A Method to Characterize <em>In Vivo</em> Binding of Morpholinos for Drug Design

Special Article - Antisense Drug Research and Development

J Drug Discov Develop and Deliv. 2016; 3(1): 1018.

A Method to Characterize In Vivo Binding of Morpholinos for Drug Design

Dou S and Liu G*

Department of Radiology, University of Massachusetts Medical School, USA

*Corresponding author: Guozheng Liu, Department of Radiology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655, USA

Received: January 28, 2016; Accepted: March 25, 2016; Published: March 28, 2016


Antisense nucleotide oligomer analogues such as morpholinos are actively investigated as drugs to treat or manage human diseases. Their binding affinity to the complementary target sequence is a crucial parameter. The in vitro measures such as association constant and melting temperature are useful but do not provide the exact information about the in vivo binding. This paper introduces a mouse model to which a circulating vehicle molecule carrying a target morpholino (t-morpholino of interest) is first injected followed by the effecting morpholino (e-morpholino). The 3-h blood concentration level of the e-morpholino is used as an indicator for its binding to the t-morpholino. Particular to this report, an amine-derivatized t-morpholino is conjugated to an NHS-activated lipid. Mixing the conjugate with albumin as a natural vehicle molecule forms a vehicle complex and the t-morpholino can circulate on the vehicle after injection. This method can be utilized for modulation of the in vivo binding affinity of e-morpholino oligomers to their targets and may be applied to other nucleotide oligomers as well.

Keywords: Antisense nucleotide oligomers; DNA analogues; Morpholinos; In vivo applications; In vivo binding affinity


DNA: Deoxyribonucleic Acid; NHS: N-Hydroxysuccinimide; DSPE: a lipid, 1,2-Distearoyl-Sn-Glycero-3-Phosphoethanolamine; PEG: Polyethylene Glycol; MAG3: Mercaptoacetyltriglycine; UV: Ultraviolet; HPLC: High Performance Liquid Chromatography; HEPES: ((4-(2-Hydroxyethyl)-1-Piperazineethanesulfonic Acid) or 2-[4-(2-Hydroxyethyl)Piperazin-1-yl] Ethanesulfonic Acid; HSA: Human Serum Albumin; %ID: Percent of Injected Dose; %ID/g: Percent of Injected Dose Per Gram of Tissue; PB: Phosphate Buffer


The in vivo use of synthetic antisense DNA analogues (antisense nucleotide oligomer analogues) as potential pharmaceuticals is the most important aspect of their applications [1-8]. In most cases, the antisense DNA analogues form duplexes in vivo with their targets. Thus, the binding affinity between two mutual complements is a crucial parameter for the success. In vitro methods measuring or calculating the binding affinity in terms of melting temperature, free energy change ΔG°, association constant, etc. have long been available [9-12]. However, these in vitro measurements or calculations may not exactly predict the in vivo binding. This is especially true for the comparatively new synthetic DNA analogues lacking in vitro-in vivo correlation.

The affinity of a DNA analogue to its complementary target is required to be sufficiently high to take effect. As said, although the quantitative in vitro metrics do provide a measure for affinity, the in vitro measures such as association constants or melting points are not a direct indicator of in vivo binding. Thus, a convenient in vivo approach that can characterize in vivo binding is needed.

In this report, we introduce an in vivo measure for the in vivo binding of morpholino oligomers. Free single strand morpholino oligomers are stable in vivo and are cleared very rapidly from the blood pool. If we load the complement of a free single strand onto a vehicle molecule of a long circulating half-life (Figure 1), the free single strand would bind to the complement and circulate with the vehicle molecule. At the time when the unbound single strand is essentially cleared , the retained portion would be due to the bound and can be used as a measure for the in vivo binding. Long circulating artificial molecules and long circulating endogenous proteins (antibodies or serum protein) may both be considered as the vehicle, but we chose the latter and employed serum albumin.