doses of the LNP-formulated VEGF siRNA (LNP-VEGF), both the reduc-tion in tumor hemorrhage and microvascular density were seen to a degree comparable with results in animals treated with the anti-VEGF antibody bevacizumab (Supplementary Fig. S2C). Following these demonstrations of anti-KSP and anti-VEGF pharmacodynamics, the antitumor activity of ALN-VSP administered as repeated doses over 3 weeks was tested in mice bearing established tumors. Animals treated with ALN-VSP had an approximately 50% improvement in median survival relative to control animals (Supplementary Fig. S2D). In addition to having an impact on hepatic tumors, parenterally administered ALN-VSP also has activity against extrahepatic tumors, as shown by the detection of tumor





Tabernero et al.RESEARCH ARTICLE

Total study population, N 41�Dose escalation (0.1–1.5 mg/kg) 30�Expansion phase at 1.0 and 1.25 mg/kg 11 (6 at 1.0 mg/kg,

5 at 1.25 mg/kg)

Median age 57 (range, 34–78)

Male:female 17:24

ECOG performance status 0/1, % 44 / 56

Average number of prior regimens for metastatic disease

4.3 (range, 0–15)

Prior chemotherapy/anti-VEGF therapy, %

88/61

Liver/extrahepatic metastases, % 98/88

Tumor types, N �Gastrointestinal 24�Gynecological 9�Genitourinary 3�Sarcoma 2�Other 3

Doses administered 277

Average number of doses/patient 6.8 (range, 1–50)

Abbreviation: ECOG, Eastern Cooperative Oncology Group.

Table 1. ALN-VSP phase I trial: demographics and dosing

Table 2. Characteristics of patients with disease control

Patient

no.

Dose level,

mg/kg Tumor type

Best

response

Number

of doses

021 0.70 Endometrial CR 50

037 1.00 Renal cell SD 17

040 1.00 Pancreatic neuroendo-crine

SD 36

041 1.00 Renal cell SD 23

Abbreviations: CR, complete response; SD, stable disease.

cell monoasters following ALN-VSP treatment in mice with abdominal peritoneal Hep3B tumor implants (Supplemen-tary Fig. S3A and S3B).

Phase I Study Design and Patient Characteristics

The dose-escalation phase of the study used a 3 + 3 design in which patients were enrolled sequentially on 1 of 7 dose levels (0.1–1.5 mg/kg). This was followed by an expansion phase at the maximum-tolerated dose (MTD). ALN-VSP was administered through either a peripheral angiocath or cen-tral line as a 15-minute i.v. infusion via a controlled infu-sion device using an extension set with a 1.2-μm fi lter every 2 weeks, with a cycle of therapy defi ned as 2 doses given over 1 month (Supplementary Fig. S4). All patients were premedi-cated before each dose with dexamethasone, 8 mg orally the night before and dexamethasone, 20 mg i.v., acetaminophen 650 mg orally, diphenhydramine, 50 mg i.v. (or hydroxyzine, 25 mg orally), and either 50 mg of ranitidine or 20 mg of famotidine i.v. 30 minutes before infusion to reduce the risk of infusion-related reactions (IRR) observed with other liposomal products ( 29 ). Tumor measurements by computed tomography (CT) scan were conducted after every 2 treat-ment cycles. Patients whose disease had not progressed by CT scan after 4 cycles of therapy were eligible to continue treatment on an extension study. A total of 41 patients were enrolled between March 2009 and August 2011, including 30 on the dose-escalation phase treated at 0.1 to 1.5 mg/kg and 11 on the expansion phase treated at 1.0 ( n = 6) or 1.25 mg/kg ( n = 5; Table 1 ). Almost all patients were heavily pretreated with either chemotherapy or anti-VEGF/VEGF

receptor (VEGFR) agents. The majority had both hepatic and extrahepatic tumors. A total of 277 doses (average of 6.8 doses/patient; range, 1–50) were administered on both the phase I and extension studies.

Antitumor and Pharmacodynamic Activity Among 37 patients evaluable for tumor response, 4 of

24 (16.7%) patients treated at ≥0.7 mg/kg had disease con-trol (stable disease or better after at least 12 doses over 6 months; Table 2 ). Seven patients receiving ALN-VSP doses ranging from 0.4 to 1.0 mg/kg whose disease did not progress after the fi rst 4 months went on to the exten-sion study (Supplementary Table S2) and were treated for an average of 11.3 months (range, 5–26 months); these included patients with VEGF-overexpressing tumors such as renal cell cancer (RCC), endometrial cancer, pancreatic neuroendocrine tumor (PNET), and angiosarcoma ( 30–33 ). A major response [complete response, defi ned per Response Evaluation Criteria in Solid Tumors (RECIST) as disappear-ance of all target and nontarget lesions] occurred in a patient with endometrial cancer and multiple hepatic metastases ( Fig. 1A ) as well as an abdominal lymph node metastasis, whose disease had progressed after prior chemotherapy and experimental therapy with a Hedgehog inhibitor. Tumor regression was observed after the fi rst 2 cycles of ALN-VSP dosed at 0.7 mg/kg, and by the end of 6 cycles the patient had achieved a partial response with complete regression of the lymph node metastasis and substantial shrinkage of the liver tumors. A complete response was obtained after 40 doses in this patient, who has remained in remission and completed treatment after receiving 50 doses over 26 months. Two patients with RCC and extrahepatic sites of disease including kidney, lung, and lymph nodes that were progressing follow-ing prior therapy with VEGF pathway inhibitors had stable disease at all sites for approximately 8 to 12 months, and a patient with PNET and multiple liver metastases continued on the extension study for 18 months (36 doses) with stable disease ( Table 2 ).

