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Interplay of polyethyleneimine molecular weight and oligonucleotide backbone chemistry in the dynamics of antisense activity.

Sundaram S, Lee LK, Roth CM - Nucleic Acids Res. (2007)

Bottom Line: Complexes were prepared between branched polyethyleneimine (PEI) of various MWs and ONs of phosphodiester and phosphorothioate chemistries.While the extent of target mRNA down-regulation was determined primarily by the polymer MW, dynamics were determined principally by the ON chemistry.Of particular importance is the strength of interactions between the carrier and the ON, which determines the rate at which the ONs are delivered intracellularly.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biochemical Engineering, Rutgers University, 599 Taylor Road, Piscataway, NJ 08854, USA.

ABSTRACT
The widespread utilization of gene silencing techniques, such as antisense, is impeded by the poor cellular delivery of oligonucleotides (ONs). Rational design of carriers for enhanced ON delivery demands a better understanding of the role of the vector on the extent and time course of antisense effects. The aim of this study is to understand the effects of polymer molecular weight (MW) and ON backbone chemistry on antisense activity. Complexes were prepared between branched polyethyleneimine (PEI) of various MWs and ONs of phosphodiester and phosphorothioate chemistries. We measured their physico-chemical properties and evaluated their ability to deliver ONs to cells, leading to an antisense response. Our key finding is that the antisense activity is not determined solely by PEI MW or by ON chemistry, but rather by the interplay of both factors. While the extent of target mRNA down-regulation was determined primarily by the polymer MW, dynamics were determined principally by the ON chemistry. Of particular importance is the strength of interactions between the carrier and the ON, which determines the rate at which the ONs are delivered intracellularly. We also present a mathematical model of the antisense process to highlight the importance of ON delivery to antisense down-regulation.

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Relationship between antisense inhibition and intracellular ON levels. Each data point represents maximum down-regulation and maximum ON levels detected when the indicated PEI MW was used to deliver (A) PO and (B) PS ONs. For both sets, results correspond to data obtained 8 h after PEI/ON complexes were introduced to CHO-d1EGFP cells.
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Figure 8: Relationship between antisense inhibition and intracellular ON levels. Each data point represents maximum down-regulation and maximum ON levels detected when the indicated PEI MW was used to deliver (A) PO and (B) PS ONs. For both sets, results correspond to data obtained 8 h after PEI/ON complexes were introduced to CHO-d1EGFP cells.

Mentions: Using the d1EGFP gene as an easily quantifiable antisense target, we screened various combinations of PEI MWs (1.2 K, 10 K, 25 K, 70 K and 600 K) and ON chemistries (PO and PS) for their ability to elicit an effective antisense response. For PO ONs, maximum antisense response was observed with intermediate MW PEI (25 K) as the carrier, while complexes of PS ONs with higher MW PEI such as 70 K and 600 K produced comparable levels of d1EGFP down-regulation. These particular PEI/ON combinations that achieved highest antisense response were also the ones that delivered the most ONs, i.e. maximum intracellular ON levels were recorded for these cases. Indeed, a monotonic relationship is apparent when the maximum antisense inhibition is plotted against the maximum ONs delivered for each combination of PEI MW and ON chemistry (Figure 8). Conditions under which no antisense inhibition was observed occurred because no ONs were delivered using those particular PEI/ON combinations. A correlation between intracellular levels of short interfering RNA and gene silencing has also been reported (39).Figure 8.


Interplay of polyethyleneimine molecular weight and oligonucleotide backbone chemistry in the dynamics of antisense activity.

Sundaram S, Lee LK, Roth CM - Nucleic Acids Res. (2007)

Relationship between antisense inhibition and intracellular ON levels. Each data point represents maximum down-regulation and maximum ON levels detected when the indicated PEI MW was used to deliver (A) PO and (B) PS ONs. For both sets, results correspond to data obtained 8 h after PEI/ON complexes were introduced to CHO-d1EGFP cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1935005&req=5

Figure 8: Relationship between antisense inhibition and intracellular ON levels. Each data point represents maximum down-regulation and maximum ON levels detected when the indicated PEI MW was used to deliver (A) PO and (B) PS ONs. For both sets, results correspond to data obtained 8 h after PEI/ON complexes were introduced to CHO-d1EGFP cells.
Mentions: Using the d1EGFP gene as an easily quantifiable antisense target, we screened various combinations of PEI MWs (1.2 K, 10 K, 25 K, 70 K and 600 K) and ON chemistries (PO and PS) for their ability to elicit an effective antisense response. For PO ONs, maximum antisense response was observed with intermediate MW PEI (25 K) as the carrier, while complexes of PS ONs with higher MW PEI such as 70 K and 600 K produced comparable levels of d1EGFP down-regulation. These particular PEI/ON combinations that achieved highest antisense response were also the ones that delivered the most ONs, i.e. maximum intracellular ON levels were recorded for these cases. Indeed, a monotonic relationship is apparent when the maximum antisense inhibition is plotted against the maximum ONs delivered for each combination of PEI MW and ON chemistry (Figure 8). Conditions under which no antisense inhibition was observed occurred because no ONs were delivered using those particular PEI/ON combinations. A correlation between intracellular levels of short interfering RNA and gene silencing has also been reported (39).Figure 8.

Bottom Line: Complexes were prepared between branched polyethyleneimine (PEI) of various MWs and ONs of phosphodiester and phosphorothioate chemistries.While the extent of target mRNA down-regulation was determined primarily by the polymer MW, dynamics were determined principally by the ON chemistry.Of particular importance is the strength of interactions between the carrier and the ON, which determines the rate at which the ONs are delivered intracellularly.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biochemical Engineering, Rutgers University, 599 Taylor Road, Piscataway, NJ 08854, USA.

ABSTRACT
The widespread utilization of gene silencing techniques, such as antisense, is impeded by the poor cellular delivery of oligonucleotides (ONs). Rational design of carriers for enhanced ON delivery demands a better understanding of the role of the vector on the extent and time course of antisense effects. The aim of this study is to understand the effects of polymer molecular weight (MW) and ON backbone chemistry on antisense activity. Complexes were prepared between branched polyethyleneimine (PEI) of various MWs and ONs of phosphodiester and phosphorothioate chemistries. We measured their physico-chemical properties and evaluated their ability to deliver ONs to cells, leading to an antisense response. Our key finding is that the antisense activity is not determined solely by PEI MW or by ON chemistry, but rather by the interplay of both factors. While the extent of target mRNA down-regulation was determined primarily by the polymer MW, dynamics were determined principally by the ON chemistry. Of particular importance is the strength of interactions between the carrier and the ON, which determines the rate at which the ONs are delivered intracellularly. We also present a mathematical model of the antisense process to highlight the importance of ON delivery to antisense down-regulation.

Show MeSH
Related in: MedlinePlus