Jon-D's blog

Following are citations of Ebola or Marburg filovirus drug development using Morpholino antisense oligos.

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Enterlein S, Warfield KL, Swenson DL, Stein DA, Smith JL, Gamble CS, Kroeker AD, Iversen PL, Bavari S, Muhlberger E. VP35 Knockdown Inhibits Ebola Virus Amplification and Protects against Lethal Infection in Mice. Antimicrob Agents Chemother. 2006 Mar;50(3):984-93.

Phosphorodiamidate morpholino oligomers (PMO) are a class of uncharged single-stranded DNA analogs modified such that each subunit includes a phosphorodiamidate linkage and morpholine ring. PMO antisense agents have been reported to effectively interfere with the replication of several positive-strand RNA viruses in cell culture. The filoviruses, Marburg virus and Ebola virus (EBOV), are negative-strand RNA viruses that cause up to 90% lethality in human outbreaks. There is currently no commercially available vaccine or efficacious therapeutic for any filovirus. In this study, PMO conjugated to arginine-rich cell-penetrating peptide (P-PMO) and nonconjugated PMO were assayed for the ability to inhibit EBOV infection in cell culture and in a mouse model of lethal EBOV infection. A 22-mer P-PMO designed to base pair with the translation start site region of EBOV VP35 positive-sense RNA generated sequence-specific and time- and dose-dependent inhibition of EBOV amplification in cell culture. The same oligomer provided complete protection to mice when administered before or after an otherwise lethal infection of EBOV. A corresponding nonconjugated PMO, as well as nonconjugated truncated versions of 16 and 19 base residues, provided length-dependent protection to mice when administered prophylactically. Together, these data suggest that antisense PMO and P-PMO have the potential to control EBOV infection and are promising therapeutic candidates.

http://aac.asm.org/content/50/3/984.abstract

mice and cell culture

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Warfield KL, Swenson DL, Olinger GG, Nichols DK, Pratt WD, Blouch R, Stein DA, Aman MJ, Iversen PL, Bavari S. Gene-Specific Countermeasures against Ebola Virus Based on Antisense Phosphorodiamidate Morpholino Oligomers. PLoS Pathog. 2006 Jan;2(1):e1. Epub 2006 Jan 13.

The filoviruses Marburg virus and Ebola virus (EBOV) quickly outpace host immune responses and cause hemorrhagic fever, resulting in case fatality rates as high as 90% in humans and nearly 100% in nonhuman primates. The development of an effective therapeutic for EBOV is a daunting public health challenge and is hampered by a paucity of knowledge regarding filovirus pathogenesis. This report describes a successful strategy for interfering with EBOV infection using antisense phosphorodiamidate morpholino oligomers (PMOs). A combination of EBOV-specific PMOs targeting sequences of viral mRNAs for the viral proteins (VPs) VP24, VP35, and RNA polymerase L protected rodents in both pre- and post-exposure therapeutic regimens. In a prophylactic proof-of-principal trial, the PMOs also protected 75% of rhesus macaques from lethal EBOV infection. The work described here may contribute to development of designer, \"druggable\" countermeasures for filoviruses and other microbial pathogens.

http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat...

Rhesus macaque, mouse, guinea pig, Vero cells

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Vander-Linden CL. Gene-Specific Ebola Therapies Protect Nonhuman Primates from Lethal Diseases. Chem-Bio Defense Quarterly. 2006 3(2):8-9.

Also this press release from USAMRIID:

http://www.usamriid.army.mil/press_releases/warfield_press_release.pdf

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Silvestri LS, Ruthel G, Kallstrom G, Warfield KL, Swenson DL, Nelle T, Iversen PL, Bavari S, Aman MJ. Involvement of Vacuolar Protein Sorting Pathway in Ebola Virus Release Independent of TSG101 Interaction. J Infect Dis. 2007 Nov 15;196 Suppl 2:S264-70.