Dynamic contrast-enhanced MRI (DCE-MRI) was con-ducted at baseline and twice during the week after the first dose of ALN-VSP (see Supplementary Methods for details) to determine whether treatment was associated with a reduction in tumor blood flow indicative of an anti-VEGF effect ( 34 ). To be evaluable by DCE-MRI, patients





Clinical Activity of an RNA Interference Therapeutic in Cancer Patients RESEARCH ARTICLE

Figure 1. Clinical activity of ALN-VSP in patients with cancer. A, complete response in endometrial cancer patient 021 with multiple liver metastases. CT images of metastatic tumors (black arrows) obtained before start of ALN-VSP (pretreatment) and following 12 and 40 doses of drug. B, decrease in blood fl ow ( K trans ) in liver tumors of patients treated with ALN-VSP. For 24 patients with evaluable DCE-MRI scans, the average peak change in K trans for one or more liver tumors occurring during the fi rst week after dose #1 is shown for each patient by dose level. C, DCE-MRI images from patient 012 with metastatic PNET treated at 0.7 mg/kg. Three tumors in right lobe of liver are seen on abdominal MRI (top left) indicated by white arrows. Top right, peak changes in K trans and IAUGC occurring during fi rst week after dose #1 in each tumor. Bottom, corresponding DCE-MRI images of tumor blood fl ow (indicated in red). D, decrease in spleen volume in patients treated with ALN-VSP. Spleen volume was measured on abdominal CT conducted for tumor measurements in 25 evaluable patients with both pre- and posttreatment scans.

were required to have at least 1 liver tumor measuring 2 cm or more in diameter. Both transfer constant ( K trans ) and initial area under the gadolinium concentration (IAUGC) time curve were measured to assess blood flow and capillary permeability. Among 28 patients across all dose levels with evaluable scans, 46% (13 of 28) had a 40% or more peak average reduction in K trans ( Fig. 1B ) although there was no evidence of dose-dependence. Changes in K trans and IAUGC tended to occur in parallel and were often seen within days of the first dose in both large and small tumors ( Fig. 1C ).

To determine whether ALN-VSP had the capability to cause the changes in spleen size observed in monkeys, serial measurements of spleen volume were undertaken retrospec-tively in 25 evaluable patients who had abdominal CT scans conducted for tumor measurements. After 2 to 16 doses of ALN-VSP (patients with more doses had remained on study longer due to lack of disease progression), an average reduction of −37% ± 25% (range, +8 to −92%) was observed in patients dosed at 0.4 to 1.5 mg/kg ( Fig. 1D ), with no clear dose response but with the largest effect seen after 12 or more doses. These splenic changes were consistent with the

preclinical fi ndings in monkeys and therefore suggestive of an anti-KSP effect.

Although no overall correlation was shown between change in K trans or spleen volume and tumor response, the patient with endometrial cancer who had a complete response had a more than 50% decrease in K trans and a 90% decrease in spleen size.

RNAi Proof of Mechanism Voluntary CT-guided core needle tumor biopsies were con-

ducted in 15 patients dosed at 0.4 to 1.5 mg/kg to measure drug levels and look for evidence of RNAi. Biopsies were taken before the fi rst dose of ALN-VSP and then again 2 to 7 days after treatment. Biopsied metastatic tumors were hepatic in 11 patients and extrahepatic in 4 patients. The biopsies were notable for the considerable degree of inter-patient heterogeneity with respect to the amount of viable tumor, necrotic tumor, fi brosis, and/or normal tissue (e.g., liver) present. This degree of heterogeneity, coupled with the different tumor types, sites of metastasis, and days from dos-ing to biopsy, placed limitations on the ability to compare quantitative assessments such as drug levels between patients.

0.2n = 1

0.4n = 4

0.7n = 6

1.0n = 7

1.25n = 6

1.5n = 2

Dose level (mg/kg): 0.1n = 2

0.2n = 2

0.4n = 6

0.7n = 4

1.0n = 8

1.25n = 3

1.5n = 1

0.1n = 1

40%↓

# of tumors:

–100

–80

–60

–40

–20

–100

–80

–60

–40

–20

0

20

Ave

rag

e K

tra

ns c

ha

ng

e

fro

m b

as

elin

e (

%)

1 3 11 3 3 11 3 21 2 11 3 23 1 33 3 31 1 13 1 1

Ktrans

Baseline (BL) predose Day 4 postdose 1 Day 7 postdose 1

T: liver tumor numberKtrans2/IAUGC2: Δ DCE-MRI #1 (BL) to DCE-MRI #2 (day 4)Ktrans3/IAUGC3: Δ DCE-MRI #1 (BL) to DCE-MRI #3 (day 7)

T1 T2 T3

Baseline MRI, coronal view Patient 012

–80

–60

–40

–20

0 Ktrans2IAUGC2Ktrans3IAUGC3

Ktr

an

s a

nd

IA

UG

C c

han

ge

fro

m b

aselin

e (

%)

T1

T2

T3

T1

T2

T3

T1

T2

T3

T1

T2

T3

C

% C

ha

ng

e in

sp

lee

n v

olu

me

# doses:

40

4 4 4 4 2 4 11 3 4 4 3 8 16 8 4 8 3 12 12 8 16 2 4 4 4

20

0

Avg. ↓: 37%

D

Dose level (mg/kg):

