Efficacy of Novel Anti-sense Treatment for Filovirus Infections

Marc Nakashima | BIOL113 | DUE 11.05.10

INTRODUCTION
DATA
Popularized by Richard Preston’s non-fiction novel The Hot Zone, Ebola and Marburg viruses are highly pathogenic and lethal to humans and non-human
primates. Five species of Ebola have been discovered; of these five, Zaire Ebola (ZEBOV) has the highest case-fatality rate, at 90 percent. Marburg virus (MARV), a FIG1. Antisense therapy significantly mitigates VHF in non-
less deadly cousin of Ebola, has a case-fatality rate of 23-25 percent. Initial diagnosis of both diseases is difficult, because early symptoms are similar to other human primates infected with Zaire-Ebola. a) Kaplan-Meier
infections. The cause of death is by systemic organ failure and viral hemorrhagic fever (VHF); patients often “bleed out” from natural openings or cuts on the survival analysis of AVI-6002-treated monkeys. Five out of
body.[1] To date, there is no known cure for either disease. eight (62.5%) AVI-6002-treated monkeys survived ZEBOV
infection. Negative control died day 7. b-d) Multiple-dose
Both Ebola and Marburg belong to a family of single-stranded, negative-sense RNA viruses known as Filoviridiae, or filoviruses.[2] The RNA basis for gene post-exposure efficacy assessment of AVI-6002 for
storage and expression makes for an excellent drug target; inhibition of viral translation via novel anti-sense technology is a recent attempt at a cure, discussed treatment of ZEBOV infection. b) Kaplan-Meier analysis
below. The antisense molecule used is a positively charged phosphorodiamidate morpholino oligomer (PMOplus). Administration of equivalent concentrations shows higher survival rate with increased dosage.
of PMOplus molecules specific to ZEBOV viral protein 24 and 35 transcripts (eVP24 and eVP35, respectively) have demonstrated great success in mice and guinea Statistically significant differences (*P < 0.05) between
pigs (≥90% mice; 83% guinea pig survival rate).[3] Furthermore, survival rate was independent of time of treatment, i.e. pre-exposure and post-exposure means of AVI-6002 treatments and the negative control
AVI-6003 treatment are indicated. Negative controls did not
administration of the PMOs had no significant effect on survival. The combination therapy of eVP24 and eVP35 specific PMOplus molecules was designated as survive the experiment. c) Mean platelet count shows
AVI-6002. The aim of the following experiment with ZEBOV is to test the efficacy of AVI-6002 in a non-human primate. Based on the success of this experiment, a steady decline, then increase in surviving subjects receiving
similar cocktail of equivalent concentrations of PMOplus molecules specific to MARV nucleoprotein and MARV VP24 was prepared, designated as AVI-6003. The AVI-6002. d) Mean peripheral blood lymphocyte count
efficacy of AVI-6003 was also tested in a non-human primate. shows steady decline, then increase in surviving subjects
receiving AVI-6002. e) Mean AST concentration reaches a
variable maxima corresponding to dosage gradient. f)
RESEARCH QUESTION: Does a cure exist for fatal filoviral infections, such as Ebola and Marburg virus? Plasma viremia assessed by quantitative real-time PCR
shows viremia increase corresponding to dosage gradient.