Budding of Ebola virus (EBOV) particles from the plasma membrane of infected cells requires viral and host proteins. EBOV virus matrix protein VP40 recruits TSG101, an ESCRT-1 (host cell endosomal sorting complex required for transport-1) complex protein in the vacuolar protein sorting (vps) pathway, to the plasma membrane during budding. Involvement of other vps proteins in EBOV budding has not been established. Therefore, we used VP40 deletion analysis, virus-like particle-release assays, and confocal microscopy to investigate the potential role of ESCRT-1 proteins VPS4, VPS28, and VPS37B in EBOV budding. We found that VP40 could redirect each protein from endosomes to the cell surface independently of TSG101 interaction. A lack of VPS4 adenosine triphosphatase activity reduced budding by up to 80%. Inhibition of VPS4 gene expression by use of phosphorodiamidite morpholino antisense oligonucleotides protected mice from lethal EBOV infection. These data show that EBOV can use vps proteins independently of TSG101 for budding and reveal VPS4 as a potential target for filovirus therapeutics.

http://jid.oxfordjournals.org/content/196/Supplement_2/S264.long

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Swenson DL, Warfield KL, Warren TK, Lovejoy C, Hassinger JN, Ruthel G, Blouch RE, Moulton HM, Weller DD, Iversen PL, Bavari S. Chemical modifications of antisense morpholino oligomers enhance their efficacy against ebolavirus infection. Antimicrob Agents Chemother. 2009 Feb 17. [Epub ahead of print].

Phosphorodiamidate morpholino oligomers (PMO) are uncharged nucleic acid-like molecules designed to inactivate specific gene expression via antisense-based steric hindrance of mRNA translation. PMOs have been successful in knockdown of viral gene expression and replication in the case of acute viral infections in animal models and are well tolerated in human clinical trials. We propose that antisense PMOs represent a promising class of therapeutic agents to combat filoviral infections. Previously we have shown that mice treated with a PMO complementary to a region spanning the start codon of VP24 mRNA were protected against lethal Ebolavirus challenge. In the present study, we report on the ability of two additional VP24-specific PMOs to reduce cell-free translation of a VP24 reporter, to inhibit in vitro replication of Ebolavirus, and to protect mice against lethal challenge when delivered prior to infection. Additionally, structure-activity relationship evaluations were conducted to assess enhancement of antiviral efficacy associated with PMO chemical modifications that included conjugation with peptides of variable length and composition, positioning of conjugated peptides to either the 5' or 3' terminus, and charge modifications conferred by addition of piperazine moieties. Conjugation with arginine-rich peptides greatly enhanced the antiviral efficacy of VP24-specific PMOs in infected cells and in mice during lethal Ebolavirus challenge.

http://aac.asm.org/content/53/5/2089.long

mice, cell cultures

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Iversen PL, Warren TK, Wells JB, Garza NL, Mourich DV, Welch LS, Panchal RG, Bavari S. Discovery and Early Development of AVI-7537 and AVI-7288 for the Treatment of Ebola Virus and Marburg Virus Infections. Viruses. 2012;4(11):2806-2830. doi:10.3390/v4112806.

There are no currently approved treatments for filovirus infections. In this study we report the discovery process which led to the development of antisense Phosphorodiamidate Morpholino Oligomers (PMOs) AVI-6002 (composed of AVI-7357 and AVI-7539) and AVI-6003 (composed of AVI-7287 and AVI-7288) targeting Ebola virus and Marburg virus respectively. The discovery process involved identification of optimal transcript binding sites for PMO based RNA-therapeutics followed by screening for effective viral gene target in mouse and guinea pig models utilizing adapted viral isolates. An evolution of chemical modifications were tested, beginning with simple Phosphorodiamidate Morpholino Oligomers (PMO) transitioning to cell penetrating peptide conjugated PMOs (PPMO) and ending with PMOplus containing a limited number of positively charged linkages in the PMO structure. The initial lead compounds were combinations of two agents targeting separate genes. In the final analysis, a single agent for treatment of each virus was selected, AVI-7537 targeting the VP24 gene of Ebola virus and AVI-7288 targeting NP of Marburg virus, and are now progressing into late stage clinical development as the optimal therapeutic candidates.

http://www.mdpi.com/1999-4915/4/11/2806

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Heald AE, Iversen PL, Saoud JB, Sazani P, Charleston JS, Axtelle T, Wong M, Smith WB, Vutikullird A, Kaye E. Safety and Pharmacokinetic Profiles of Phosphorodiamidate Morpholino Oligomers with Activity against Ebola Virus and Marburg Virus: Results of Two Single Ascending Dose Studies. Antimicrob Agents Chemother. 2014 Aug 25. pii: AAC.03442-14. [Epub ahead of print]