PretreatmentAfter 12 doses

ALN-VSPAfter 40 doses

ALN-VSP

BA






Nonetheless, among 12 patients who had biopsies evaluable for both VEGF and KSP siRNAs using quantitative reverse transcriptase PCR (qRT-PCR; Table 3 ; ref. 35 ), all had detect-able VEGF siRNA after treatment (average concentration of 21.3 ± 39.1 ng/g tissue; range, 0.45–142), and 11 of 12 had detectable KSP siRNA (average, 13.6 ± 20.8 ng/g tissue; range, 0.4–73.3; difference between VEGF and KSP siRNA concentrations not signifi cant). Two of these patients had biopsies that were 96% to 100% viable tumor (#025 with sarcoma muscle metastasis and #042 with melanoma liver metastasis), thereby suggesting drug delivery to tumor itself. Because biopsies from the other 10 patients contained vary-ing amounts of viable tumor mixed with fi brosis/necrosis and/or normal tissue ( Table 3 ), the proportion of KSP and/or VEGF siRNA distributed to tumor versus other tissue types within the biopsy could not be determined.

The 5′ RACE assays for VEGF and KSP were conducted on biopsies from all 15 patients. The basal level of specifi c cleav-age product, predicted by the siRNA sequence for each target mRNA and determined by evaluating all of the pretreatment biopsy samples as well as multiple banked tumor and nor-mal liver samples from untreated subjects not on the clinical trial, was 0.7% of total sequences. Among the posttreatment biopsies, 2 patients biopsied 48 hours after dosing, whose liver tumor biopsies were composed predominantly of normal liver and had little to no viable tumor, had a substantial increase in the specifi c cleavage product for VEGF mRNA by sequencing compared with basal level, going as high as 29.2% and 27.9% for patients 016 (metastatic adenocarcinoma of the tongue) and 017 (metastatic ovarian cancer), respectively ( Fig. 2A ; P < 0.000001). The average of 3 separate measurements con-ducted on the same samples for these 2 patients was 19.9%

Table 3. Drug levels and changes in target mRNA in tumor biopsies after first dose

Patient

no.

Dose,

mg/kg

Tumor type

(biopsy site,

day of postdose

biopsy)

Postdose biopsy, % Drug levelsa (ng/g tissue) Change in target mRNA (qRT- PCR)

Viable

tumor Liver

Fibrosis/

necrosis

VEGF

siRNA KSP siRNA

VEGF

mRNA KSP mRNA

007 0.40 Colorectal (liver, d7)

17 80 3 25.7 12.5 20%↓e No Δ

017 0.40 Ovarian (liver, d2)

0 95 5 28.9 17.2 N/A N/A


20 5 75 142 73.3 No Δ No Δ

022 1.00 Colorectal (adre-nal, d2)

10 0 78b 9.8 3.8 No Δ No Δ

025 1.00 Sarcoma (muscle, d2)

96 0 4 0.45 0.40 No Δ No Δ


56 0 35c 5.4 2.2 No Δ No Δ

041 1.00 Renal cell (liver, d2)

70 15 15 6.0 6.8 No Δ No Δ

042 1.00 Uveal melanoma (liver, d2)

100 0 0 7.0 5.8 No Δ No Δ


14 0 71d 0.32 <LLOQ No Δ No Δ

031 1.25 Ovarian (abdomen, d4)

30 0 70 4.9 3.6 74%↓e No Δ

035 1.25 Ovarian (lymph node, d5)

40 0 60 20.4 20.3 37%↓e 25%↓e

032 1.50 Small bowel (liver, d6)

42 0 58 4.9 3.6 No Δ No Δ

NOTE: N/A indicates no sample for analysis. a Measured by qRT-PCR, LLOQ (lower limit of quantitation) = 0.14 ng/g tissue. All pretreatment biopsy samples were <LLOQ. b Remaining 12% was normal adrenal (6%) and fat (6%). c Remaining 9% was skeletal muscle. d Remaining 15% was skeletal muscle. e P values by Relative Expression Analysis Tool (REST) analysis: Pt 007 VEGF: P < 0.001; Pt 031 VEGF: P = 0.001; Pt 035 VEGF: P < 0.001;KSP: P = 0.015.






None of the 15 posttreatment biopsies were positive by KSP 5′ RACE; this result was not unexpected, as the levels of KSP mRNA in banked tumor and normal liver were sub-stantially lower than VEGF mRNA (Supplementary Fig. S5), making detection of the specifi c cleavage product in biopsies with small amounts of viable tumor more diffi cult. However, the biopsy of the ovarian cancer lymph node metastasis that showed the 37% reduction in VEGF mRNA also showed a 25% reduction in KSP mRNA ( P = 0.015; Table 3 ). Unipolar mitotic spindles were not seen in tumor biopsies. In contrast with the rapidly growing Hep3B tumors with synchronously dividing cells in the mouse orthotopic liver tumor model, the more slowly growing tumors in patients treated with ALN-VSP rarely had any identifi able mitoses in the small amounts of viable tumor present in most biopsies (data not shown). This fi nding was consistent with the 2-log lower KSP mRNA expression in banked tumors relative to Hep3B (Supplemen-tary Fig. S5) and may have limited the ability to detect tumor cell monoasters indicative of an anti-KSP effect. Alternatively, the absence of monoasters may have been due to inadequate drug penetration into tumor and/or insuffi cient tumor cell uptake of the KSP siRNA.

