Invention and Early History of Gapmers.

Gapmers are antisense oligonucleotides composed of a central DNA segment flanked by nucleotides of modified chemistry. Hybridizing with transcripts by sequence complementarity, gapmers recruit ribonuclease H and induce target RNA degradation. Since its concept first emerged in the 1980s, much work has gone into developing gapmers for use in basic research and therapy. These include improvements in gapmer chemistry, delivery, and therapeutic safety. Gapmers have also successfully entered clinical trials for various genetic disorders, with two already approved by the U.S. Food and Drug Administration for the treatment of familial hypercholesterolemia and transthyretin amyloidosis-associated polyneuropathy. Here, we review the events surrounding the early development of gapmers, from conception to their maturity, and briefly conclude with perspectives on their use in therapy.

Phosphorothioates, essential components of therapeutic oligonucleotides.

Phosphorothioates have found their usefulness in the general area of oligonucleotide therapeutic applications. Initially this modification was introduced into the antisense methodology because of the nuclease resistance of the phosphorothioate linkage in comparison with that of the phosphate linkage. However, as experimental data accumulated, it was detected that this chemical modification also facilitates cellular uptake and bioavailibity in vivo. Thus, today the majority of therapeutic oligonucleotides contain this modification. This review will discuss the historical development of this modification and present some of its chemical properties where they differ from those of the phosphate group. The antisense application will be discussed in the original context with cleavage of the target mRNA, but other target RNAs such as microRNAs and long noncoding RNAs will also be covered. It continues with applications where the target RNA should not be cleaved. A brief presentation of decoy oligonucleotides will be included, as well as some miscellaneous applications. Cellular uptake is a crucial step for oligonucleotides to reach their target and will be briefly reviewed. Lastly, a most surprising recent observation is the presence of phosphorothioate groups in bacterial DNA where functions still remain to be fully determined.

From protein synthesis to genetic insertion.

In 1946, (14)C-cyanide made its appearance as an offshoot of the Atomic Energy Program. Our colleague Robert Loftfield built it into (14)C-alanine by the Strecker synthesis, and a lusty program directed toward uncovering the unknown mechanism of protein synthesis grew out of this beginning. The necessity for an undiscovered series of steps and enzymes was soon evident. A cell free system was developed, and a succession of components necessary for this new pathway tumbled out. ATP dependence, amino acid activation, the ribosome as the site of polypeptide formation, discovery of tRNA as the translation molecule linking the gene and protein sequence, and GTP as the essential energy ingredient in peptide chain extension all appeared from our laboratory within the next decade. A little later the AP(4)N family, whose functions remain imperfectly defined, of intracellular molecules was discovered. Isolation of specific species of RNA became a high priority, and we sequenced a small segment of the 3' end of the Rous sarcoma virus, just inside the poly(A) tail, at the same time the Gilbert group at Harvard was sequencing the 5' end. The sequence identity and polarity of the two ends suggested a circular intermediate in replication and predicted correctly that a synthetic antisense oligonucleotide targeted against this sequence might be a specific inhibitor of replication. More recently, we have evolved a technique that appears to achieve a trinucleotide insertion into tissue culture cells bearing a specific Delta508 mRNA triplet deletion, resulting in phenotypic reversion in the tissue culture.