or the close analogue phosphorothioate(Section 4.4.3). Phosphorothioates are considerably more resistant
than phosphodiesters to nuclease degradation and are well tolerated in humans. Thus, first generation thera-
peutic oligonucleotides contained only 2-deoxyribonucleotide phosphorothioates, such as the clinically
approved drug Vitravene, which is a 26-mer used for treatment of CMV-induced retinitis in AIDS patients.
Second-generation oligonucleotides employ the principle of a gapmer. Such oligonucleotides contain
a section of 6–10 residues (usually centrally placed) of 2-deoxyribonucleoside phosphorothioates, but the
flanking regions on each side comprised of other analogues that enhance binding to the RNA target and
further increase the oligonucleotide stability to nuclease, but which do not direct RNase H cleavage. Such
analogues are generally ribonucleoside analogues, such as 2-O-methyl, 2-O-methoxyethyl or locked
nucleic acids (LNA), where the sugar conformation is 3-endo. Overall, gapmers are recognised and direct
RNA cleavage by RNase H. Whereas several first generation antisense oligonucleotides, such as ISIS
3521 (Affinatak) targeted to the mRNA for protein kinase C ,^37 failed clinical trials, there is more hope
of clinical benefit for some higher potency gapmers against viral and cancer targets (Figure 5.23).26,31
For steric block applications, there is no restriction in principle to the type or placement of an analogue
within a sequence as long as other antisense considerations are met. The variety of analogues that have been
investigated is very large. In some cases analogues can be combined in one oligonucleotide to give mixmers.
In addition to 2-O-methyl and 2-O-methoxyethyl ribonucleotides described above, other important ana-
logues used in steric block applications fall into two classes: (a) those that contain a phosphate group, such
as LNA, tricyclo DNA, 3-amino phosphoroamidate and phosphorothioamidate, and (b) non-phosphate con-
taining analogues such as peptide nucleic acids (PNA) and morpholinodiamidates. In class (b) it was hoped
that the absence of the negative charges on the oligonucleotide would enhance cell uptake, but this is not the
case. Attachment of a cationic or other cell penetrating peptide appears to improve cell uptake, but the uni-
versality of this approach is still under study. A steric blocking, phosphorothioamidate oligonucleotide tar-
geted to the essential RNA involved in the enzyme telomerase (Section 6.6.5) is moving close to clinical
trials as an anti-cancer agent.^38
5.7.1.4 Non-Duplex Therapeutic Activities of Single-Stranded Oligonucleotides. Recently,
other biological activities of oligonucleotides have been found that are sequence-dependent but which are
independent of duplex formation with an RNA target.
5.7.1.4.1 Immune Modulation. Single-stranded oligodeoxynucleotide phosphodiesters and phos-
phorothioates that contain the dinucleotide sequence CpG can trigger an immune response when adminis-
tered to humans and animals.39,40The response is mediated through binding to a ‘toll-like receptor’TLR9
that is present in cytosolic vesicles and the binding stimulates signalling pathways that activate transcrip-
tion factors. By contrast, double-stranded RNA and siRNA (Section 5.7.2) binds to another receptor TLR7
and may stimulate a different immune response. The context of the CpG determines the immune modula-
tion specificity, such that mouse TLR9 prefers CpG when flanked at 5by two purines and at 3by two
pyrimidines, while human TLR9 is recognised optimally by GTCGTT and TTCGTT sequences. Such activ-
ities are now recognised to have contributed being harnessed for therapeutic applications and as vaccine
196 Chapter 5
Figure 5.23 First and second generation clinically used phosphorothioate-containing oligodeoxyribonucleotides