Safety and Pharmacokinetic Profi le ALN-VSP was generally well tolerated, with predominantly

low-grade fatigue/asthenia, nausea/vomiting, and fever

Figure 2. Demonstration of RNAi in liver biopsies. A, sequencing from VEGF 5′ RACE analysis. The predicted specifi c VEGF mRNA cleavage product is indicated by the cyan bar; other VEGF fragments are shown in orange. *, P values compared with basal or predose level derived from t test. Liver, normal liver; CRC, colorectal cancer metastasis; patient 016, adenocarcinoma of the tongue (had pre- and posttreatment biopsies); patient 017, ovarian cancer (had posttreatment biopsy only); pre, pretreatment with ALN-VSP; post, 2 days after treatment with dose #1 ALN-VSP. B, Agilent microfl uidic bioana-lyzer DNA 1000 results from VEGF 5′ RACE second-round PCR conducted on untreated normal liver and on liver tumor biopsies from patients 016 and 017. Arrow points to predicted specifi c VEGF (230 bp) mRNA cleavage product.

% T

ota

l re

ad

s

05

10152025

05

10152025

05

10152025

05

10152025

05

10152025

Position

5� Location along VEGF transcript 3�

Patient 017 Post

Patient 016 Post

Patient 016 Pre

CRC

Liver 0.102%

0.64%

1.41%

29.2%

27.9%

P < 0.000001*

P < 0.000001

1319

1344

1369

1394

1419

1444

1467

Ladd

er

400

300

200

150

100

Nor

mal

live

r

Pre

016

Post

016 017

A BSpecific cleavage

and 23.9%, respectively ( P = 0.019 and 0.007 by ANOVA). This increase in specifi c cleavage product was also shown by micro-fl uidic chip analysis of the second-round PCR products ( Fig. 2B ). Neither of these patients had material available for mRNA quantitation by qRT-PCR. A third patient with an ovarian cancer peritoneal metastasis that contained 30% viable tumor, who was treated at 1.25 mg/kg and biopsied 96 hours after dosing, had a more modest average increase in the specifi c cleavage product for VEGF mRNA (4.3%, third highest among all patients analyzed) that did not reach statistical signifi cance relative to basal level and was not seen on microfl uidic chip analysis (data not shown). Measurement of VEGF mRNA by qRT-PCR in this same patient (031) showed a 74% reduction relative to pretreatment biopsy ( P = 0.001; Table 3 ), and a liver metastasis showed a 64% reduction in K trans after treatment as measured by DCE-MRI. Samples from 2 additional patients (035 and 007, with ovarian cancer lymph node metastasis and colorectal cancer liver metastases, respectively) biopsied 5 and 7 days after treatment showed 37% and 20% reductions in VEGF mRNA ( P < 0.001; Table 3 ), respectively, but were negative for specifi c cleavage product by the 5′ RACE assay. Although these results provide evidence for VEGF mRNA downregulation by ALN-VSP in biopsies of both hepatic and extrahepatic tumors, the presence of normal liver and/or necrosis/fi brosis in these samples does not permit a determination of whether target downregulation was occurring in viable tumor tissue.






occurring in 15% to 24% of patients without any clear dose dependence (Supplementary Table S3). No clinically signifi -cant changes in liver function tests were observed in 40 of 41 patients, including 1 patient who received 50 doses at 0.7 mg/kg and 6 others dosed at 0.4 to 1.0 mg/kg who also went onto the extension study and received more than 8 doses. One patient with a PNET and more than 70% liver involvement with metastatic disease who had undergone prior splenectomy and partial hepatectomy tolerated the fi rst dose well but developed hepatic failure several days after the second dose of ALN-VSP at 0.7 mg/kg and subsequently died. A CT scan conducted at the time of liver failure showed an increase in tumor necrosis relative to the pretreatment scan. As this patient’s DCE-MRI after the fi rst dose showed a 50% to 60% decrease in K trans in the 3 liver tumors evalu-ated ( Fig. 1C ), overwhelming anti-VEGF–mediated tumor necrosis was one possible explanation for the liver failure, in addition to disease progression and/or drug-induced injury to the small amount of remaining normal liver. The prior splenectomy and partial hepatectomy were unique to this patient among all those enrolled onto the study. Given the biodistribution of LNPs to liver and spleen, this could have led to a greater exposure of the remaining liver to drug and contributed to the liver toxicity that was not seen in any other subjects treated at doses as high as 1.5 mg/kg. In addition, as an increase in hepatic VEGF production has been observed following liver resection and is thought to be involved in liver regeneration ( 36, 37 ), it is possible that this patient’s liver may have been less able to tolerate local VEGF inhibi-tion by ALN-VSP. In response to this serious adverse event, the protocol was subsequently amended to exclude patients with more than 50% liver involvement with tumor or prior splenectomy. Other dose-limiting toxicities (DLT) occurring during the study included transient grade 3 thrombocyto-penia in 2 of 11 patients treated at 1.25 mg/kg (fi rst episode during dose escalation and a second on the expansion phase, neither of which required platelet transfusion) and tran-sient grade 3 hypokalemia in 1 patient treated at 1.5 mg/kg (Supplementary Table S4).

