Advanced antisense therapies for postexposure protection ... - Nature

b r i e f c o m m u n i c at i o n s

Advanced antisense therapies for postexposure protection against lethal filovirus infections

© 2010 Nature America, Inc. All rights reserved.

Travis K Warren1,4, Kelly L Warfield1,3,4, Jay Wells1, Dana L Swenson1,3, Kelly S Donner1, Sean A Van Tongeren1, Nicole L Garza1, Lian Dong1, Dan V Mourich2, Stacy Crumley2, Donald K Nichols1, Patrick L Iversen2 & Sina Bavari1 Currently, no vaccines or therapeutics are licensed to counter Ebola or Marburg viruses, highly pathogenic filoviruses that are causative agents of viral hemorrhagic fever. Here we show that administration of positively charged phosphorodiamidate morpholino oligomers (PMOplus), delivered by various dosing strategies initiated 30–60 min after infection, protects >60% of rhesus monkeys against lethal Zaire Ebola virus (ZEBOV) and 100% of cynomolgus monkeys against Lake Victoria Marburg virus (MARV) infection. PMOplus may be useful for treating these and other highly pathogenic viruses in humans. Ebola viruses and Marburg viruses are highly virulent emerging pathogens of the family Filoviridae and are causative agents of viral hemorrhagic fever (VHF). Nonhuman primate infection models closely reproduce the clinical, histopathological and pathophysiological aspects of fatal filovirus hemorrhagic fever in humans1,2. Primary human ZEBOV and MARV isolates are highly lethal in nonhuman primate models of infection. Initial clinical signs following laboratory infections of nonhuman primates occur approximately 3–4 d after infection and include fever and lethargy as the virus replicates to levels detectable in the blood3. Clinical signs typically progress rapidly and include coagulation abnormalities such as hemorrhage and thrombocytopenia4, alteration of blood chemistry parameters (liver transaminases in particular)1,5 and immunological responses characterized by lymphocyte apoptosis1,2 and late induction of uncontrolled proinflammatory mediators6. Death most often occurs 6–10 d after ZEBOV infection and 8–12 d after MARV infection2. Few reports describe promising outcomes from postexposure therapeutic interventions in nonhuman primate models of filovirus infection. Although administration of recombinant vesicular stomatitis Indiana virus–based vaccine platforms, within 24 h after virus infection, have provided complete or high-level protection against ZEBOV, Sudan Ebola virus and MARV infections in monkeys7–10, the safety and efficacy of this therapeutic modality is unproven in human subjects. Postexposure administrations (initiated within 60 min of infection) of recombinant human activated protein

C and recombinant nematode anticoagulant protein c2, strategies designed to mitigate symptoms of sepsis and coagulopathy, have failed to provide substantive protection of infected monkeys11,12. Although a recent proof-of-concept study showed that a pool of liposomally formulated siRNAs targeting ZEBOV L polymerase, viral protein 2, and viral protein 35 (administered beginning 30 min after infection with ZEBOV) completely protected rhesus monkeys, additional, well-controlled studies are required to determine the extent to which nonspecific, off-target mechanisms may have contributed to protection13. We have previously protected three out of four rhesus monkeys against lethal ZEBOV infection through pre-exposure treatment with neutrally charged PMOs14. Additionally, in a previous report, we introduced a new class of positively charged PMOs (PMOplus) containing piperazine linkages within the molecular backbone and showed that PMOplus constructs targeting ZEBOV viral protein 24 (eVP24) inhibit translation of VP24 in cell-free translation assays and protect mice against lethal ZEBOV challenge15. Although rodent models (laboratory mouse16 and guinea pig17) do not always reliably reproduce the VHF disease course seen in humans and nonhuman primates, these models are useful for screening new therapeutics that directly target viral replication. In the guinea pig model of lethal ZEBOV infection, treatment with a combination of PMOplus agents targeting eVP24 and eVP35 transcripts substantially enhanced protection relative to treatment with eVP24 PMOplus alone. Using the mouse model of ZEBOV infection, we screened the efficacy of eVP24- and eVP35-specific PMOplus combination treatments containing chemical variations in the number and placement of piperazine moieties. From these evaluations, we observed a high degree (≥90% in mice; 83% in guinea pigs) of survival after either pre- or postexposure administration of a combination of PMOplus molecules (each containing five piperazine moieties) designed with sequence complementarity to eVP24 and eVP35 transcripts (data not shown). This combination therapy, containing equivalent concentrations (wt/vol basis) of eVP24- and eVP35-specific PMOplus molecules, is designated as AVI-6002 (Supplementary Table 1). Encouraged by the efficacy of AVI-6002 in the rodent models of infection, we evaluated the efficacy of AVI-6002 in rhesus monkeys in two proof-of-concept studies, in which nine monkeys were challenged with a lethal dose of ZEBOV by intramuscular injection (Supple mentary Methods). PMOplus treatments were initiated 30–60 min after viral exposure. In the initial experiments, we administered treatments using combined subcutaneous and intraperitoneal delivery routes to reproduce the efficacious delivery strategy used in our previous nonhuman primate challenge experiments14. Whereas the untreated macaque developed progressive clinical signs consistent with ZEBOV VHF and succumbed to infection on day 7, five out of