METHODS
ZEBOV Experiment 1 (FIG1a) FIG2. Antisense therapy significantly mitigates VHF in non-
1) Infect rhesus monkeys (n = 9) with a lethal dose of ZEBOV (~1 000 ZEBOV-Kikwit plaque-forming units) by intramuscular injection. human primates infected with Marburg virus. a) Combined
survival and viremia plots of AVI-6003-treated monkeys. All
2) Deliver 40 mg per kg body weight AVI-6002 by intraperitoneal and subcutaneous injection 30-60 min. post-exposure to experimental group (n = 8). Four AVI-6003-treated monkeys, regardless of means of
monkeys receive treatment for 10 d and the remaining four receive treatment for 14 d. Negative control (n = 1) receives no treatment. treatment, survived MARV infection. No controls survived.
3) Observe mortality over time (t = 30 d) and analyze by Kaplan-Meier estimation. b-d) Multiple-dose post-exposure efficacy assessment of
AVI-6003 for treatment of MARV infection. b) Kaplan-Meier
ZEBOV Experiment 2 (FIG1b-f) analysis shows higher survival rate with increased dosage.
Statistically significant differences (*P < 0.05) between
1) Test efficacy of AVI-6002 dosage on rhesus monkeys, post-exposure to ZEBOV. AVI-6002 administered to experimental group at four dose levels: 40 (6002-40; means of AVI-6003 treatments and the negative control AVI-
n = 5), 28 (6002-28; n = 5), 16 (6002-16; n = 5), or 4 (6002-4; n = 5) mg per kg body weight. Negative control: Four monkeys received 40 mg per kg body 6002 treatment are indicated. Negative controls did not
weight AVI-6003-40, and one monkey PBS. All treatments administered by daily intravenous injection through day 14 after infection. survive the experiment. c) Mean platelet count shows
2) Observe monkeys and monitor vital signs (platelet count, lymphocyte count, AST concentration, viremia concentration) over time (t = 28-30 d) steady decline, then increase in surviving subjects receiving
AVI-6003. d) Mean peripheral blood lymphocyte count
shows steady decline, then increase in surviving subjects
MARV Experiment 1 (FIG2a) receiving AVI-6003. e) Mean AST concentration reaches a
1) Infect cynomolgus monkeys with a lethal dose of MARV (~1 000 MARV-Musoke plaque-forming units) by subcutaneous injection. variable maxima corresponding to dosage gradient. f)
2) Deliver AVI-6003 30-60 min. post-exposure to experimental group at doses of 40 (n -= 4) or 30 (n = 3) mg per kg body weight via intraperitoneal and Plasma viremia assessed by quantitative real-time PCR
subcutaneous injection or at 40 mg per kg body weight by subcutaneous (n = 3) or intravenous (n = 3) injection. Negative controls received no treatment. shows viremia increase corresponding to dosage gradient.
3) Observe mortality and viremia over time.

MARV Experiment 2 (FIG2b-f)

