Perspectives in MicroRNA Therapeutics - Pharmaceutical Technology

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Perspectives in MicroRNA Therapeutics
The authors provide further insight into microRNA biology, and the simplicity of anti-miR oligonucleotide drug delivery.


Pharmaceutical Technology
Volume 35, pp. a18-s24

Anti-miR oligonucleotide drug delivery

Up until nearly a decade ago, insufficient in vivo stability, limited methods of delivery and tissue distribution of oligonucleotides hampered successful clinical development for several promising oligonucleotide therapeutic agents. As high molecular weight, highly charged polyanionic molecules, oligonucleotides faced many hurdles in reaching their target organ or target cell type. First-generation antisense phosphorothiolated oligodeoxynucleotide clinical candidates administered into the bloodstream had a low affinity for their target, poor stability because of nuclease degradation, unfavorable immunostimulatory properties, and rapid excretion by renal clearance, resulting in shortened half-lives (11). To increase their metabolic stability and tissue half-life, antisense and anti-miR oligonucleotides from second-generation nucleoside chemistries were developed that dramatically altered the pharmacokinetic properties of these molecules (10,12).


Figure 4: Similar pattern of tissue distribution for chemically modified anti-miRs in mouse and monkey. Anti-miR oligonucleotide quantitation was performed on tissues by either mass spectrometry analysis or capillary gel electrophoresis. IP is Intraperitoneal, SC is subcutaneous.
The introduction of chemical modifications such as 2' methoxyethyl (MOE) and 2', 4'-constrained 2'O-ethyl (cEt) into the ribose sugar ring significantly improved both the pharmacokinetic and safety profile of antisense oligonucleotides. Once delivered systemically, these second-generation compounds rapidly partition from the plasma and are taken up by cells of multiple tissues without the need of formulation or a delivery vehicle. Benefitting from nearly 20 years of oligonucleotide chemistry advances at companies such as Isis Pharmaceuticals, leading developers of anti-miR therapies have garnered a tremendous advantage in improved delivery strategies. The high water solubility of anti-miR oligonucleotides due to their polyanionic chemical structure has allowed anti-miR formulation in simple aqueous solutions such as buffered saline (13). The only limiting factor is the viscosity of the solution, which is generally concentration-dependent for single-stranded oligonucleotides (13). This simple anti-miR formulation is in contrast to the requirements for double-stranded siRNA drug delivery, which must fully encapsulate the siRNA in a lipid nanoparticle to systemically deliver its contents to a target tissue (14).

Anti-miR route of administration and tissue distribution


Figure 5: The distributions of anti-miRs in mice and monkey are highly correlated. Quantitative analysis of drug concentration by mass spectrometry revealed a good correlation of drug tissue distribution across multiple species.
Bioavailability and tissue distribution of anti-miRs have been studied extensively in rodents and nonhuman primates. The preferred route of administration for most therapeutic anti-miR compounds is subcutaneous systemic delivery, because it provides efficient dissemination of the drug to different tissues including the liver, kidney and adipose tissue without the need of a drug delivery system. Additionally, the biodistribution of anti-miRs in multiple animal species following subcutaneous administration provides valuable information regarding organs that may be successfully treated, as well as those organs unlikely to be affected. Multiple studies were performed in mice and monkeys with second generation anti-miR 1 and anti-miR 2 compounds given subcutaneously once weekly over several weeks. A quantitative analysis of tissues demonstrated broad biodistribution of modified anti-miRs among multiple tissue types including the kidney, liver, lymph nodes, adipose tissue, and spleen, as demonstrated by mass spectrometry analysis (see Figure 4). These organs have been previously shown to be target sites for oligonucleotide distribution after parenteral administration (13). Additionally, the similar pharmacokinetics and correlated tissue distribution of each anti-miR in different preclinical animal models provide important guidance for selection of different disease indications and may assist in better clinical trial designs with anti-miR therapies (see Figure 5). Effective delivery of anti-miR oligonucleotides has also been demonstrated in different species through multiple routes of administration including: intravenous, intraperitoneal, intratracheal, intranasal, and intracerebral. A more detailed analysis of anti-miR tissue distribution using quantitative whole body autoradiography to provide additional quantitative information is in progress.

Anti-miR delivery and function


Figure 6: Functional drug delivery of anti-miRs in mouse peritoneal macrophages. Flow-cytometry studies (a) and gene-regulation studies (b) demonstrate the internalization of anti-miR and target engagement in macrophages (Sylamer analysis). Potential targets containing heptamer 1-7 (GCATTAA) or heptamer 2-8 (AGCATTA) are enriched. X-axis is ranked genes by fold change. Y axis is -log (P-value enrichment). PBS is Phosphate-buffered saline.
mRNA expression profiling methods coupled with statistical techniques that can measure small changes in the expression of many genes have become powerful tools to further our understanding of the biological role and function of microRNAs. Relying on the scientific findings that some microRNAs are capable of regulating hundreds of messenger RNAs, studies were performed in mice to determine anti-miR delivery to different cell types. Mice were treated with a specific anti-miR (intraperitoneal injection) and multiple cell types were harvested to for mRNA expression studies using Sylamer enrichment analysis (15). Anti-miR oligonucleotides are distributed to peritoneal macrophages as evidenced by flow cytometry analysis and target gene up-regulation (see Figure 6). An analysis identifying an overrepresented set of genes associated with a specific anti-miR biological effect was conducted and a data plot from the isolated macrophages was generated that demonstrated the most up-regulated sets of genes after anti-miR treatment. P values generated for this dataset suggest statistically significant preferential up-regulation of genes matched to their target sequence after anti-miR treatment.


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