Gene Therapy: Principles and Applications | Genetics

In this article we will discuss about the principles and applications of gene therapy based on targeted inhibition of gene expression in vivo.

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 such cases, the basis of the therapy is to knock out the expres­sion of a specific gene that allows the cancer­ous cells, infection, allergy, inflammation, etc., 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, etc.

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 possible using conventional gene augmentation therapy.

However, since hetero- zygotes with 50% of normal gene product can be severely affected, successful gene therapy for heterozygotes requires efficient expression of the introduced genes. However, dominantly inherited disorders which arise because of gain-of-function mutations may not be amenable to simple addition of normal genes.

Instead, it may be possible, in some cases, to specifically inhibit the expression of the mutant gene, but the expression of the normal allele must be maintained. Such allele-specific inhibition of gene expression is 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 function. Gene targeting by homologous recombination offers the possibility of site-specific mutagenesis to inactivate a gene.

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 are current­ly 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).

Therapy by selective inhibition of gene expression is technically possible at all three expression levels (see Fig. 23.7):

(i) Triple helix therapeutics (involves binding of gene-specific oligonucleotides to double-stranded DNA in order to inhibit trans­cription of a gene).

(ii) Antisense therapeutics (involves binding of gene-specific oligonucleotides or polynucleotides to the RNA; in some cases, the binding agent may be a specifically engi­neered ribozytne, a catalytic RNA molecule that can cleave the RNA transcript).

(iii) Use of intracellular antibodies (intra-bodies) and oligonucleotide aptamers (involves the construction of antibodies that can be directed to specific locations within cells in order to bind a specific protein, or oligonucleotide aptamers, which can bind specifically to a selected polypeptide).

Triple helix therapeutics relies on bind­ing of gene-specific oligonucleotides to the major groove of the double helix:

Synthetic short oligonucleotides (15-27 nucleotides long) are capable of specifically binding to a sequence of double-stranded DNA, forming a triple helix. The oligonu­cleotide binds by Hoogsteen hydrogen bonds to the double-stranded DNA, without disrupting the original Watson-Crick hydrogen bonding.

The most stable Hoogsteen-bonded structures are G bound to a GC base pair and a T bound to an AT base pair (see Fig. 23.8). Although such structures can inhibit DNA replication in vitro, helicases can unwind triple strand structures in vivo.

However, triplex for­mation has been shown to block binding of transcription factors in vitro, and also, at least in some cases, evidence has been obtained for gene-specific inhibition of transcription in intact cells.

Oligonucleotides are large polyanionic hydrophilic structures and so are not ideally suited to diffusing across the highly hydropho­bic plasma membrane. Direct delivery into the cytoplasm using cell permeabilization tech­niques provides the most efficient approach to enable subsequent transfer into the nucleus, and delivery using liposomes is a popularly used method.

Thereafter, the oligonucleotides can migrate rapidly to the nucleus (by passive diffusion through the pores of the nuclear envelope). Inside the cell, the oligonucleotides are exposed to nuclease attack, notably from exonucleases, and the half-life of conventional oligonucleotides with phosphodiester bonds is typically about 20 min.

Accordingly, it is usual for the 3′ and 5′ ends of the oligonucleotides to be chemically modified to protect against nuclease attack. Often, chemical modification involves incorporation of sulfur-containing phosphorothioate bonds to generate so- called S-oligonucleotides.

Although the technology is improving rapidly, some general difficulties need to be overcome. Inhibition of gene expression requires comparatively large amounts of oligonucleotide. More worrying is the limita­tion imposed by Hoogsteen hydrogen bond­ing; the target sequences need to carry virtual­ly all their purine bases on one DNA strand. Preliminary attempts to solve this problem include replacement of the phosphate groups by different chemical groupings that allow triplex-forming oligonucleotides to ‘hop’ from one strand of the bound DNA duplex to the other.

Antisense oligonucleotides or poly­nucleotides can bind to a specific mRNA, inhibiting its translation and, in some cases, ensuring its destruction:

During transcription, only one of the two DNA strands in a DNA duplex, the template strand, serves as a template for making a complementary RNA molecule. As a result, the base sequence of the single-stranded RNA transcript is essentially identical (except that U replaces T) to the other DNA strand, com­monly called the sense strand.

Any oligonu­cleotide or polynucleotide which is comple­mentary in sequence to an mRNA sequence, including the templates strand of the gene, can, therefore, be considered to be an anti- sense sequence.

Binding of an antisense sequence to the corresponding mRNA sequence would be expected to interfere with translation, and thereby inhibit polypeptide synthesis. Indeed, naturally occurring antisense RNA is known to provide a way of regulating the expression of genes in some plant and animal cells, as well as in some microbes. Synthetic oligonu­cleotides can be designed to be complemen­tary in sequence to a specific mRNA and, when transferred into cells, show evidence of inhibition of expression of the corresponding gene.