Two identical single ascending dose (SAD) studies evaluated the safety and pharmacokinetics (PK) of AVI-6002 and AVI-6003, two experimental combinations of phosphorodiamidate morpholino oligomers with positive charges (PMOplus®) that target viral messenger RNA (mRNA) encoding Ebola virus and Marburg virus proteins, respectively. Both AVI-6002 and AVI-6003 were found to suppress disease in virus-infected nonhuman primates (NHPs) in previous studies. AVI-6002 (a combination of AVI-7537 and AVI-7539) or AVI-6003 (a combination of AVI-7287 and AVI-7288) were administered as sequential intravenous (IV) infusions of a 1:1 fixed dose ratio of the two subcomponents. In each study, 30 healthy male and female subjects between 18 and 50 years of age were enrolled in 6 dose escalation cohorts of 5 subjects each and received a single IV infusion of active study drug (0.005, 0.05, 0.5, 1.5, 3, and 4.5 mg/kg per component) or placebo in a 4:1 ratio. Both AVI-6002 and AVI-6003 were safe and well tolerated at the doses studied. A maximum tolerated dose (MTD) was not observed in either study. The four chemically similar PMOplus components exhibited generally similar PK profiles. The mean peak plasma concentration (Cmax) and area under the concentration curve (AUC) values of the four components exhibited dose-proportional PK. The estimated plasma half-life of all four components was 2 to 5 hours. The safety of the two combinations and the PK of the four components were similar, regardless of the target RNA sequence.

http://aac.asm.org/content/early/2014/08/19/AAC.03442-14.long

human iv infusion

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Patrick Reid S, Shurtleff AC, Costantino JA, Tritsch SR, Retterer C, Spurgers KB, Bavari S. HSPA5 is an essential host factor for Ebola virus infection. Antiviral Res. 2014 Sep;109:171-4. doi: 10.1016/j.antiviral.2014.07.004. Epub 2014 Jul 11.

Development of novel strategies targeting the highly virulent ebolaviruses is urgently required. A proteomic study identified the ER chaperone HSPA5 as an ebolavirus-associated host protein. Here, we show using the HSPA5 inhibitor (-)- epigallocatechin gallate (EGCG) that the chaperone is essential for virus infection, thereby demonstrating a functional significance for the association. Furthermore, in vitro and in vivo gene targeting impaired viral replication and protected animals in a lethal infection model. These findings demonstrate that HSPA5 is vital for replication and can serve as a viable target for the design of host-based countermeasures.

http://www.sciencedirect.com/science/article/pii/S0166354214002034

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Clinical trials

http://clinicaltrials.gov/ct2/show/NCT01353027
http://clinicaltrials.gov/ct2/show/NCT01353040
http://clinicaltrials.gov/ct2/show/NCT01566877

Preitner N, Quan J, Nowakowski DW, Hancock ML, Shi J, Tcherkezian J, Young-Pearse TL, Flanagan JG. APC Is an RNA-Binding Protein, and Its Interactome Provides a Link to Neural Development and Microtubule Assembly. Cell. 2014 Jul 17;158(2):368-82. doi: 10.1016/j.cell.2014.05.042.

This paper reports rat brain in utero injection then electroporation of Morpohlinos as well as mouse experiments with peptide nucleic acids.

This paper provides some experimental support for targeting steric-blocking oligos to alter splicing and trigger nonsense-mediated decay. The paper compared uniform 2'-methoxyethyl phosphorothioate oligos with 2'-MOE phosphorothioate gapmers (steric blocking versus RNase-H competent, respectively).

Nonsense-mediated decay as a terminating mechanism for antisense oligonucleotides.
Ward AJ, Norrbom M, Chun S, Bennett CF, Rigo F.
Nucleic Acids Res. 2014 May 1;42(9):5871-9. doi: 10.1093/nar/gku184. Epub 2014 Mar 3.

http://nar.oxfordjournals.org/content/42/9/5871.long

Peptide-conjugated Morpholinos were administered intranasally to mice to alter the immune response to subsequent intranasal influenza doses. "BALB/c mice were anesthetized with ketamine, followed by i.n. administration with 100 mg (approximately 5 mg/kg) PPMO (in 40 ml of PBS)." Repeat dosing at 24 hours, influenza inoculation at 48 hours.

"TRIM6 expression was silenced in the lungs of mice via peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO). In line with our results from hDCs and cell lines, the designed TRIM6 PPMOs efficiently and specifically downregulated TRIM6 protein levels in MEFs, resulting in a reduction of ISG54 protein expression upon IFN-b treatement."

"Treatment with PPMOs resulted in 50% reduction in TRIM6 mRNA and protein compared to nontargeting PPMO control in the lungs of infected and noninfected mice."

