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Evaluation of adjuvants that enhance the effectiveness of antisense oligodeoxynucleotides
J A Hughes1, A I Aronsohn, A V Avrutskaya
1School of Pharmacy, Department of Pharmaceutics, University of Florida, Gainesville 32610, USA.
Purpose:
A factor limiting the effectiveness of antisense (AS) deoxyoligonucleotides (ODNs) is inefficient transport to their sites of action in the cytoplasm and in the nucleus. The extent of ODN transfer from endosomes to cytosol seems to be an important determinant of ODN effects. Consequently, the development of compounds (adjuvants) that enhance endosome to cytosol transfer may be vital in AS ODN therapeutics.
Methods:
In this report, we evaluated compounds for their potential to enhance the effects of phosphorothioate ODNs. The test system used a CHO cell line expressing the enzyme chloramphenicol acetyl-transferase (CAT) under the control of an inducible promoter. Several potential endosomal disrupting adjuvants were screened, including: (a) fusogenic peptides; (b) a pH sensitive polymer; (c) polymeric dendrimers, (d) cationic liposomes and (e) a pH sensitive surfactant N-dodecyl 2-imidazole-propionate (DIP). ODN effects were evaluated at the protein level by quantitating levels of CAT.
Results:
The use of AS ODN in co-incubation with the GALA peptide, cationic liposomes or 5th generation dendrimers resulted in a 35-40% reduction in CAT expression. The mis-matched ODN had no effect on CAT expression. Only modest effects were observed with the other adjuvants. DIP did not increase ODN activity by itself; however, when the liposomal form was used a significant reduction (48%) in CAT activity was seen.
Conclusions:
We found the fusogenic peptide GALA, dendrimers, as well as the liposomal form of DIP, could significantly enhance the effects of ODNs.
Insights
Antisense deoxyoligonucleotides (ODNs) efficacy is limited by cellular transport. Adjuvants like GALA peptide, dendrimers, and liposomal DIP significantly enhance ODN delivery and therapeutic effects.
Area of Science:
- Biotechnology
- Molecular Biology
- Drug Delivery
Background:
- Antisense (AS) deoxyoligonucleotides (ODNs) show therapeutic promise but face challenges in cellular uptake.
- Inefficient transport to cellular targets limits AS ODN effectiveness.
- Enhancing endosome-to-cytosol transfer is crucial for AS ODN therapeutics.
Purpose of the Study:
- To evaluate compounds that enhance the effects of phosphorothioate ODNs.
- To identify adjuvants that improve AS ODN delivery and efficacy.
- To investigate strategies for overcoming cellular barriers to ODN action.
Main Methods:
- Screened potential endosomal-disrupting adjuvants including fusogenic peptides, pH-sensitive polymers, dendrimers, cationic liposomes, and a pH-sensitive surfactant (DIP).
- Utilized a CHO cell line expressing chloramphenicol acetyl-transferase (CAT) to assess ODN effects.
- Quantitated CAT protein levels to evaluate the impact of adjuvants on ODN activity.
Main Results:
- Co-incubation with GALA peptide, cationic liposomes, or 5th generation dendrimers reduced CAT expression by 35-40%.
- Liposomal N-dodecyl 2-imidazole-propionate (DIP) significantly reduced CAT activity by 48%.
- Other tested adjuvants showed only modest effects on ODN activity.
Conclusions:
- Fusogenic peptide GALA, dendrimers, and liposomal DIP effectively enhance ODN effects.
- These adjuvants show potential for improving AS ODN therapeutic delivery.
- Targeting endosomal escape is a viable strategy for enhancing ODN efficacy.