As a result, the concept of antisense therapeutics was developed; unwanted expression of a specific gene in disease tissues could be selectively inhibited using an artificia­lly gene-specific antisense sequence. A variety of different types of antisense sequence can be used.

Antisense Oligodeoxynucleotides:

The use of artificial antisense oligonu­cleotides is often favored, simply because they can be synthesized so simply. They can be transferred efficiently into the cytoplasm of cells using liposomes, and their intracellular stability is improved by using chemically modi­fied oligonucleotides, notably S-oligonucleo­tides (see above; note that although antisense oligonucleotides migrate to the nucleus, they do not bind the double-stranded DNA because they are not designed to participate in Hoogsteem hydrogen bonding).

Antisense oligodeoxynucleotides (ODNs) are preferred to oligoribonucleotides because they are general­ly less vulnerable to nuclease attack, and importantly because they have the additional advantage of inducing the destruction of an mRNA to which they bind.

This is so because an ODN-mRNA hybrid, like all DNA-RNA hybrids, is vulnerable to attack and selective cleavage of the RNA strand by a specific class of intracellular ribonuclease, RNase H. Despite teething problems in early studies, refinement of the technology has meant that antisense ODNs are now considered to have great therapeutic potential, and clinical trials are now in progress for several human diseases

Antisense Genes:

Antisense oligonucleotides, even when chemically modified, are not stable indefini­tely. One way of ensuring a continuous supply of antisense sequence is a form of expression cloning in which a specially designed antisense gene is transferred into the relevant cells. Such a gene can be engineered simply by constructing a mini- gene in which an inverted coding sequence is placed downstream of a powerful promoter.

The DNA strand that normally serves as the sense strand is now transcribed to give an antisense RNA which can be synthesized repeatedly (Fig. 23.9). If the antisense gene is provided using an integrative vector, long- term production of antisense RNA may be obtained.

Ribozymes:

Increasingly, it is becoming clear that RNA molecules are functionally different from DNA molecules; collectively they can serve diverse functions, rather than simply being involved in transfer of genetic informa­tion. Some RNA molecules are able to lower the activation energy for specific biochemical reactions, and so effectively function as enzymes (ribozymes). For example, the tran­scripts of group 1 introns are autocatalytic and self-splicing.

Other ribozymes which cleave RNA, are trans-acting, that is they cleave an RNA sequence on a different molecule. They contain two essential compo­nents; target recognition sequences (which base-pair with complementary sequences on target RNA molecules), and a catalytic com­ponent, much like the active site of an enzyme (which cleaves the target RNA molecule while the base-pairing holds it in place).

The cleavage leads to inactivation of the RNA, presumably because of subsequent recognition by intracellular nucleases of the two unnatural ends. Examples include human ribonuclease P and various ribozymes obtained from plant viroids (virus-like parti­cles).

Genetic engineering can be employed to custom design the recognition sequence so that it contains antisense sequences that can base-pair to a specific mRNA molecule, while retaining the catalytic site (see Fig. 23.10). Engineered genes which can be transcribed to produce the desired ribozyme can then be transfected into suitable cells.

One applica­tion has been the design of ribozymes against specific oncoproteins. Pilot studies have shown that transfection of an anti-Ras ribozyme gene into human bladder carcino­ma cells with the ras mutation resulted in blocking of Ras production and reversal of the metastatic, invasive and tumorigenic properties of the cells. Early problems in the efficiency of targeting to their targets inside the cell are currently being addressed and clinical trials have already been initiated in some cases, such as in gene therapy for AIDS.

Artificially designed intracellular antibo­dies (intra-bodies), oligonucleotides (aptamers) and mutant proteins can inhibit the function of a specific polypeptide:

Intracellular Antibodies (Intra-bodies):

Antibody function is normally conducted extracellular: upon synthesis, antibodies are normally secreted into the extracellular fluid or remain membrane bound on the B-cell surface as antigen receptors. Recently, however, anti­body engineering has been extended to the design of genes encoding intracellular antibod­ies, or intra-bodies.

This achievement raises the possibility of using antibodies within cells to block the construction of viruses or harmful proteins, such as oncoproteins. The first exam­ple of this approach involved engineering the antibody F105 which binds to gp120, a crucial human immunodeficiency virus (HIV) envelope protein that the AIDS virus uses to attach to and infect its target cells.

This envelope protein is derived from a larger precursor gp160 which is synthesized in the endoplasmic reticulum. Marasco and co-work­ers designed a novel F105 gene which encod­ed an antibody that was stably expressed and retained in the endoplasmic reticulum without being toxic to the cells. The engineered anti­body binds to the HIV envelope protein within the cell and inhibits processing of the gpl60 precursor, thereby substantially reducing the infectivity of the HIV-1 particles produced by the cell.

