Antisense Oligonucleotides and RNA Interference - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 338

receptors that bind to conserved structures called pathogen-associated molecular pat-
terns (PAMPs), common to many pathogens [71] . Thus, innate immunity is of major
concern in case of viral vector-based antisense drug delivery. On the other hand,
adaptive immunity an individual acquires after birth, on exposure to disease-caus-
ing pathogens, and it is mediated by T and B lymphocytes. When a pathogen invades
the body, it interacts with specific surface receptors on T and B lymphocytes, caus-
ing their activation and production of effector T cells and antibodies that neutralize
the pathogen. Many researchers have reported dangerous immune responses with
AS ODNs [72] . When phosphorothioates were administered to monkeys as a large,
one-time injection, they triggered a systemic and lethal inflammation by activating
complement. They also stimulated a dramatic increase in immunoglobulin secretion
within 24 h and increased the expression of activation markers such as MHC class II.
CpG (cytosine and guanine) separated by phosphate-containing phosphorothioates
augment natural killer (NK) cell activity, modulate T cell function, and may stimu-
late the release of several members of the interleukin family.
7.5 Molecular Mechanisms of AS ODN Interactions
AS ODNs bind to specific mRNA, thereby downregulating its expression and that
of the encoded protein. However, the mechanisms by which these oligonucleotides
interact with the complementary mRNA and induce a biological effect are complex
and difficult to elucidate completely. The ultimate goal of an antisense agent is to
suppress or completely block the production of the related gene product. This means
that, in the process of transition from DNA sequence to amino acid sequence, the
normal transcription and translation apparatus must be affected. The formation of a
protein product involves three distinct steps ( Fig. 7.3 ).
In the first step, the sense strand of the DNA is transcribed into a pre-mRNA. In
the second, the pre-mRNA is converted into a mature mRNA via the simultaneous
action of three separate processes viz . 5 capping, intron excision, and polyadenyl-
ation. Finally, in the third step the mRNA is transported to the ribosomes for transla-
tion into the appropriate polypeptide. Thus, antisense drugs can act by inhibiting any
of these steps involved in normal protein production. Although activation of RNase
H enzyme activity is thought to be the mechanism of action for the majority of the
AS ODNs, many still exert their biological effect via other reported mechanisms, and
next we discuss these mechanisms in detail.
7.5.1 Induction of RNase H
RNase H is an endogenous enzyme that cleaves the RNA strand of an RNA-DNA
duplex [73] . This is the most widely used and validated mechanism for the knock-
down of mRNA, resulting in more than 80% reduction in mRNA and protein expres-
sion. However, the precise mechanism by which the RNase H enzyme recognizes a
duplex has not been elucidated completely. The RNase H cleavage sites are found