RESULTS/DISCUSSION
1) Test efficacy of AVI-6003 dosage on cynomolgus monkeys, post-exposure to MARV. AVI-6003 administered to experimental group at three dose levels: 30
Protective efficacy gradient observed against ZEBOV; 60% survival rate among recipients of either 28 or 40 mg per kg body weight AVI-6002, 20%
(6003-30; n = 5), 15 (6003-15; n = 5), or 7.5 (6003-7.5; n = 5) mg per kg body weight. Negative control: Four monkeys received 30 mg per kg body weight AVI-
survival among 16 mg per kg body weight recipients, and no survivors among 4 mg per kg body weight recipients suggests direct correlation with
6002-30, and one monkey PBS.
dosage and protection (FIG1b).
2) Observe monkeys and monitor vital signs (platelet count, lymphocyte count, AST concentration, viremia concentration) over time (t = 28-30 d)
Thrombocytopenia (FIG1c) and lymphocytopenia (FIG1d) unaffected by treatment among all treatment groups through day 8; increase in platelet
and lymphocyte count suggests disease resolution.
KEY TERMS Relatively low AST concentration among AVI-6002-28 and AVI-6002-40 recipients suggests mitigation of liver damage by ZEBOV.
Negative-sense RNA virus: An RNA virus that must use an RNA polymerase to convert its RNA into positive-sense RNA, which can be read as mRNA and translated by host ribosomes
Antisense therapy: The use of molecules complementary to nucleic acids that modify or block gene expression via irreversible binding or competitive inhibition. At the peak of plasma viremia (day 8) the mean viral load in AVI-6002-40 recipients was suppressed approx. 100 times that of AVI-6003 recipients
Morpholino oligomer: An antisense molecule designed to block small (~25 base pair) regions of RNA. (analysis not shown).
Kaplan-Meier survival analysis: An estimate of the survival function from life-time data. Viremia in monkeys that survived to day 8 generally correlates with survival, suggesting that suppression of viral replication may be a crucial
Intramuscular (IM) injection: Injection into a muscle.
Intraperitoneal injection: Injection into the body cavity. mechanism of AVI-6002-mediated protection against ZEBOV.
Intravenous (IV) injection: Injection into a vein.
Subcutaneous (subcut) injection: Injection just beneath the surface of the skin. Protective efficacy gradient observed against MARV; 100% survival rate among recipients of 30 mg per kg body weight AVI-6003 and 60% survival
Aspartate aminotransferase (AST): An enzyme found primarily in the liver, and in lesser quantities in the brain, heart, kidneys, and skeletal muscle. Elevated levels of AST is an indication of severe damage to these
organs. among 7.5 mg per kg body weight and 15 mg per kg body weight recipients suggests direct correlation with dosage and protection (FIG2b).
Platelet/thrombocyte: Anucleate cells which circulate in the mammalian bloodstream and are responsible for hemostasis by providing clotting and growth factors. Thrombocytopenia (FIG2c) and lymphocytopenia (FIG2d) unaffected by treatment among all treatment groups.
Thrombocytopenia: A medical condition where platelet count falls below 50 000 per microliter. Low platelet concentration increases the risk of uncontrolled bleeding. Significant reductions in circulating concentrations of AST relative to control treatments (FIG2e).
Lymphocyte: Cells involved in the immune system. Includes T cells, B cells, and natural killer (NK) cells.
Lymphocytopenia: A medical condition where lymphocyte count is abnormally low. Analysis of peak viremia in individual monkeys suggests AVI-6003 mediated protection by reducing viral burden (FIG2f).
Viremia: A medical condition where viruses have entered the bloodstream.
CONCLUSIONS

 PMOplus antisense therapy is an effective treatment for lethal Ebola and Marburg
filoviruses in non-human primates
 Protective gradient correlates with dosage of PMOplus drugs
 Treatment may be given by different delivery routes with no significant effect on
efficacy
 Future studies needed to assess window for effective intervention in non-human
primates
 Clinical studies for AVI-6002 and AVI-6003 recently approved by US Food and Drug
Administration
REFERENCES
Ebola virus (SEM) [4] [1] Viral Hemorrhagic Fevers. CDC Special Pathogens Branch http://www.cdc.gov/ncidod/dvrd/spb/mnpages/dispages/vhf.htm
[2] Filoviruses. CDC Special Pathogens Branch http://www.cdc.gov/ncidod/dvrd/spb/mnpages/dispages/filoviruses.htm
[3] Warren TK, Warfield KL, Wells J, Swenson DL, Donner KS, Van Tongeren SA, Garza NL, Dong L, Mourich DV, Crumley S, Nichols DK, Iversen PL, Bavari S. “Advanced antisense therapies for postexposure
Marburg virus (SEM 10^6 X; negative stain) [3] protection against lethal filovirus infections.” Nat Med. 2010 Sep;16(9):991-4. Epub 2010 Aug 22.
Phosphorodiamidate morpholino oligomer (PMO) bound to RNA [6] [4] Marburg Hemorrhagic Fever. CDC Special Pathogens Branch http://www.cdc.gov/ncidod/dvrd/spb/mnpages/dispages/marburg/qa.htm#marburgem
[5] Ebola Hemorrhagic Fever. CDC Special Pathogens Branch http://www.cdc.gov/ncidod/dvrd/spb/mnpages/dispages/ebola/qa.htm
[6] Morpholino. Wikipedia. http://en.wikipedia.org/wiki/Morpholino