Poliovirus replicons for targeting the CNS

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MOLECULAR MEDICINE TODAY, DECEMBER 2000 (VOL. 6)

Reference standard for gene therapy closer On both sides of the Atlantic, new initiatives to establish gene therapy benchmarks could stimulate research into rare conditions and allow researchers to compare their results with unprecedented accuracy. Certainly, there is a need to standardize gene therapy vectors. Terence Flotte (University of Florida, Gainesville, FL, USA) notes that there was a consistent fiveto-tenfold difference in the potency of adeno-associated virus (AAV), one of the most common gene therapy platforms, between his lab and just one of his collaborators. ‘The range is probably much more than this,’ he admits. ‘There is no comprehensive data. However, assays taken on the spot suggest a 100-fold difference in potency. And that doesn’t take into account differences in the biological potency – the virus’s ability to infect cells.’ Clearly, this makes comparing studies difficult. To accurately compare studies, Dominic Wells (Imperial College School of Medicine, London, UK) proposes that researchers need to know three key measurements: (1) the number of virus particles, obtainable from DNA based measurements; (2) the number of infectious units, based on cell culture assays; and (3) the number of in vivo infectious units. ‘Cell culture and in vivobased assays are difficult to standardize due to individual differences in handling cells and in vivo experiments,’ he notes. ‘In addition, gene therapy reagents show different efficiencies of infection of different cells and tissues. These differences can be compensated for by parallel testing of a known reference standard with a known outcome.’ This principle underlines attempts on both sides of the Atlantic to standardize gene delivery. For instance, researchers at the University of Florida, supported by a $75 000 grant from the National Institutes of Health, are culturing a batch

of high-concentration, high-purity AAV that will be stored at a national repository after ‘extensive scrutiny’ by universities and biotechnology companies. The benchmark will allow scientists to compare the strength of their investigational therapies. Flotte predicts that the new reference standard could have its greatest impact in the management of rare diseases using gene therapy. Taking a vector through phase I, II and III trials is impractical in such cases – there is neither the patient population nor an adequate return on investment. ‘The new benchmarked vector offers a platform for the management of rare disease that has an established, characterized toxicological profile,’ he says. ‘This is an important step forward in safety testing and will allow a more direct comparison of the results from different groups,’ Wells comments. But he adds: ‘Clearly, reference standards will be needed for each gene delivery system. The University of Florida work just deals with AAV. As each vector system is developed, there will be a need to produce new reference standards. However, AAV is the agent with the most immediate need for a standard as quantitation of this vector has been the most variable.’ The European initiative aims to standardize a wider range of vectors. Over the last few years, Généthon (Evry, France) provided standardized gene therapy reagents, such as cell lines, plasmids and tailored viral vector preparations both within France, via the Gene Vector Production Network (GVPN*), and across Europe (Gene vector Database and Repository: EGDR†). But, while there is considerable demand, standardization remains a problem.

‘The quantity of gene therapy products delivered is doubling each year,’ says Mauro Mezzina, Research Director at Généthon. ‘We are still far from standard methods to produce reagents and standard samples to be used as internal positive controls. Even though the three GVPN laboratories in France produce vectors and other related material using similar procedures, these are still non-homogeneous, and no reference material has been yet produced or validated. Therefore, gene therapy medicine is still very far from establishing ‘‘which dose of vector and how often’’ to obtain a therapeutic benefit.’ The next stage, which is just underway, will make standardized preparations and calibrated vectors available to European researchers. EGDR brings together 15 European laboratories, spanning academia and biotechnology, to establish reference standard samples and procedures for gene therapy, covering, for example, all viral and non-viral vectors, cell lines, other reagents, production and purification procedures. Olivier Danos, Scientific Director of Généthon and President of the European Society of Gene Therapy, adds that EGDR also acts as a repository for knowledge in the literature as well as unpublished practical details (see also Nevin and Spink, this issue). Eventually, there is likely to be a single repository for the entire world. Danos notes there is already collaboration across the Atlantic and he predicts that EGDR will probably use the American vector as the standard. Eventually, he expects systems to merge. ‘We would like to have a common world reference,’ he says. ‘The European initiative is the first step towards this.’

*Gene Vector Production Network: http://www.genethon.fr/gvpn †

European Gene vector Database and Repository:

http://www.egdr.org

Mark Greener Freelance science writer

Poliovirus replicons for targeting the CNS The ability of type 1 poliovirus to target the central nervous system (CNS) is being exploited by scientists at the University of Alabama at Birmingham (UAB; Birmingham, AL, USA) to deliver gene therapy directly to the spinal cord. In theory, poliovirus genomes can be constructed with genes for therapeutic proteins in place of genes encoding viral capsid proteins. These specially 454

constructed recombinant genomes, known as replicons, deliver genes directly to neurons and, because they do not possess a capsid, they are unable to leave the cell and cause widespread polio infection. ‘We believe that replicons can be used to deliver therapeutic or anti-inflammatory molecules to the spinal cord that could benefit patients with spinal cord injuries and neurological diseases,’ says

the head of the research team, Casey Morrow (Department of Microbiology, UAB). The Alabama team tested the theory by administering poliovirus replicons encoding murine tumour necrosis factor-a (mTNF-a) directly into the spines of transgenic mice1. The mice were transgenic for the human poliovirus receptor, which enabled them to be infected by the virus. After a few hours,

