Gene therapy approaches

Gene Therapy Approaches

What is gene therapy?
The term gene therapy includes all techniques used to treat inherited disorders. It can refer to two different strategies:
• the replacement of a defective gene with the correct version
• expression of a transgene whose product supplants its defective counterpart.
Both approaches are limited by the clinical applicability of the therapeutic agent, which depends on the ability to deliver the vector efficiently and exactly to its area of action.

The importance of RNA in biological systems and in transferring the information in DNA to cellular processes and phenotypes raises the question of whether it is possible to use these fascinating molecules as therapeutic tools.

To answer this question we have to first of all understand the basic molecular biology of RNA and then to examine its role in different kinds of disease.

EURASNET members are engaged in generating knowledge on alternative splicing and its regulation. Furthermore, many laboratories world-wide are trying to find novel strategies for using RNA molecules in gene therapy and several EURASNET members are active in this area.

RNA strategies for gene therapy
One potential strategy for gene therapy includes the use of antisense oligonucleotides. This method opens the way to various therapeutic approaches; to target genes involved in cancer, inflammatory diseases and viral infections, to specifically knock down targets and the disease. However, these down-regulation approaches are not applicable to diseases caused by the loss of a functional protein.

The search for potential therapies to tackle diseases where a protein is lost due to gene mutation focuses on the use of antisense oligonucleotides to alter gene expression through the manipulation of pre-mRNA splicing. This antisense oligonucleotide approach is, for example, of use for diseases which are caused by mis-splicing and include a large number of genetic disorders.

Antisense Oligonucleotides (AONs) for knock-downs
Antisense Oligonucleotides (AONs) are single-stranded DNA sequences with a variable length between 16 and 22 nucleotides. They hybridise to sense RNA target sequences in the cell and RNase H cleaves the RNA- DNA hybrid leading to a specific gene expression knockdown. This mechanism offers useful opportunities to knock out genes in vivo, since AONs can be expressed in specific cells ensuring continuous expression.

For the modulation of splicing the RNA-DNA hybrid must be made to be resistant to degradation by RNases. To achieve this AONs are chemically modified to block degradation by cellular RNase H. AONs can be designed to bind to the specific regions of pre-mRNA and therefore to disrupt signals important for splicing like splice sites, intronic branch point sequences or exonic splicing enhancer elements. This subsequently leads to disruption of splicing in a particular region of the pre-mRNA and, for instance, causes the removal of the targeted exon from the processed mRNA.

Antisense oligonucleotides are becoming more widely accepted as potential therapeutics for various diseases. Reports of restoration of normal splicing by blocking aberrant splice sites, down-regulation of the expression of targeted genes as well as the change of ratios in spliced mRNA variants have been reported. Clinical benefits can be achieved without or with only marginal toxicity effects of the provided drug.

The long road to the use of antisense oligonucleotides in therapy
Methods to cause variations in alternative splicing by using antisense oligonucleotides have been gaining increased interest in the last decade. The ultimate aim is to provide RNA molecules as drugs to the patient. The medication has to be applied frequently, be effective and have low or moderate toxicity.

However, the key question is how to develop a safe and efficient delivery system to specifically reach the target cells and molecular targets. For instance, in the case of Spinal Muscular Atrophy (SMA) patients, skeletal or cardiac muscle cells would have to be targeted. Additionally, the long-term effects of an AON treatment and the potential immunological reaction after repeated treatments have to be investigated.

β-thalassemia and Cystic Fibrosis showed the way
Antisense oligonucleotides were first used to restore normal splicing of the β-globin gene in β-thalassemia patients. In this context, the antisense sequences were targeted to block the use of cryptic splice sites in β-thalassemia patients. Splicing of the cystic fibrosis transmembrane conductance regulator (CFTR), a protein which is spliced incorrectly in some Cystic Fibrosis patients, could likewise be re-established via AONs. Both approaches do offer therapeutic potential by the redirection of normal splicing in patients.

