Figure 22.

   Targeted inhibition of gene expression in vivo

Gene therapy based on selective inhibition of a predetermined gene in vivo can

be achieved at several levels. In principle, it is possible to mutate the gene via

homologous recombination-mediated gene targeting to a nonfunctional form

(1). In practice, however, it is more convenient to block expression: at the

level of transcription by binding a gene-specific triplex-forming oligonucleotide

(TFO) to the promoter region (2), or at the mRNA level by binding a gene-

specific antisense oligonucleotide or RNA (3). In the latter case, an antisense

gene is normally provided which can encode a simple antisense RNA or a

ribozyme (Figure 22.9). In each case, the binding interferes with the ability of
 the mRNA to direct polypeptide synthesis, and may ensure its destruction: a

bound oligodeoxynucleotide makes the mRNA susceptible to cleavage by RNase

H, while a bound ribozyme cleaves the RNA directly. The technology for specific

inhibition at the polypeptide/protein level (4) is less well developed but is

possible using genes which encode intracellular antibodies or oligonucleotide

aptamers which specifically bind to the polypeptide and inhibit its function.

Vvvv

22.3. Therapeutics based on targeted inhibition of

gene expression and mutation correction in vivo

22.3.1. Principles and applications of therapy based on

targeted inhibition of gene expression in vivo

One way of treating certain human disorders is to selectively inhibit the expression of a

predetermined gene in vivo. In principle, this general approach is particularly suited to treating

cancers and infectious diseases, and some immunological disorders. In these cases, the basis of

the therapy is to knock out the expression of a specific gene that allows disease cells to flourish,

without interfering with normal cell function. For example, attention could be focused on

selectively inhibiting the expression of a particular viral gene that is necessary for viral

replication, or an inappropriately activated oncogene.

In addition to the above, targeted inhibition of gene expression may offer the possibility of

treating certain dominantly inherited disorders. If a dominantly inherited disorder is the result of

a loss-of-function mutation, treatment may be difficult using conventional gene augmentation

therapy: given that heterozygotes with 50% of normal gene product can be severely affected,

very efficient expression of the introduced genes would be required for the gene therapy to be

successful. However, dominantly inherited disorders which arise because of a gain-of-function

mutation may not be amenable to simple addition of normal genes. Instead, it may be possible,

in some cases, to inhibit specifically the expression of the mutant gene, while maintaining

expression of the normal allele. Such allele-specific inhibition of gene expression would be

facilitated if the pathogenic mutation results in a significant sequence difference between the

alleles.

The expression of a selected gene might be inhibited by a variety of different strategies. One

possible type of approach involves specific in vivo mutagenesis of that gene, altering it to a form

that is no longer functional. Gene targeting by homologous recombination offers the possibility of

site-specific mutagenesis to inactivate a gene (Section 21.2.3). However, this technique has only

very recently become feasible with normal diploid somatic cells and is still very inefficient.

Instead, methods of blocking the expression of a gene without mutating it have been preferred.

In principle, this can be accomplished at different levels: at the DNA level (by blocking

transcription); at the RNA level (by blocking post-transcriptional processing, mRNA transport or

engagement of the mRNA with the ribosomes); or at the protein level (by blocking post-

translational processing, protein export or other steps that are crucial to the function of the

protein).

Figure 22.8
Targeted inhibition of gene expression in (more...)

Figure 22.8

   Targeted inhibition of gene expression in vivo

Gene therapy based on selective inhibition of a predetermined gene in vivo can

be achieved at several levels. In principle, it is possible to mutate the gene via

homologous recombination-mediated gene targeting to a nonfunctional form

(1). In practice, however, it is more convenient to block expression: at the

level of transcription by binding a gene-specific triplex-forming oligonucleotide

(TFO) to the promoter region (2), or at the mRNA level by binding a gene-

specific antisense oligonucleotide or RNA (3). In the latter case, an antisense

gene is normally provided which can encode a simple antisense RNA or a

ribozyme (Figure 22.9). In each case, the binding interferes with the ability of
 the mRNA to direct polypeptide synthesis, and may ensure its destruction: a

bound oligodeoxynucleotide makes the mRNA susceptible to cleavage by RNase

H, while a bound ribozyme cleaves the RNA directly. The technology for specific

inhibition at the polypeptide/protein level (4) is less well developed but is

possible using genes which encode intracellular antibodies or oligonucleotide

aptamers which specifically bind to the polypeptide and inhibit its function.

Various techniques for selectively inhibiting expression of a specific gene have been devised, and

include examples where expression is inhibited at all three major levels (see Figure 22.8

):

 Targeted inhibition of expression at the DNA level. Under certain

conditions, DNA can form triple-stranded structures, as occurs naturally in

the case of a portion of the mitochondrial genome (Section 7.1.1). The

rationale of triple helix therapeutics is to design a gene-specific

 oligonucleotide that will have a high chance of base-pairing with a defined

double-stranded DNA sequence of a specific target gene in order to inhibit

transcription of that gene (Vasquez and Wilson, 1998). Binding of the

single-stranded oligonucleotide to a pre-existing double helix occurs by

Hoogsteen hydrogen bonds and certain bases are preferred. The most

stable of such bonds are formed by a G binding to the G of a GC base pair

and a T binding to the A of an AT base pair.

 Targeted inhibition of expression at the RNA level. Antisense therapeutics

involves binding of gene-specific oligonucleotides or polynucleotides to the

RNA; in some cases, the binding agent may be a specifically engineered

ribozyme, a catalytic RNA molecule that can cleave the RNA transcript

(Section 22.3.2).

 Targeted inhibition of expression at the protein level. Oligonucleotide

aptamers and intracellular antibodies can be designed to specifically bind

to and inactivate a selected polypeptide/protein (Section 22.3.3).