IRRs occurred after the fi rst dose in 15% of patients dosed at ≥0.4 mg/kg and were readily managed by temporarily

interrupting the infusion and giving the remainder of the dose over 30 to 60 minutes. These IRRs typically occurred within the fi rst minutes of the infusion and usually included some combination of facial fl ushing, chest tightness, back/abdominal pain, elevated heart rate, or sweating. While increases in Bb complement were seen in most patients treated at ≥0.2 mg/kg, those patients with IRRs at 0.4 to 1.25 mg/kg tended to have more Bb induction ( Fig. 3A ). Total C3, C4, and CH50 did not change in patients with IRRs. Dose-dependent proinfl ammatory cytokine induction peaking after either the fi rst or third dose was fi rst observed at 0.4 mg/kg, where several patients were noted to have a modest, transient increase in serum IP-10 and interleukin (IL)-1RA peaking at 6 hours postdose and normalizing by 24 hours. Additional cytokines seen at ≥0.7 mg/kg that also peaked at 6 hours and normalized by 24 hours post-dose included IL-6 ( Fig. 3B ), granulocyte colony-stimulating factor (G-CSF) and, less consistently, TNF-α. Cytokines not induced included IL-1β, IFN-α, and IFN-γ, with IFN-α being most commonly associated with Toll-like receptor (TLR) activation by single- or double-stranded RNA ( 18–20 ). Symptoms related to cytokine induction (grade 1–2 chills/rigors) were seen predominantly at 1.25 mg/kg in patients with peak IL-6 elevations of ≥1,000 pg/mL ( Fig. 3B ). These symptoms typically occurred 4 to 8 hours after the infusion, around the time of peak cytokine induction. There was no cytokine induction in the patient with endometrial cancer with the complete response treated at 0.7 mg/kg. In light of the DLTs seen at 1.25 and 1.5 mg/kg and the magnitude of cytokine induction with associated symptoms occurring at 1.25 mg/kg, the recommended phase II dose of ALN-VSP was determined to be 1.0 mg/kg.

Analysis of plasma pharmacokinetics for the VEGF and KSP siRNAs showed that maximum concentration ( C max ) and area under the curve (AUC) were similar for both and dose-proportional ( Fig. 4 ), with similar profi les after the fi rst and third doses and no evidence of accumulation (data not shown). Approximately 97% of the siRNA detected in plasma was encapsulated. The pharmacokinetic profi le in man was similar to what was observed in non-human primates (Sup-plementary Fig. S6).

Figure 3. Complement and cytokines in patients treated with ALN-VSP. Bb complement (A) and IL-6 (B) induction by dose level. Peak fold-increase following the fi rst and third doses of ALN-VSP is shown for individual patients. P values are derived from linear regression used to model the parameter response as a function of dose level.

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Dose (mg/kg)

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IL-6

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IL-6P < 0.0001 for dose response

Grade 1 chills Grade 1–2 chills and rigors MeanAcute infusion reaction Mean

B






DISCUSSION

The results of this fi rst-in-humans study using LNP-formu-lated siRNAs to treat human disease have established the safety of chronic i.v. dosing in patients with advanced cancer and liver involvement. The study has also demonstrated the abil-ity to measure drug in hepatic and extrahepatic tumor biop-sies and provided evidence for RNAi-mediated target mRNA cleavage in liver, on-target pharmacodynamic effects in liver metastases, and antitumor activity at both hepatic and extra-hepatic sites of disease, including a complete response.

From a safety standpoint, ALN-VSP was generally well tol-erated, with an adverse event profi le that compares favorably with chemotherapy and with other orally or intravenously administered targeted therapies in oncology. The IRRs seen in a minority of patients dosed at ≥0.4 mg/kg were readily managed by slowing the infusion rate. Although C3a and C5a were not measured, the IRRs occurring with ALN-VSP seemed to be complement mediated, as they were not temporally related to cytokine induction and were associated with higher induction of Bb complement. Transient cytokine induction and mild to moderate chills and rigors occurring in a delayed manner after completion of dosing, while not DLTs, were

seen in some patients predominantly at the higher doses of 1.0 and 1.25 mg/kg. Although the 2 siRNAs were chemically modifi ed with 2′-O-methyl groups to minimize immunos-timulation through RNA sensor pathways including TLRs ( 18–20 ), it is possible that either the siRNAs or one or more of the lipid components were involved in proinfl ammatory cytokine induction. Although the cause of the transient grade 3 thrombocytopenia occurring in 2 patients at 1.25 mg/kg is not known, the rapid onset within days of dosing and the swift recovery, as well as the absence of other cytopenias, makes temporary platelet activation and/or sequestration far more likely than marrow suppression. Notably, toxicities seen with systemic VEGF and KSP inhibition, such as hyper-tension/proteinuria ( 38–40 ) and myelosuppression/diarrhea ( 25, 26 ), respectively, were not observed with ALN-VSP. Although this could refl ect a lower level of activity compared with antibodies and/or small molecules targeting these pro-teins, it could also be due to the more limited biodistribution of the LNP formulation to liver, spleen, and tumors, which is consistent with the absence of such toxicities in preclinical monkey toxicology studies using high doses of ALN-VSP. The one instance of hepatotoxicity seen in the patient dosed at 0.7 mg/kg with a particularly heavy tumor burden in the liver

10 200 400 600 800 1,000 1,200 1,400 1,600

Time (min)

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KS

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iRN

A(n

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10,000

100,000

0.100.200.400.70

1.001.251.50

Dose (mg/kg)