1United

States Army Medical Research Institute of Infectious Diseases (USAMRIID), Fort Detrick, Maryland, USA. 2AVI Biopharma, Corvallis, Oregon, USA. address: Integrated BioTherapeutics, Germantown, Maryland, USA. 4These authors contributed equally to this work. Correspondence should be addressed to S.B. ([email protected]) or P.L.I. ([email protected]). 3Current

Received 22 March; accepted 28 July; published online 22 August 2010; doi:10.1038/nm.2202

nature medicine VOLUME 16 | NUMBER 9 | SEPTEMBER 2010

991

B r i e f c o m m u n i c at i o n s

Percentage survival

25

0

10 20 Time after infection (d)

PBS

* *

9 8 7 6

6002-4

6002-40

6002-16

6003-40

6002-28

PBS

0

10 20 Time after infection (d)

e 2,000

5 4 3

6002-4

6002-40

600

6002-16

6003-40

500

6002-28

PBS

400 300 200

6002-4

6002-40

6002-16

6003-40

6002-28

PBS

f

1,000

1 0 0

3

5

8

10

14

Time after infection (d)

21

28

0

3

5

8

10

0

30

2 0

700

100

30

AST (U l –1)

Lymphocytes (×103 µl –1)


6002-28

c

0

0

d

6002-16

6003-40

14


21

28

10

0

3

5 8 10 14 Time after infection (d)

21

28

Survivor Nonsurvivor

8 6 4 2 0 6002-40 6002-28 6002-40 6002-40 6002-28 6002-40 6002-28 6002-4 6002-16 6003 6002-16 6002-4 6002-28 6002-40 6002-4 6002-28 6002-16 6002-4 6002-16 6003

50

6002-40

Viremia log10 (copies per ml)

Percentage survival

75

6002-4

–1

100

6002

3

b

Naive

Platelets (×10 ml )

