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Lessons learned from vivo-morpholinos: How to avoid vivo-morpholino toxicity
 
David P. Ferguson1,3, Lawrence J. Dangott2, and J. Timothy Lightfoot3
1Children's Nutrition Research Center, Baylor College of Medicine, Houston, TX
2Protein Chemistry Laboratory, Dept. of Biochemistry/Biophysics, Texas A&M University, College Station, TX
3Biology of Physical Activity Laboratory, Dept. of Health & Kinesiology, Texas A&M University, College Station, TX
BioTechniques, Vol. 56, No. 5, May 2014, pp. 251–256
Full Text (PDF)
Abstract

Vivo-morpholinos are a promising tool for gene silencing. These oligonucleotide analogs transiently silence genes by blocking either translation or pre-mRNA splicing. Little to no toxicity has been reported for vivo-morpholino treatment. However, in a recent study conducted in our lab, treatment of mice with vivo-morpholinos resulted in high mortality rates. We hypothesized that the deaths were the result of oligonucleotide hybridization, causing an increased cationic charge associated with the dendrimer delivery moiety of the vivo-morpholino. The cationic charge increased blood clot formation in whole blood treated with vivo-morpholinos, suggesting that clotting could have caused cardiac arrest in the deceased mice. Therefore, we investigate the mechanism by which some vivo-morpholinos increase mortality rates and propose techniques to alleviate vivo-morpholino toxicity.

The ability to silence genes in vivo provides the opportunity to study gene function in whole animal models and could potentially be used to treat a variety of genetic disorders. Attempts to develop tools for silencing genes in vivo have had limited success. Initially, siRNA and phosphorodiamidate morpholino oligomers (PMOs) were thought to be potential mediators of in vivo gene silencing; however, their use resulted in limited gene silencing and significant associated toxicity (1).

Morcos et al. (2) developed vivo-morpholinos as an alternative to siRNA and PMOs. Vivo-morpholinos are anti-sense oligonucleotide analogs that bind to complementary RNA sequences and block translation of a targeted gene (2). Vivo-morpholinos are unique in that the delivery moiety consists of eight guanidinium head groups (a dendrimer) of arginine-rich peptides, conferring resistance to proteases and nucleases and thus preventing or slowing the degradation of the oligos, increasing the efficiency of their uptake into the cell by endocytosis (2).

A variety of studies have used vivo-morpholinos (2-31), and all have reported at least a 50% knockdown of the target gene with no adverse side effects. Fifteen of these studies used a mouse model (2, 4, 11, 13, 14, 16, 21, 23-30), with the remaining studies using rats (17-19), newts (22), chicken embryos (7), zebrafish (3, 5, 6), and amphibians (8, 10, 20). In the mouse model, it has been shown that intravenous (IV) and intraperitoneal (IP) administration of vivo-morpholinos were equally efficacious, and recent studies have shown success with direct injection in target tissue (17-19). To this point, no toxicity of vivo-morpholinos has been reported in any published study.

Our lab has evaluated the gene silencing ability and washout effects of vivo-morpholinos in various tissues in a mouse model (29). We have published the effects of three different vivo-morpholinos in addition to a vivo-morpholino cocktail (a combination of two vivo-morpholinos) and achieved significant knockdown of the targeted proteins (Drd1, Glut4, Vmat2) with no observable adverse side effects, even when using the vivo-morpholino cocktail (29). Furthermore, we previously established that the use of a scrambled vivo-morpholino (i.e., a vivo-morpholino that does not correspond to any genetic sequence) does not evoke responses different from saline injection (29). Thus, our previous work, as well as the entire vivo-morpholino literature base at this time, has shown vivo-morpholinos to be effective and non-toxic.

METHOD SUMMARY

The purpose of this study was to determine the mechanism by which vivo-morpholino treatment resulted in the death of treated mice. The nucleotide sequences of vivo-morpholinos used in our lab were evaluated for base pairing, and the hybridization of vivo-morpholinos was evaluated in whole blood to determine the influence on blood clot formation. Based on the results, we propose several techniques for preventing vivo-morpholino toxicity.

Recently, we initiated a study with the goal of using vivo-morpholinos to silence the annexin A6 (Anxa6) and calsequestrin 1 (Casq1) genes via IV injection in the mouse model. Both genes are involved in skeletal muscle calcium regulation. Following our previous success in using vivo-morpholino cocktails (29), we combined the Anxa6 and Casq1 vivo-morpholinos in an attempt to silence both genes at the same time. The initial results were disconcerting in that the treatments resulted in numerous fatalities. Thus, the purpose of this report is to characterize the cause of death, mechanism of action, and propose possible solutions for the toxicity we observed with vivo-morpholinos in this model.

Materials and methods

Observation of the problem

Vivo-morpholinos (2000 nmol batches) designed to target Anxa6 and Casq1 were obtained from GeneTools LLC (Philomath, OR). GeneTools provides a vivo-morpholino design service whereby the investigator provides the gene of interest along with the NCBI accession number. Previous work from our lab has utilized the 400 nmol batch of vivo-morpholinos due to the smaller sample sizes of our studies (29). Male C57L/J mice (9 weeks of age) received a tail vein injection of a single vivo-morpholino (11 mg/kg or ~56 µL of Anxa6 or Casq1 vivo-morpholino) or a cocktail treatment (11 mg/kg of both Anxa6 and Casq1 vivo-morpholino or ~112 µL). These dosages have been recommended by GeneTools (2) and have been used in published studies (31), including our previous work (29), without reports of toxicity. All mice used in this study were C57L/J male mice provided by the Jackson Laboratory (Bar Harbor, ME). Based on the homozygous genetic background of the mice and the fact they were kept in a climate controlled vivarium, the only difference between mice that could explain the differing mortality rates would be the treatment and type of vivo-morpholino given.

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