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Pharmaceutical Journal Vol 263 No 7065 p542-543
October 2, 1999 The Conference

Science medal lecture 1999

Antisense and chips: a novel way to put an end to cancer?

Two emerging forms of gene therapy for brain tumours were presented on September 13, both of which prevent tumour cell proliferation, the first by inhibiting telomerase production and the second by down-regulating the epidermal growth factor gene

The Science Medal is awarded each year to a young academic or industrial scientist (young meaning under 35) who has a proven record of research carried out in such a manner that future high-calibre work can be virtually guaranteed. The recipient of the award is chosen each year and gives the medal lecture the next, when they receive a cheque for £500.
The 1998 winner, and so the 1999 lecturer, was Dr Saghir Akhtar, reader in pharmaceutical sciences at the University of Aston. He obtained his PhD at Bath University, spent some time in Carolina, was appointed to the Aston staff in 1991 and was promoted to his present post in 1997. He won the Lilly Prize in 1996 and the Pfizer Academic Award in 1997. His lecture was entitled "Antisense and Chips: a novel way to put an end to cancer?" and was an account of two newly emergent forms of gene therapy for brain tumours. This type of tumour was difficult to treat, particularly by the oral route, not least because of the blood-brain barrier, Dr Akhtar said.

Telomerase inhibition

The basis of the first new therapeutic method was to construct an oligodeoxynucleotide (a short synthetic DNA or RNA segment), which had the ability to disrupt the process of protein synthesis by attaching itself firmly to pre-messenger RNA, for example, by means of a complementary or antisense linkage. It used the normal C-G (cytosine-guanine) and A-T (adenine-thymine) affinities to attach itself to a specific nucleotide sequence of the target molecule (see figure 1). However, such antisense oligonucleotides were not very stable, so chemical analogues such as phosphorothioates were prepared and employed instead.
In normal eukaryotic chromosomes, there was a so-called telomere sequence at the end of the DNA strand. This was a series of repeats of the grouping TTAGGG, reading from the 5¢ towards the 3¢ end. At each cell division, the telomere was shortened due to incomplete replication at the 5¢ end. When it reached a critical length, cell senescence and death were signalled. Human telomerase was a ribonucleoprotein enzyme that prevented this shortening and was a form of DNA polymerase. Since it was found in excess in 90 per cent of human tumours, it was thought to be implicated in the cell proliferation and potential immortality of such malignant tissue.

Figure 1
Figure 1: The potential sites of action of sequence-specific antisense oligonucleotides and ribozymes. Unlike traditional drugs, which usually interact with proteins, these molecules target complementary sequences in mRNA or pre-mRNA. Other forms of oligonucleotides can even interact with duplex-DNA in a sequence-specific manner

Dr Akhtar described how an antisense oligonucleotide might physically block the progression of a ribosome along an m-RNA strand (see figure 2a), or, a fairly recent discovery, it could be designed to act as an enzyme. In this case it cleaved the m-RNA at a specific site, then released itself to move on and attack another m-RNA molecule. Such an oligonucleotide was a ribozyme (see figure 2b).
Dr Akhtar's group synthesised a "hammerhead" ribozyme, so named because it had three strands. Two strands anchored it to RNA at a UN location in a sequence (where U is uracil and N is any base but G), while the third performed the cleavage. Thus, they created a sequence-specific inhibitor of gene expression. Having produced (with considerable labour) a hammerhead ribozyme with a suitable constitution to target a template region with the arrangement AAUCCC for attachment, it turned out, luckily, that there was a cleavable site nearby. By using polymerase chain reaction amplification methods, Dr Akhtar showed that the production of telomerase, which was present in lysates from tumour cells but not from normal cells, was inhibited in a dose-dependent manner by the ribozyme.
Gliomas were an extremely invasive form of brain tumour. The worst — glioblastoma multiforme — typically had a prognosis of six months to death, despite the best that could be done in the way of surgery, radiotherapy and chemotherapy. It affected around 4,000 adults and children in the UK each year. There was thus a great need for any promising line of attack. Telomerase inhibition was well worth investigating, therefore, as it was likely to develop into a much-needed therapy.
Gliomas were also characterised by many deletions, amplifications and rearrangements of chromosomal regions. One particular gene, responsible for the production of epidermal growth factor receptor (EGFr), was over-expressed in a large proportion of them. The second therapeutic approach described by Dr Akhtar was the synthesis of an oligonucleotide to down-regulate this EGFr gene.

Figure 2a
Figure 2A
Figure 2b
Figure 2B
Figure 2: Mechanisms of action of antisense oligonucleotides and ribozymes. (A) Antisense oligonucleotides can arrest the translation of mRNA into the encoded protein by either blocking readthrough of the message by ribosomes or by activating ribonuclease H (RNAseH), which selectively hydrolyses the targeted mRNA. (B) Ribosomes, by means of a catalytic core of ribonucleotides, can directly cleave the mRNA target, without the need for RNAseH.

Epidermal growth factor receptor down-regulation

A new technique, initiated by Professor E. Southern (Oxford University, was used, in which an array of oligonucleotides attached to a polymeric substrate was produced, the nucleotides being fastened by imino groups. Each oligonucleotide molecule was slightly different from its neighbour and a vast number of them were made. This was so that they contained, at known geographical positions within the array, all the possible combinations of nucleotide sequences that might be of use in attacking the gene expression pathway. The technique was called the "scanning combinatorial oligonucleotide array".
Because the RNA that had to be attacked was very convoluted, it proved difficult to find an attachment site, but some of the synthesised molecules proved to have the required ability. Even when an active antisense molecule had become available, there still remained the problem of administration.
Local delivery of the oligonucleotide to rat brain proved effective, as could be shown by cerebellar cross-section examination. The active molecule was taken up by the cell bodies, although not by the myelinated bundles. However, Dr Akhtar said, the degradative half-life was too short, so it was decided to try entrapment in biodegradable polymeric microspheres. The polymer used was poly-(D,L-lactide-co-glycolide), which was commercially used in products such as Zoladex from Astra Zeneca. An injectable formulation was made by a double emulsification-deposition method. This contained phosphorothioate oligonucleotides that were either radiolabelled with 32P or carried a fluorescent marker. Direct injection of this preparation into specific regions of the rat brain gave much improved results. Whereas unprotected oligonucleotide gave a punctate, cellular distribution of drug that was almost cleared after 12 hours, the microspheres were visible, by fluorescence, within cells. Here, they occupied both cytosolic and nuclear regions, with the occurrence of considerable spreading of the drug by diffusion over the residence period, which could be from 5 days to as long as 28 days.
Dr Akhtar's team, largely funded by the Cancer Research Campaign, expects to reach clinical trial stage in two to five years' time.