a 100

Figure 1 Postexposure protection of ZEBOV-infected rhesus monkeys by AVI-6002. In all experiments, monkeys were challenged with approximately 1,000 plaque-forming units of ZEBOV-Kikwit by intramuscular injection, and PMOplus was administered in PBS beginning 30–60 min after challenge. (a) Combined Kaplan-Meier survival analysis from two proof-of-concept experiments in which monkeys (n = 8) were treated daily with 40 mg per kg body weight AVI-6002. Doses were divided into equal volumes and administered at intraperitoneal and subcutaneous sites. Four monkeys received treatments for 14 d, and four received treatment for 10 d. A single untreated monkey served as an infection control. (b–f) Multiple-dose postexposure efficacy assessment of AVI-6002 for treatment of ZEBOV infection in rhesus monkeys. Monkeys were randomly assigned to treatment groups, and the in-life portion of the experiment was conducted under single-blind experimental conditions. AVI-6002 was delivered at one of four dose levels: 40 (6002-40; n = 5), 28 (6002-28; n = 5), 16 (6002-16; n = 5) or 4 (6002-4; n = 5) mg per kg body weight. Four monkeys were treated with 40 mg per kg body weight negative-control PMOplus formulation (AVI-6003; 6003-40), and one monkey was treated with PBS. All treatments were administered by bolus intravenous injection daily through day 14 after infection. Statistically significant differences (*P < 0.05) between means of AVI-6002 treatments and the AVI-6003 treatment are indicated. (b) Kaplan-Meier survival curves. (c) Mean platelet counts. (d) Mean peripheral blood lymphocyte counts. (e) Mean aspartate aminotransferase (AST) concentration (instrument interference prevented lymphocyte quantification of day 28 6002-16 sample). (f) Plasma viremia assessed by quantitative real-time PCR; results from samples collected from individual monkeys on day 8 are shown.

eight (62.5%) of AVI-6002–treated macaques survived ZEBOV infection (Fig. 1a). In surviving macaques, plasma viremia (Supplementary Fig. 1a) and circulating concentrations of aspartate aminotransferase (Supplementary Fig. 1b), interleukin-6 (Supplementary Fig. 1c) and monocyte chemotactic protein-1 (Supplementary Fig. 1d) were reduced, suggesting that administration of AVI-6002 successfully suppressed virus replication, liver damage and potentially harmful inflammatory responses. Given the promising results obtained from these initial pilot experiments, we further explored the efficacy of AVI-6002 after viral exposure in a multiple-dose evaluation. To control for possible off-target effects, we treated four of these monkeys with AVI-6003, a negative-control PMOplus combination containing two MARV-specific molecules (described in greater detail herein) and that lacks complementarity to ZEBOV targets (Supplementary Table 1). In this experiment, we chose to deliver treatments by intravenous administration to mirror treatment approaches that may be used after accidental needle-stick injuries occurring in research or medical settings. All PBS- and AVI6003–treated monkeys succumbed by day 8 after infection (Fig. 1b) after developing characteristic VHF signs such as fever and petechia. In contrast, 60% (three out of five) of the monkeys in each of the groups treated with either 28 or 40 mg per kg body weight AVI-6002 survived (Fig. 1b). We observed a gradient of protective efficacy, as there was 20% survival in the treatment group receiving 16 mg per kg

992

body weight AVI-6002 and no survivors in the group receiving 4 mg per kg body weight AVI-6002 (Fig. 1b). Although the severity of thrombocytopenia (Fig. 1c) and lymphocytopenia (Fig. 1d) observed in all treatment groups through day 8 was unaffected by AVI-6002 treatments, platelet and lymphocyte counts rebounded in the survivors, indicative of disease resolution. Administration of 28 or 40 mg per kg body weight AVI-6002 mitigated indices of liver damage, such as serum aspartate aminotransferase concentration (Fig. 1e). At the peak of plasma viremia (occurring on day 8 after infection in all monkeys) the mean viral load in AVI-6002–treated monkeys (at a 40 mg per kg body weight dose) was suppressed approximately 100 times that of AVI-6003–treated monkeys (analysis not shown). Viremia in monkeys that survived to day 8 generally correlated with survival (Fig. 1f), suggesting that suppression of viral replication may be a crucial mechanism of AVI-6002–mediated protection. We sought to determine whether the PMOplus therapeutic s trategy might be applied to counter other highly pathogenic filoviruses, such as MARV. Using mouse 18 and guinea pig 19 lethal MARV infection models, we screened the efficacy of PMOplus antisense molecules t argeting MARV VP24, VP35, nucleoprotein and RNA-dependent RNA polymerase L protein. In these models, a combination therapeutic containing equivalent concentrations (wt/vol) of PMOplus molecules specific to MARV nucleoprotein and MARV VP24, designated AVI-6003 (Supplementary

