Inhibition of DNA damage repair by artificial activation of PARP with siDNA.

One of the major early steps of repair is the recruitment of repair proteins at the damage site, and this is coordinated by a cascade of modifications controlled by phosphatidylinositol 3-kinase-related kinases and/or poly (ADP-ribose) polymerase (PARP). We used short interfering DNA molecules mimicking double-strand breaks (called Dbait) or single-strand breaks (called Pbait) to promote DNA-dependent protein kinase (DNA-PK) and PARP activation. Dbait bound and induced both PARP and DNA-PK activities, whereas Pbait acts only on PARP. Therefore, comparative study of the two molecules allows analysis of the respective roles of the two signaling pathways: both recruit proteins involved in single-strand break repair (PARP, XRCC1 and PCNA) and prevent their recruitment at chromosomal damage. Dbait, but not Pbait, also inhibits recruitment of proteins involved in double-strand break repair (53BP1, NBS1, RAD51 and DNA-PK). By these ways, Pbait and Dbait disorganize DNA repair, thereby sensitizing cells to various treatments. Single-strand breaks repair inhibition depends on direct trapping of the main proteins on both molecules. Double-strand breaks repair inhibition may be indirect, resulting from the phosphorylation of double-strand breaks repair proteins and chromatin targets by activated DNA-PK. The DNA repair inhibition by both molecules is confirmed by their synthetic lethality with BRCA mutations.

Pharmacokinetics and Toxicity in Rats and Monkeys of coDbait: A Therapeutic Double-stranded DNA Oligonucleotide Conjugated to Cholesterol.

Increased DNA repair activity in cancer cells is one of their primary mechanisms of resistance to current radio- and chemotherapies. The molecule coDbait is the first candidate in a new class of drugs that target the double-strand DNA break repair pathways with the aim of overcoming these resistances. coDbait is a 32-base pair (bp) double-stranded DNA molecule with a cholesterol moiety covalently attached to its 5'-end to facilitate its cellular uptake. We report here the preclinical pharmacokinetic and toxicology studies of subcutaneous coDbait administration in rodents and monkeys. Maximum plasma concentration occurred between 2 to 4 hours in rats and at 4 hours in monkeys. Increase in mean AUC0-24h was linear with dose reaching 0.5 mg·h/ml for the highest dose injected (32 mg) for both rats and monkeys. No sex-related differences in maximum concentration (Cmax) nor AUC0-24h were observed. We extrapolated these pharmacokinetic results to humans as the subcutaneous route has been selected for evaluation in clinical trials. Tri-weekly administration of coDbait (from 8 to 32 mg per dose) for 4 weeks was overall well tolerated in rats and monkeys as no morbidity/mortality nor changes in clinical chemistry and histopathology parameters considered to be adverse effects have been observed.