Rajsbaum R, Versteeg GA, Schmid S, Maestre AM, Belicha-Villanueva A, Martínez-Romero C, Patel JR, Morrison J, Pisanelli G, Miorin L, Laurent-Rolle M, Moulton HM, Stein DA, Fernandez-Sesma A, tenOever BR, García-Sastre A. Unanchored K48-Linked Polyubiquitin Synthesized by the E3-Ubiquitin Ligase TRIM6 Stimulates the Interferon-IKKε Kinase-Mediated Antiviral Response. Immunity. 2014;[Epub ahead of print] doi:10.1016/j.immuni.2014.04.018

Vivo-Morpholinos were fed to mosquitos. The oligos decreased the amount of MAPK protein in the midgut and the number of Plasmodium oocysts in the mudgut.

"The feeding protocol described in the present study provides several advantages over current methods for gene knockdown in insects. The method is easy to apply, requires no special skills for delivery, and is highly target specific. ... This protocol should therefore be considered as an efficient alternative in studies requiring target specific protein knockdown in mosquitoes and other arthropods."

Pietri JE, Cheung KW, Luckhart S. Knockdown of mitogen-activated protein kinase (MAPK) signalling in the midgut of Anopheles stephensi mosquitoes using antisense morpholinos. Insect Mol Biol. 2014 May 28. doi: 10.1111/imb.12103. [Epub ahead of print]

http://onlinelibrary.wiley.com/doi/10.1111/imb.12103/abstract

Michel M, Wilhelmi I, Schultz A-S, Preussner M, Heyd F. Activation-induced Tumor Necrosis Factor Receptor-associated Factor 3 (Traf3) Alternative Splicing Controls the Noncanonical Nuclear Factor κB Pathway and Chemokine Expression in Human T Cells. J Biol Chem. 2014;289:13651-60. doi:10.1074/jbc.M113.526269

"Furthermore, the interferon response, which can be regulated by Traf3 (35), was not altered in Traf3E8 MO-treated cells (data not shown)" supports that the Morpholino did not induce an innate immune response.

Shen H-w, Scofield MD, Boger H, Hensley M, Kalivas PW. Synaptic Glutamate Spillover Due to Impaired Glutamate Uptake Mediates Heroin Relapse. J Neurosci. 2014;34(16):5649-57 doi:10.1523/JNEUROSCI.4564-13.2014

http://www.jneurosci.org/content/34/16/5649.abstract

Vivo-Morpholinos targeting the glial excitatory amino acid transporter 2 (GLT-1) were administered to adult male Sprague Dawley rats by infusion through microdialysis guide cannulas (26 ga) implanted bilaterally in their nucleus accumbens (position details in paper). Vivo-Morpholino stocks (0.5 mM) were diluted 50-fold in PBS. 1 ul was infused at 0.5 ul/min though 33ga microinjectors extending 2mm below the cannula, with the injectors left in place for 60 seconds after infusion was completed. Injections were done once per day for three days and GLT-1 assessed from tissue taken seven days later. Their protocol was shown to reduce GLT-1 expression by 56% (+/- 10%) in the nucleus accumbens seven days after the last infusion.

Jayakumar MK, Bansal A, Huang K, Yao R, Li BN, Zhang Y. Near-Infrared-Light-Based Nano-Platform Boosts Endosomal Escape and Controls Gene Knockdown in Vivo. ACS Nano. 2014 Apr 14. [Epub ahead of print]

http://www.cell.com/current-biology/abstract/S0960-9822%2812%2901327-9

Nanoparticles of about 30 nm diameter were constructed based on β-NaYF₄ crystals doped with Yb³⁺ and Tm³⁺ inside and Yb³⁺ and Er³⁺ in the shell. When illuminated with a 980nm IR laser, the particles emitted in the visible and UV bands. The particles were coated with mesoporous silica and loaded with TPPS2a (mesotetraphenylporphine with two sulfonate groups on adjacent phenyl rings) and Photo-Morpholino duplexes. The TPPS2a is a photosensitizer for photochemical internalization and the Photo-Morpholino duplexes consisted of antisense Morpholino targeting STAT3 and a complementary sense Photo-Morpholino strand to cage the antisense activity.

In B16F0 cells, which express STAT3, the STAT3 expression decreased to about 50% after incubation with nanoparticles and irradiation with the IR laser. Uptake was temperature dependent.

To set up a melanoma tumor model in mice, B16F0 cells were injected subcutaneously on day 0. Nanoparticles were injected day 5 and day 8 and tumors were irradiated with the IR laser. The treatment reduced the tumor volume relative to controls by about eightfold. The mice maintained their weight through the treatments.

I've skipped describing many controls and characterizations of materials. The bottom line is that their system delivered antisense activity to the IR-illuminated tumors. For more detail, see their fascinating paper.