VOLUME 16 | NUMBER 9 | SEPTEMBER 2010 nature medicine

B r i e f c o m m u n i c at i o n s

4

20

2

6003-7.5 6003-15 6003-30

10

N ai

ve

0

e 1,600

6003-7.5

6003-15

1,400

6003-15

7

6003-30

6003-30

6002-30

1,200

6

–1

PBS

4 3

1

200

0 8

10

14

–1

f

PBS

** *

0 5

0

21

28

0

3


5

8

3

5

8

10

14

21

28


600 400

3

30

800

2

0

0

20

6002-30

1,000

** *

200 100

–1

6003-7.5

8

6003-30 6002-30 PBS

300


9

5

400

PBS

0

AST (U l )

Lymphocytes (×103 µl –1)


* *

50

500

6002-30

0

40 i.p mg .a p nd er 30 s. kg c m i.p g . .a p nd er s. kg 40 c. m g pe r s. kg 40 c. m g pe rk i.v g .

0

6003-15

600

* * *

10

Survivor Nonsurvivor

8 6 4 2 0

10

14


21

28

6003-15 6003-30 6003-15 6003-30 6003-7.5 6003-15 6003-30 6003-15 6003-7.5 6003-30 6002-30 6003-7.5 6003-15 6002-30 6003-7.5 6002-30 6003-7.5 PBS 6002-30

40

6003-7.5

700

3

6

c

*

Platelets (×10 ml )

60

100

Viremia log10 (PFU ml )

Percentage survival

8

Viremia log10(PFU ml –1)

80

d

b

10

Percentage survival

a 100

Figure 2 Postexposure protection of MARV-infected cynomolgus monkeys by AVI-6003. In all experiments, monkeys were challenged with approximately 1,000 plaque-forming units of MARV-Musoke by subcutaneous injection, and treatments were initiated beginning 30–60 min after challenge and were administered daily through day 14 after infection. PMOplus was formulated in PBS for delivery. (a) Combined survival and viremia from two independent proof-of-concept evaluations. AVI-6003 was administered at doses of 40 (n = 4) or 30 (n = 3) mg per kg body weight via intraperitoneal and subcutaneous injections or was delivered at 40 mg per kg body weight by subcutaneous (n = 3) or intravenous (n = 3) injection. Treatments delivered at intraperitoneal and subcutaneous sites were administered by injecting equal volumes at each site. Viremia results from day 8, obtained by standard plaque assay (Vero cells), are presented and are depicted as the range and geometric mean. PFU, plaque-forming units. (b–f) Multiple-dose assessment of AVI-6003 for treatment of MARV infection after exposure in cynomolgus monkeys. In-life study components were conducted under single-blind experimental conditions, and monkeys were randomized to treatments. AVI-6003 was delivered intravenously at one of three doses: 30 (6003-30; n = 5), 15 (6003-15; n = 5) or 7.5 (6003-7.5; n = 5) mg per kg body weight. Four monkeys were treated with 30 mg per kg body weight negative-control PMOplus formulation (AVI-6002; 6002-30), and one monkey was treated with PBS. Statistically significant differences (*P < 0.05) between means of AVI-6003 treatments and the AVI-6002 treatment are indicated. (b) Kaplan-Meier survival curves. (c) Mean platelet counts. (d) Mean peripheral blood lymphocyte counts. (e) Mean aspartate aminotransferase concentration. (f) Plasma viremia assessed by standard plaque assay; the maximum viremia value (occurring at either at day 8 or day 10 after infection in all monkeys) obtained during the course of infection is shown for each monkey. This research was conducted in compliance with the US Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals, and it adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, National Research Council. The studies were approved by the USAMRIID Institutional Animal Care and Use Committee, and USAMRIID is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International.