1,000

10,000

100,000

VE

GF

siR

NA

(ng

/mL

)

KSP siRNA

ALN-VSPdose (mg/kg)

0.10(n = 3)

0.20(n = 3)

0.40(n = 5)

0.70(n = 7)

1.00(n = 4)

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Cmax (μg/mL)

AUC0-last (min.μg/mL)

0.76 ± 0.36 2.26 ± 0.54 3.92 ± 1.12 7.99 ± 3.39 15.31 ± 5.35 21.94 ± 7.64 18.04

30.83 ± 20.86 133.02 ± 46.66 252.38 ± 97.9 523.18 ± 306.18 923.59 ± 360.13 2,035.74 ± 1,100.08 1,491.03

ALN-VSPdose (mg/kg)

Cmax (μg/mL)

AUC0-last (min.μg/mL)

0.86 ± 0.42 2.51 ± 0.56 4.31 ± 1.10 8.81 ± 2.75 15.84 ± 7.01 21.58 ± 6.80 18.19 ± 1.09

69.95 ± 66.12 143.11 ± 58.58 272.21 ± 98.16 626.37 ± 272.66 984.26 ± 436.81 2,006.58 ± 1,113.54 1,514.63 ± 150.24

VEGF siRNA

0.100.200.400.70

1.001.251.50

Dose (mg/kg)

Figure 4. ALN-VSP phase I pharmacokinetic data. Mean plasma pharmacokinetic results for KSP (left) and VEGF (right) siRNAs shown for each dose level following the fi rst dose of ALN-VSP.






who had undergone prior partial hepatectomy and splenec-tomy was of unclear etiology and underscores the importance of further evaluating safety across a range of disease presenta-tions in future studies of patients with cancer involving the liver. However , it is notable that the liver safety profi le was benign in the other 40 patients treated during the study at doses as high as 1.5 mg/kg, including those on the extension study who received biweekly treatments for an average of 11.3 months (with 2 patients at 0.7 and 1.0 mg/kg treated for 26 and 18 months, respectively).

Although the ALN-VSP phase I trial did require that patients have at least one measurable tumor in the liver, a notable fi nding of the study was the detection of drug in both hepatic and extrahepatic tumor biopsies. Both siRNAs in ALN-VSP were detected in liver tumor biopsies and in metastases involving the adrenal gland, lymph nodes, and abdominal cavity, across multiple different tumor types. In several instances, these biopsies were composed entirely of viable tumor, thereby showing that the drug was delivered to the tumor itself. However, other biopsies contained varying amounts of necrosis and fi brosis or normal tissue in addition to tumor, and therefore localization to the tumor could not be determined in those samples. The demonstration of RNAi-mediated VEGF mRNA cleavage in the liver of 2 patients, albeit in biopsy samples that predominantly showed normal liver with little or no tumor, establishes proof of mechanism for RNAi in humans using this novel siRNA-LNP formula-tion. The demonstration of VEGF mRNA downregulation in a liver metastasis from 1 patient and in extrahepatic metastases from 2 patients, though not conclusive for target downregulation in tumor due to the presence of other tissue types in the biopsy samples, nonetheless provides additional examples of VEGF mRNA modulation in vivo by ALN-VSP supportive of proof of mechanism for RNAi.

A possible clinical correlate of the observed changes in VEGF mRNA was the substantial decrease in tumor blood fl ow seen on DCE-MRI in nearly half of the patients with evaluable liver tumors. Although qualitatively these changes in blood fl ow resembled the reported changes observed with anti-VEGF antibodies and small-molecule inhibitors of the VEGFR ( 41, 42 ), no dose response was observed. One therefore cannot exclude the possibility that these fi ndings were the result of a nonspecifi c effect of the LNP formula-tion or premedication regimen rather than a specifi c effect of the VEGF siRNA in ALN-VSP. Although we were not able to detect tumor cell monoasters indicative of an anti-KSP effect in tumor biopsies, changes in spleen volume were observed that were indicative of an anti-KSP effect based on preclinical data in monkeys. This fi nding, combined with the demonstration of KSP mRNA reduction in an ext-rahepatic tumor biopsy, suggests that pharmacodynamic changes consistent with both VEGF and KSP inhibition were observed with ALN-VSP.

Major tumor responses, especially complete responses, are infrequently seen in phase I studies involving heavily pretreated patients with progressive disease. Therefore, it is notable that the ALN-VSP phase I trial included a complete response in a patient with nodal and extensive liver metas-tases, as well as prolonged disease stabilization for as long as 1 to 1.5 years in patients with hepatic and extrahepatic

metastases. With respect to the mechanism underlying the observed antitumor activity, there is no evidence that this was an off-target effect of the drug. While transient cytokine induction was observed after the fi rst and third doses of drug in some patients treated at the higher dose levels, this change was not seen in the patient who achieved the complete response. Therefore, it is unlikely that the tumor regression resulted from an antitumor immune response. In the mouse Hep3B tumor model, a control siRNA formulated in the same LNP as ALN-VSP had no antitumor activity, further showing that the activity of ALN-VSP was not due to an off-target effect of the LNP. Given the heterogeneity of tumor types and prior therapies (including prior anti-VEGF therapies) and the relatively small number of patients treated across various dose levels, it is unlikely that this phase I study would have been able to establish correlations between on-target pharmacodynamic changes and clinical outcome. Subsequent phase II trials with ALN-VSP focusing on a single tumor type, enrolling patients with fewer prior therapies, and treating all patients at the same active dose level will be better suited for exam-ining the relationship between target modulation and clini-cal response to therapy.