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MOLECULAR MEDICINE TODAY, DECEMBER 2000 (VOL. 6)

high levels of mTNF-a were expressed and the mice began to show the neurological symptoms of ataxia and tail atony. Histology also revealed neuronal damage. In contrast, mice inoculated with replicons encoding a control protein (green fluorescent protein; GFP) had no ill effects. ‘I believe these studies represent a ground-breaking effort to utilize a novel gene delivery vector for the spinal cord,’ says Morrow. After six days, gene expression returned to normal and the symptoms of the animals had all but disappeared. Only transient gene expression was expected as the replicons are RNA molecules and do not generate DNA intermediates during replication. For this reason, they are also not integrated into host cell chromosomes. It is not yet known why the replicons are expressed for no more than six days. However, Morrow believes this is simply the result of gradual RNA degradation and, therefore, diminished protein expression after replication. ‘We are not sure what would happen if the genes were expressed for longer. Undoubtedly, the expression of some of these genes over a long period of time could result in deleterious effects within

the cell or neighbouring cells,’ he adds. In any case, he explains, ‘many of the genes we are targeting, such as growth factors, cytokines, and so on, are only expressed transiently under natural conditions and generally are not expressed over long periods of time. Thus, expression of these proteins from the replicon mimics natural gene expression.’ Transient expression of genes encoding therapeutic molecules will allow doctors to control the dose and length of exposure to these molecules. ‘In recent studies, we have shown that it is possible to give multiple doses of GFP replicons to a single animal without any observable deleterious effects, thus establishing the foundation for delivery of replicons encoding therapeutic proteins or anti-inflammatory proteins using multiple doses,’ says Morrow. The team are now working on delivering therapeutic proteins, such as nerve growth factor, to promote nerve regeneration. Other genes they aim to express in the spinal cord include genes for cytokines, such as interleukin-10, to reduce inflammation following spinal cord injury, genes that might prevent neuron death (Bcl-2 protein),

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and genes that encode enzymes such as super oxide dismutase, which could prevent the generation of free radicals and, therefore, neuronal damage. Morrow and his team will test the therapeutic potential of expressing these molecules in a mouse spinal cord injury model they have developed in conjunction with other researchers at UAB. ‘Ultimately, we hope to use replicons encoding different therapeutic proteins and anti-inflammatory molecules to provide benefit for the injured animals in the model,’ says Morrow. The team will then extend its work to humans. Morrow adds: ‘When we have established that we can produce replicons under GMP conditions, we will seek FDA approval for testing replicons in people with spinal cord injuries or relevant neurological diseases.’ 1 Bledsoe, A.W. et al. (2000) Cytokine production in motor neurons by poliovirus replicon vector gene delivery. Nat. Biotechnol. 18, 964–969

Sharon Dorrell Freelance science writer

An early start to ill health Maternal undernutrition at the earliest stages of pregnancy, before implantation of the embryo, might affect health later in life, reports Tom Fleming (University of Southampton, UK). These new results1 give further support to the foetal origins of adult disease (FOAD) or ‘Barker’ hypothesis and, says Fleming, emphasize the importance of periconceptional diet in the control of mammalian development. The idea that growth in utero might be an important determinant of adult disorders, including coronary heart disease, diabetes and hypertension, was first proposed in 1986 by David Barker (also from the University of Southampton). ‘We were looking at the way in which coronary disease is more common in the poorer places in Britain,’ he explains, ‘and we found some amazingly strong correlations between the death of newborn babies 70 years ago, when the certified cause of death was usually given as low birthweight, and coronary heart disease today.’ Three years later, Barker and his team published the Hertfordshire study2, in which recorded low birthweight correlated with an increased risk of heart disease. ‘The whole field really lit up then,’ says Barker. The FOAD hypothesis proposes that some adult disorders result from adaptations the embryo and foetus make in

response to a deleterious intrauterine environment. These adaptations – for example, changes in the vasculature – become set or programmed and can cause ill health in adult life. But how foetal programming occurs is largely unknown, says Fleming, ‘so we thought it would be logical to start at the beginning of gestation in our search for mechanisms.’ It is known that female rats fed a low protein diet during pregnancy produce offspring with a lower birthweight and higher blood pressure than do rats fed a normal protein diet. In their experiments, Fleming and colleagues fed pregnant rats these two diets for the first 4.25 days of pregnancy only. ‘The birthweight of the female offspring was reduced and hypertension at different stages of life in the male offspring was increased when the mothers were given the low protein diet,’ explains Fleming. When the researchers looked at the embryos at the blastocyst stage, they found fewer cells in the inner cell mass and in the trophectoderm of the embryos exposed to the low protein diet. These changes in cell number were caused by changes in proliferation rates. In addition, there was a reduction in insulin levels in the maternal serum around day 4. ‘We know that insulin present in the

1357-4310/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.

reproductive tract preferentially affects the inner cell mass lineage,’ explains Fleming. ‘Thus we have circumstantial evidence that the diet might be affecting embryonic growth through insulin. A similar, but more severe, phenomenon has been identified in animal models of diabetes.’ ‘These results, which extend previous studies, show that programming can occur very early and they give some insight into how it might occur,’ says epidemiologist Matthew Gillman (Harvard Medical School, Boston, MA, USA). ‘There is now plausible evidence that programming occurs in human disease but,’ he warns, ‘we cannot say what the causative factors are. Birthweight is an amalgam of thousands of determinants and we have to figure out which are the important ones. Until we do, I don’t think we can really estimate the public health impact of programming.’ In addition to Gillman’s caveat, Fleming also cautions about extrapolating too far from animal work. ‘Although I would argue that all preimplantation embryos are very sensitive to the environment and that this can have long-term consequences, it would be folly to immediately consider rats as equivalent to people from our work,’ he says. One area where caution might be advisable, however, is in vitro fertilization 455