Table 1), consistently conferred a high degree of efficacy (≥90% survival) in both infection models (data not shown). Two independent nonhuman primate pilot experiments were conducted to assess the efficacy of AVI-6003 against lethal MARV challenge. Treatments were initiated 30–60 min after infection, and AVI-6003 was delivered by one of four treatment strategies (Fig. 2a). Untreated, infected control subjects became moribund and were killed on day 9 after infection after developing clinical signs characteristic of MARV infection. In contrast, treatment with AVI-6003 completely protected all 13 monkeys, regardless of dose or route of administration, and suppressed plasma viremia in all groups relative to controls (Fig. 2a). To determine the postexposure therapeutic dose range of AVI-6003 and to rule out possible protection by nonspecific mechanisms, we conducted a randomized, multiple-dose, negative-PMOplus controlled study. Twenty cynomolgus monkeys were infected with a lethal dose of MARV, and the monkeys were intravenously treated with

nature medicine VOLUME 16 | NUMBER 9 | SEPTEMBER 2010

either PBS, AVI-6002 or one of three doses of AVI-6003 beginning 30–60 min after infection. Control monkeys treated with either PBS or AVI-6002 died on days 9–12 (Fig. 2b) with clinical signs characteristic of MARV VHF including petechia, weight loss, fever and/or lethargy. One monkey, treated with 30 mg per kg body weight AVI-6003, died unexpectedly due to complications of anesthesia on day 12 and has been excluded from the survival analysis. All four remaining monkeys treated with 30 mg per kg body weight AVI-6003 survived the infection, and three out of five (60%) of monkeys in each of the groups treated with 7.5 or 15 mg per kg body weight survived (Fig. 2b). In AVI-6003–treated monkeys that succumbed to infection, the mean time to death was delayed by >4 d relative to that of control subjects (Fig. 2b). Although administration of AVI-6003 generally provided little protective effect against MARV-induced thrombocytopenia (Fig. 2c) and lymphocytopenia (Fig. 2d), it significantly reduced circulating concentrations of aspartate aminotransferase (Fig. 2e) relative to control treatments at days 8 and 10 after infection. Analysis

993


B r i e f c o m m u n i c at i o n s of peak viremia in individual monkeys suggested that AVI-6003 mediated protection, at least in part, by reducing viral burden (Fig. 2f). Taken together, these studies provide a major advancement in therapeutic development efforts for treatment of filovirus hemorrhagic fever. Although the 30–60-min interval between virus exposure and initiation of treatment we used in these experiments reflects a timing useful for treating accidental exposures in research or medical settings, the efficacy of delayed treatment we observed in the ZEBOV mouse model of infection (Supplementary Fig. 2) suggests that the window for effective treatment in primates may be wider than we have yet tested. To determine the applicability of PMOplus therapies for treatment of infected individuals during naturally occurring outbreaks in Africa or in possible emergency-response situations after deliberate release of pathogens, future evaluations will focus on defining the window for effective intervention in nonhuman primate models of infection. PMOplus agents possess multiple drug properties favorable for further development for use in humans to counter filoviruses or other highly virulent emerging viruses. They are highly stable and can be rapidly synthesized, purified and evaluated for quality20. They are readily amenable to formulation in isotonic solutions and, as shown in this report, are efficacious by a number of delivery routes. Moreover, PMOplus agents are well tolerated in primates and multiple laboratory animal species, and they possess favorable pharmacokinetic properties (Supplementary Tables 2 and 3). The investigational new drug applications for AVI-6002 and AVI-6003 were submitted to the US Food and Drug Administration, and they are now safe to proceed with clinical studies. Note: Supplementary information is available on the Nature Medicine website. Acknowledgments We thank C. Rice, D. Reed, N. Posten and J. Stockman for technical assistance and M. Ait Ichou and J. Hardick for their help in the initial viral quantification studies. S. Norris and D. Fisher provided statistical assistance and conducted animal randomization procedures. L. Welch, S. Bradfute, R. Panchal and J. Kuhn provided support and discussions and critically reviewed the manuscript. These studies were supported by the US Defense Threat Reduction Agency awarded to AVI BioPharma (HDTRA1-07-C-010) and S.B. (4.10022-08-RD-B and TMTI0048-09-RD-T).