Overall, these fi ndings show the safety, pharmacokinetics, RNAi mechanism of action, and clinical activity with a novel fi rst-in-class RNAi therapeutic in humans. These fi ndings build on the results previously reported with CALAA-01 ( 9 ), a targeted cyclodextrin-based nanoparticle similar in size to ALN-VSP that contains an siRNA targeting ribonucleoside-diphosphate reductase subunit M2 (RRM2). In that report, CALAA-01 was shown to localize in cutaneous melanoma metastases following i.v. administration and to mediate target mRNA cleavage and downregulation. Here, the data with ALN-VSP suggest that dual targeting of VEGF and KSP was attained at both the molecular and clinical level. This study also provides the basis for further development of ALN-VSP in malignancies responsive to anti-VEGF drugs, such as endometrial cancer ( 43 ), PNET ( 32 , 39 ), RCC ( 30 , 38 ) and hepatocellular carcinoma ( 44 ), in which more effective and better-tolerated therapies are needed.

METHODS

Animal Experiments Intrahepatic tumors were generated by injection of 1 × 10 6 human

Hep3B hepatocellular carcinoma cells (American Type Culture Col-lection; catalog no. HB-8064; cells were authenticated through analysis of protein expression, presence of HepB viral DNA by PCR, and demonstration of tumorigenicity in nude mice), suspended in 0.025 mL PBS, directly into the livers of SCID/beige mice. Surgery was carried out under isofl uorane anesthesia and according to Institutional Animal Care and Use guidelines. Tumor growth was monitored using AFP ELISA (Sierra Resources International). Ani-mals were randomly selected into groups with similar mean tumor size before treatment, and only tumor-bearing animals were used in experiments. To determine the antitumor effect of ALN-VSP, tumor-bearing animals began treatment 4 weeks after implantation. Animals were euthanized on the basis of humane endpoints. At the end of the study all remaining animals were euthanized. For histol-ogy and imaging, tumors were fi xed in buffered formalin and paraf-fi n embedded. Whole tumor sections were stained with hematoxylin






and eosin (H&E) to quantify monoasters or hemorrhage or with anti-CD34 (Abcam) to quantify microvasculature. Regions of intra-tumoral hemorrhage were outlined in H&E-stained sections, and total areas of hemorrhage were quantifi ed in each tumor. CD34-stained areas were quantifi ed as a percentage of total tumor area.

Bioanalytical Methods RNA was purifi ed from blinded biopsy samples following the

standard RNAeasy Micro Kit (Qiagen; catalog no. 74004) proto-col with minor modifi cations. Quality and quantity of RNA was assessed with a Bioanalyzer2000 (Agilent; catalog no. G2940CA). qRT-PCR was conducted using LightCycler 480 Probe Master Mix (Roche Applied Science; catalog no. 04887301001) and VEGF-A (Life Technologies; catalog no. Hs00173626_m1), KIF11 (Life Technolo-gies; catalog no. Hs00189698_m1), glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ; Life Technologies; catalog no. 4326317E) or CEACAM5 (Life Technologies; catalog no. hs00237075_m1) gene-spe-cifi c TaqMan probes on a LightCycler 480 system (Roche). qRT-PCR data analysis was conducted using the Relative Expression Analysis Tool (REST). 5′ RACE was conducted for either KIF11 or VEGF-A as per the GeneRacer Kit protocol (Life Technologies; catalog no. L1500-01) with minor modifi cations. Standard Illumina genomic sequenc-ing adaptors were added to the second round 5′ RACE PCR primers and sequenced using a custom Illumina sequencing primer. Data were processed using custom Perl programs. Sequences were aligned to the appropriate transcript ( VEGF-A = NM_001025368.3 and KIF11 = NM_004523.3). The cleavage position in KIF11 is 1249 and for VEGF-A is 1393. See Supplementary Methods for more details on 5′ RACE assay.

Phase I Clinical Trial Patients ages 18 years or older with advanced solid tumors con-

fi rmed by histologic or cytologic examination that had recurred or progressed following standard therapy, had not responded to stand-ard therapy, or for whom there was no standard therapy, were eligible to participate in this study if they had at least one measurable tumor (≥1 cm by spiral CT or ≥2 cm by standard CT) in the liver. Other eligi-bility criteria included Eastern Cooperative Oncology Group perform-ance status of 0 to 1; at least 28 days out from prior systemic therapy, radiotherapy, or major surgery; aspartate and alanine aminotrans-ferase levels ≤2.5 times the upper limit of normal; total bilirubin within normal limits; albumin >3.0 g/dL; international normalized ratio ≤1.2; absolute neutrophil count ≥1,500 cells/mm 3 ; platelet count ≥100,000 cells/mm 3 ; hemoglobin ≥9 g/dL; and serum creatinine ≤1.5 times the upper limit of normal. Patients were excluded if they had brain or leptomeningeal metastases, known infection with hepatitis B or C or HIV, or more than 50% involvement of the liver by tumor, or if they had previously undergone splenectomy. No limit was placed on the number of prior therapies. The study was approved by each site’s Institutional Review Board and registered on ClinicalTrials.gov (NCT00882180). Written informed consent was obtained from patients. To be eligible for the extension study (NCT01158079), patients had to have completed 4 cycles of treat-ment and have stable disease or better by RECIST, in addition to meeting the same laboratory and performance status eligibility criteria. Patients were monitored for adverse events using version 3.0 of the National Cancer Institute Common Toxicity Criteria.