Oligonucleotide Aptamers:

Fully degenerate oligonucleotides can be synthesized by delivering 25% each of the four bases A, C, G and T at each base position dur­ing oligonucleotide synthesis. As a result, the number of sequence permutations which can be generated (4″ where n is the chosen length of oligonucleotide) can be enormous.

The result­ing mixture of oligonucleotides can be used to screen for the ability to bind to a selected target protein (protein epitope targeting). In prac­tice, the use of partial degenerate oligonu­cleotides is preferred so that the concentration of individual oligonucleotides is not too low.

In effect, this means simultaneous screening of many thousands of oligonucleotides, and so the chance of at least one epitope of the target pro­tein being specifically bound by an oligonu­cleotide can be high. The bound oligonucleotide (sometimes known as an adaptamer or aptamer) can be eluted from the protein and sequenced to identify the specific recognition sequence. Transfer of large amounts of a che­mically stabilized aptamer into cells can result in specific binding to a predetermined polypep­tide, thereby blocking its function.

An initial success was the identification of oligonucleotides that could bind to and inhibit the protease thrombin, which is part of the blood coagulation cascade .Thrombin functions in serum and extracellular applications of this type are no different, in principle, from standard drug therapy. However, the future use of oligonucleotide aptamers to inhibit specific intracellular pro­tein targets will inevitably involve genetic modification of cells, and can, therefore, be considered as a form of gene therapy.

Mutant Proteins:

Naturally occurring gain-of-function muta­tions can involve the production of a mutant polypeptide that binds to the wild-type pro­tein, inhibiting its function. In many such cases, the wild-type polypeptides naturally associate to form multimers, and incorpora­tion of a mutant protein inhibits this process.

In some cases, gene therapy may be possible by designing genes to encode a mutant protein that can specifically bind to and inhibit a pre­determined protein, such as a protein essential for the life-cycle of a pathogen. For example, one form of gene therapy for AIDS involves artificial production of a mutant HIV-1 protein in an attempt to inhibit multimerization of the viral core proteins.

Artificial correction of a pathogenic mutation in vivo is possible, in princi­ple, but is very inefficient and not read­ily amenable to clinical applications:

Certain disorders are not easy targets for gene therapy. For example, dominantly inhe­rited disorders where a simple mutation results in a pathogenic gain of function cannot be treated by gene augmentation therapy, and targeted inhibition of gene expression may be difficult to achieve. Target inhibition is best suited to inhibiting novel or inappropriate gene expression in human cells, for example expression of viral genes, oncogenes, etc. Expression of a gain-of-function mutant allele may need to be inhibited while retaining expression of a very similar wild-type allele.

If the mutant allele carries a significant change in sequence at the site of the mutation, it may be possible to achieve selective inhibition but if the change is a simple mutation, say a single nucleotide substitution, other approaches may be needed. One possible approach is targe­ted mutation correction by inserting some reagents into cells in order to change the mutant sequence back to a form that is compatible with normal function.

In principle, there are several different ways in which a specific mutation can be cor­rected selectively in vivo, mostly at the DNA level. One way is to use gene targeting tech­niques based on homologous recombination. Because this approach offers the ability to make site-specific modifications of endoge­nous genes, it represents a potentially power­ful method for gene therapy: both acquired and inherited mutations could be corrected, and novel alterations could be engineered into the genome.

Thus far, this technique has been limited largely to pluripotent mouse embryon­ic stem cells, although recently it has been applied to normal diploid somatic cells .However, the enor­mous inefficiency of this procedure (even when using the ideal target of cells cultured in vitro) and the need to correct the defect in many different cells in vivo has meant that clinical applications are a long way off.

An alternative approach is to repair the genetic defect at the RNA level. One possibil­ity is to use a therapeutic ribozyme. One method envisages using a class of ribozymes known as group I introns, which are distin­guished by their ability to fold into a very spe­cific shape, capable of both cutting and splic­ing RNA.

If a transcript has, for example, a nonsense or a missense mutation, it may be possible to design specific ribozymes that can cut the RNA upstream of the muta­tion and then splice in a corrected transcript, a form of trans-splicing (see Fig. 23.11). Thus far, this technology is in its infancy, and cat­alytic efficiency needs to be improved. Another possibility is therapeutic RNA edit­ing.

This involves using a complementary RNA oligonucleotide to bind specifically to a mutant transcript at the sequence containing the pathogenic point mutation, and an RNA editing enzyme, such as double-stranded RNA adenosine deaminase, to direct the desired base modification . Again this technology is in its infancy and formidable technical difficulties need to be overcome before clinical applications can be envisaged.