994

Disclaimer: the opinions, interpretations, conclusions and recommendations are those of the authors and are not necessarily endorsed by the U.S. Army. AUTHOR CONTRIBUTIONS T.K.W. designed and supervised multiple-dose primate evaluations, evaluated results and wrote the manuscript. K.L.W. designed, supervised and conducted proof-of-concept nonhuman primate and rodent investigations and evaluated results. J.W., D.L.S., K.S.D., S.A.V.T. and N.L.G. conducted the nonhuman primate and rodent studies and analyzed samples. L.D. conducted quantitative PCR analysis. D.K.N. conducted post-mortem analyses of all nonhuman primate subjects. D.V.M. and S.C. were responsible for synthesis of PMOplus agents. P.L.I. and S.B. designed experiments, evaluated results and provided project oversight. All authors read and approved the final version of the manuscript. COMPETING FINANCIAL INTERESTS The authors declare competing financial interests: details accompany the full-text HTML version of the paper at http://www.nature.com/naturemedicine/. Published online at http://www.nature.com/naturemedicine/. Reprints and permissions information is available online at http://npg.nature.com/ reprintsandpermissions/. 1. Bente, D., Gren, J., Strong, J.E. & Feldmann, H. Dis. Model. Mech. 2, 12–17 (2009). 2. Warfield, K. et al. in Biodefense: Research Methodology and Animal Models (ed. Swearengen, J.R.) 227–258 (CRC Press, Boca Raton, Florida, 2006). 3. Bowen, E.T., Platt, G.S., Simpson, D.I., McArdell, L.B. & Raymond, R.T. Trans. R. Soc. Trop. Med. Hyg. 72, 188–191 (1978). 4. Fisher-Hoch, S.P. et al. J. Infect. Dis. 152, 887–894 (1985). 5. Fisher-Hoch, S.P. et al. Lancet 2, 1055–1058 (1983). 6. Mohamadzadeh, M., Chen, L. & Schmaljohn, A.L. Nat. Rev. Immunol. 7, 556–567 (2007). 7. Feldmann, H. et al. PLoS Pathog. 3, e2 (2007). 8. Daddario-DiCaprio, K.M. et al. Lancet 367, 1399–1404 (2006). 9. Geisbert, T.W. et al. J. Virol. 82, 5664–5668 (2008). 10. Geisbert, T.W. et al. Emerg. Infect. Dis. 16, 1119–1122 (2010). 11. Hensley, L.E. et al. J. Infect. Dis. 196 Suppl 2, S390–S399 (2007). 12. Geisbert, T.W. et al. J. Infect. Dis. 196 Suppl 2, S372–S381 (2007). 13. Geisbert, T.W. et al. Lancet 375, 1896–1905 (2010). 14. Warfield, K.L. et al. PLoS Pathog. 2, e1 (2006). 15. Swenson, D.L. et al. Antimicrob. Agents Chemother. 53, 2089–2099 (2009). 16. Bray, M., Davis, K., Geisbert, T., Schmaljohn, C. & Huggins, J. J. Infect. Dis. 178, 651–661 (1998). 17. Connolly, B.M. et al. J. Infect. Dis. 179 Suppl 1, S203–S217 (1999). 18. Warfield, K.L. et al. J. Virol. 83, 6404–6415 (2009). 19. Hevey, M., Negley, D., Pushko, P., Smith, J. & Schmaljohn, A. Virology 251, 28–37 (1998). 20. Iversen, P. in Antisense Drug Technology (ed. Crooke, S.T.) 375–389 (Marcel Dekker, New York, 2001).

VOLUME 16 | NUMBER 9 | SEPTEMBER 2010 nature medicine