Cytokines and Complement Cytokines were measured in serum samples obtained before dosing

and 2, 6, and 24 hours after infusion for the fi rst and third doses. Measurements of IFN-α, IFN-γ, IL-6, IL-12, TNF-α, IL-1β, IL-1RA, G-CSF, and IP-10 were conducted at Charles River Laboratories. Complement factors C3, C4, CH50, and Bb were measured before dosing and 30 minutes, 2 hours, and 24 hours after infusion for the

fi rst and third doses. C3, C4, and CH50 were measured locally; Bb measurement was conducted at Charles River Laboratories.

Pharmacokinetics For pharmacokinetic analyses, plasma and plasma fi ltrate from

specifi ed time points were analyzed for total and unencapsulated siRNA, respectively, using a validated hybridization ELISA for each of the 2 siRNAs (Charles River Laboratories). Analysis of the concentra-tion–time data was conducted by Charles River Laboratories, and the pharmacokinetic profi le of each patient was characterized by noncom-partmental analysis of each siRNA plasma concentration using vali-dated computer software (WinNonlin, version 3.2, Pharsight Corp.).

Tumor Biopsies Core biopsies of tumors were carried out under CT guidance.

Where possible, 3 separate cores were obtained from the same tumor at each time point. Two cores obtained for siRNA quantitation and for VEGF/KSP qPCR and 5′ RACE were snap frozen in liquid nitrogen and sent to Alnylam Pharmaceuticals for processing; the third core was fi xed in formalin and sent to DCL Medical Laboratories, where it was imbedded in paraffi n before sectioning and staining with H&E for histopathologic evaluation.

Disclosure of Potential Confl icts of Interest R.M. Hutabarat and J. Celesky are employed as Senior Directors

for Alnylam Pharmaceutical, Inc. S.V. Nochur is employed as Vice President for Regulatory Affairs, and J.A. Gollob is employed as Vice President for Clinical Research, both for Alnylam Pharmaceuticals, Inc. C. Gamba-Vitalo has ownership interest (including patents) in Alnylam 401K. A.K. Vaishnaw is employed as Chief Medical Offi cer for Alnylam Pharmaceuticals, Inc. G. Weiss has received honoraria and served on the speakers’ bureau for Genentech and Pfi zer. No potential confl icts of interest were disclosed by the other authors.

Authors’ Contributions Conception and design: J. Tabernero, I. Toudjarska, V.A. Clausen, J. Cehelsky, S.V. Nochur, A.K. Vaishnaw, D.W.Y. Sah, J.A. Gollob, and H.A. Burris III Development of methodology: J. Tabernero, G.I. Shapiro, P.M. LoRusso, A. Cervantes, G.K. Schwartz, G.J. Weiss, L. Paz-Ares, R. Falzone, J. Harrop, A.C.S. White, I. Toudjarska, R.E. Meyers, N. Svrzikapa, V.A. Clausen, J. Cehelsky, S.V. Nochur, A.K. Vaishnaw, J.A. Gollob, and H.A. Burris III Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): J. Tabernero, G.I. Shapiro, P.M. LoRusso, A. Cervantes, G.K. Schwartz, G.J. Weiss, L. Paz-Ares, D.C. Cho, J.R. Infante, M. Alsina, M.M. Gounder, I. Toudjarska, D. Bumcrot, C. Gamba-Vitalo, A.K. Vaishnaw, J.A. Gollob, and H.A. Burris III Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J. Tabernero, G.I. Shapiro, P.M. LoRusso, A. Cervantes, G.K. Schwartz, G.J. Weiss, L. Paz-Ares, R. Falzone, J. Harrop, A.C.S. White, I. Toudjarska, D. Bumcrot, R.E. Meyers, G. Hinkle, N. Svrzikapa, R.M. Hutabarat, V.A. Clausen, J. Cehelsky, S.V. Nochur, A.K. Vaishnaw, D.W.Y. Sah, J.A. Gollob, and H.A. Burris III Writing, review, and/or revision of the manuscript: J. Tabernero, G.I. Shapiro, P.M. LoRusso, A. Cervantes, G.K. Schwartz, G.J. Weiss, L. Paz-Ares, I. Toudjarska, R.E. Meyers, G. Hinkle, N. Svrzikapa, R.M. Hutabarat, V.A. Clausen, J. Cehelsky, S.V. Nochur, C. Gamba-Vitalo, A.K. Vaishnaw, J.A. Gollob, and H.A. Burris III Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): G. Hinkle, N. Svrzikapa, C. Gamba-Vitalo, J.A. Gollob, and H.A. Burris III Study supervision: A.K. Vaishnaw, J.A. Gollob, and H.A. Burris III






Grant Support This study was funded by Alnylam Pharmaceuticals, Inc.

Received September 24, 2012; revised December 26, 2012; accepted January 24, 2013; published OnlineFirst January 28, 2013.

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2013;3:406-417. Published OnlineFirst January 28, 2013.Cancer Discovery Josep Tabernero, Geoffrey I. Shapiro, Patricia M. LoRusso, et al. VEGF and KSP in Cancer Patients with Liver InvolvementFirst-in-Humans Trial of an RNA Interference Therapeutic Targeting

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