Antisense Oligonucleotides in Duchenne Muscular Dystrophy
The dystrophin gene contains more that 20 exons which encode a repeated region in the dystrophin protein. DMD patients carry mutations in the dystrophin gene, which change the reading frame of the mRNA leading to the formation of premature terminated protein products which cannot carry out the function of dystrophin.

One current application of AONs is to induce the specific skipping of exons in order to restore the correct open reading frame of a mutated transcript. Transcripts can then be translated into a partially or even fully functional protein and ameliorate symptoms of muscular dystrophy. Some mutations even require the deletion of two exons in order to gain the correct reading frame. Translation can be performed subsequently. In fact, antisense-mediated exon skipping is one of the most promising therapeutic approaches for Duchenne Muscular Dystrophy (DMD).

A first human trial using optimised antisense oligonucleotides in order to induce exon skipping was completed recently in the Netherlands; a second trial is about to start in the United Kingdom. Local intramuscular injection of optimised AON sequences resulted in dystrophin levels of up to 20% compared to wild type dystrophin. This observation is accompanied by improvement in muscle histology and function.

The exon skipping approach would be suitable for up to 90% of all DMD patients. However, a disadvantage of the therapy is that different mutations require skipping of different exons to restore the reading frame and currently the focus is concentrated on two hotspots where the deletions take place. The results of the human trials mean that antisense-mediated exon skipping could become a standard method in the clinical treatment of DMD in the near future.

Spinal Muscular Atrophy
Another field of application for antisense oligonucleotides is the treatment of Spinal Muscular Atrophy (SMA) patients. SMA is caused by a deletion or inactivation of the SMN1 gene. The nearby SMN2 gene carries the same genetic information but a single nucleotide exchange impairs inclusion of one exon. Therefore the product is only an incomplete substitute for SMN. In an analogous approach to AONs, the U7 small RNA which is usually involved in production of other RNAs, namely the histone mRNAs, can be converted to induce exon skipping in several – also including medically important - genes and therefore in the case of SMN as well.

The research group of Daniel Schümperli, a EURASNET member in Bern, Switzerland has pioneered the use of modified U7 RNAs as a delivery vehicle to modify splicing. The U7 RNA constructs carry a sequence allowing them to bind to exon 7 of SMN2 and a splicing enhancer sequence that will improve the recognition of the targeted exon. The strategy could be optimised in a way that nearly all SMN2 mRNAs become spliced correctly. In SMA patient fibroblasts, this technique led to the induction of a prolonged restoration of SMN protein and its correct localisation to dot-like nuclear structures called gems. This approach is being tested in animal models, to examine whether the method can be used in nerve cells whose death causes SMA.

Antisense oligonucleotides in cancer therapy?
Several pre-mRNAs derived from cancer-associated genes can produce splice variants with antagonistic effects. The ratio of the splice variants changes in a number of cancers and cancer cell lines with the splice variant which stops cell death predominating and promoting cancer cell proliferation. Being able to reverse this change in production of the different splice variants to increase the apoptotic (promoting cell death) version in cancerous cells would therefore offer an approach to cancer treatment and a form of therapy.

In the case of the Bcl-x gene, a member of the family of apoptosis regulators, AONs have been demonstrated to be effective in altering the ratio of alternatively spliced variants. The Bcl-x gene has two 5’ splice sites in exon 3. The use of both splice sites results in the generation of two different splice variants: Bcl-xL and Bcl-xS, the former giving rise to a protein with strong anti-apoptotic functions. This protein has been found in over 60% of invasive human breast carcinomas and is thought to play a major role in the pathogenesis of various lymphomas.

Transfection of cancer cell lines with the antisense oligonucleotides specific to one of the alternative 5’ splice sites caused a shift of splicing from the Bcl-xL to the Bcl-xS variant. As a consequence, the cells exhibited increased apoptosis and also increased sensitivity towards apoptosis-inducing agents. These results suggest that antisense oligonucleotides might be used for modification of the splicing pattern of genes involved in cancer as a therapeutic tool.