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Sequence-dependent cytotoxicity of second-generation oligonucleotides

Denis Drygin1, Stephen Barone, C Frank Bennett

  • 1Isis Pharmaceuticals, 2292 Faraday Avenue, Carlsbad, CA 92008, USA.

Nucleic Acids Research
|December 18, 2004
PubMed

Insights

Second-generation antisense oligonucleotides (ONs) can inhibit cell proliferation via non-antisense mechanisms. Cytotoxicity is sequence-dependent and linked to lysosomal disruption by aspartic proteases, not apoptosis.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Oligonucleotide Therapeutics

Background:

  • Second-generation antisense oligonucleotides (ONs) are investigated for therapeutic applications.
  • Non-antisense mechanisms of ONs, including cytotoxicity, require thorough investigation.
  • Understanding ON-induced cytotoxicity is crucial for optimizing drug development and minimizing off-target effects.

Purpose of the Study:

  • To investigate the non-antisense mechanisms by which second-generation antisense oligonucleotides affect cell growth.
  • To identify sequence-dependent factors contributing to ON-induced cytotoxicity.
  • To elucidate the cellular pathways involved in ON-mediated inhibition of cell proliferation.

Main Methods:

  • Evaluation of a series of chimeric 2'-O-(2-methoxy)ethyl/DNA phosphorothioate oligonucleotides (ONs) for cytotoxicity.
  • Assessment of caspase-3 activation, chromatin condensation, and lactate dehydrogenase release to distinguish between apoptosis and necrosis.
  • Investigation of lysosomal integrity and the role of aspartic proteases, including cathepsin D, in ON-induced cytotoxicity.

Main Results:

  • A subset of ONs exhibited cytotoxicity at concentrations relevant to antisense activity.
  • Toxicity was sequence-dependent and influenced by base and backbone modifications.
  • Cytotoxicity was associated with lysosomal disruption and caspase-3 activation secondary to necrosis, not apoptosis.
  • Intracellular delivery was necessary for observed toxicity.
  • Pepstatin A reduced ON cytotoxicity, while cathepsin D-specific ONs did not, indicating a role for other aspartic proteases.

Conclusions:

  • Second-generation antisense oligonucleotides can inhibit cell proliferation through non-antisense mechanisms.
  • ON-induced cytotoxicity is sequence-dependent and linked to lysosomal disruption mediated by aspartic proteases.
  • Caspase-3 activation is a consequence of necrosis, not apoptosis, and is not required for cell proliferation inhibition by cytotoxic ONs.

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