News

28 October 2005The European Parliament (EP), in their latest sitting, has agreed the text of a resolution on patenting biotechnological inventions. The resolution confirms Parliament’s rejection of research on human embryos and calls for the European Patent Office (EPO) and Members States to grant patents on human DNA only for a ‘concrete application’ and for the scope of the patent to be limited to that application. It also calls for the European Commission (EC) to file a notice of opposition to patent EP1257168, which covers a method for cryopreserving sperm cells that would enable the sex of the child to be chosen during the artificial insemination process. The EEP-ED Group within the European Parliament has dubbed this the ‘designer baby patent.’

 

In August the EC published their second report looking at Member States’ implementation of the Directive on the legal protection of biotechnological inventions (98/44/EC). The report noted that some States have granted patents on DNA sequences that cover the original disclosure in the patent application as well as possible future uses of the sequence. Other countries have taken a more conservative approach, only granting a patent for the specific application, while France has totally banned patenting of DNA sequence. The EP, in this resolution, has given its support to the more restricted ‘purpose-bound protection.’

 

The contentious patent was awarded to a US company, XY Inc., in November 2002. According to an EPO press release, some Members of the EP claim that the patent, entitled ‘Method of cryopreserving selected sperm cells,’ violates the Directive as it covers non-patentable human germ cells. The Directive states “Inventions shall be considered unpatentable where their commercial exploitation would be contrary to ordre public or morality.” The EPP-ED Group believes that “…the spirit of the Directive opposes patents on embryonic stem cells.” The European Patent Convention, which incoporates relevant portions of the Directive into European patent law, confirms that the human body and its elements cannot be patentable inventions. But “…an element isolated from the human body or otherwise produced by means of a technical process may constitute a patentable invention, even if the structure of that element is identical to a natural element.” The EPO, while acknowledging the right of anyone to oppose a patent, “…emphasises that it follows an extremely cautious approach in patenting biotechnological inventions.” The EPO had agreed in June 2005 to stop making decisions on patent applications involving human embryonic stem cell technologies [Schubert, S. Nature (2005) 435:720-1].

 

The EP will contest the grant of the patent by initiating an opposition procedure. The EPO’s Technical Board of Appeal is expected to make a first decision on the subject on 18 November. However, this Board is able to refer the issue to the Enlarged Board of Appeal, the EPO’s highest instance, for a definitive legal ruling.

20 October 2005A new international collaboration in stem cell banking has been established in Seoul, South Korea this week. The initiative forms part of the new World Stem Cell Hub, which has been launched by the South Korean government. The Hub will be lead by Professor Woo Suk Hwang, whose group have been responsible for deriving the first embryonic stem cell lines by therapeutic cloning. The group also recently announced the production of the first patient specific stem cell lines (see previous news item in PHGU newsletter).

The Hub will be based at the Seoul National University Hospital but there are plans for other centres based around the globe. These will provide training for researchers as well as a bank of cell lines and, where it is permitted, assist in the creation of new patient-specific embryonic stem cell lines. The first branches of the Hub are due to be established in the UK and California, with the possibility of further centres in other parts of Europe such as Sweden and Spain. However, although South Korea will create a foundation to fund the Hub’s headquarters and to support Korean scientists travelling abroad, the other international centres will be expected to source their own funding.

It is hoped that the new international stem cell bank and the wider Hub initiative will facilitate global cooperation in stem cell research by assisting in the production of stem cell lines with a range of defects that cause diseases such as diabetes. These lines could then be distributed and used by researchers across the globe. Commenting on the plan, in an interview with Associated Press, Professor Hwang said, “When the use of these stem cells is limited to a particular country, it takes much too long to create technologies usable for the whole of humanity. By creating a global network, we plan to share stem cells created in each country and share information on those stem cells." The Hub has also received support in the UK from the Medical Research Council. As reported by the BBC, Professor Christopher Higgins, director of the MRC Clinical Sciences Centre, said: "It is a very positive step forward. If we are going to make use of embryonic stem cells for therapeutic purposes it is very important that there is access for all the researchers who need them."

The world’s first stem cell bank was established in the UK in 2004 and currently holds over thirty adult and embryonic stem cell lines.

17 October 2005Researchers have reported in Science that nearly 20% of all protein-coding human genes have been patented, with the majority patented by private biotechnology companies [Jensen K. and Murray F. (2005) Science 310, 239-240]. This accounts for 4382 of the 23,688 genes published in the US National Center for Biotechnology Information’s database. The authors investigated United States patents, but it is likely that the same genes will have been patented in Europe, according to a Guardian newspaper report [Ravilious, K, Guardian 14 October 2005].

 

The genes are claimed in 4270 patents that are owned by 1156 different assignees. Approximately 63% of the assignees are private firms. One firm, Incyte Pharmaceuticals/Incyte Genomics, has patents on 2,000 human genes. A majority of the patented genes, at least 3000, have only a single intellectual property (IP) rights holder. But some genes related to human health and disease, such as BRCA1 that is linked to early onset breast cancer, have multiple patents covering rights to various uses of the gene. Researchers who want to work on a multi-patented gene may have to spend large amounts on complex licensing agreements in order to gain access.

 

The authors note that “…the classic argument in support of gene patenting is that strong IP protection provides incentives crucial to downstream investment and the disclosure of inventions.” On the other hand, critics of gene sequence patents fear that broad patents will hinder downstream research activities. Also, innovators may be deterred by an ‘anticommons’ effect, where “…people underuse scarce resources because too many owners can block each other” [Heller M. and Eisenberg R. (1998) Science 280, 698-701].   Little empirical research has been done on the extent of gene patenting, according to Jensen and Murray.  They suggest that the current patent examination system could be reviewed to see if the process is responsible for multiple conflicting patents being granted on the same gene. Genes that have multiple patents provide an opportunity “…to explore the variety of arrangements used to facilitate or block access to gene-based research and the impact of these arrangements on future innovators.” Such studies could provide beneficial information to inform the patent debate.

14 October 2005Seventy-three Members of the European Parliament (MEPs) have sent an open letter to Commission President Barroso asking the European Commission to not fund human embryonic stem cell (hESC) research under the Seventh Research Framework Programme (FP7). In the letter the MEPs state that Member States where hESC research is prohibited should not have to share the cost of funding research that is in conflict with their national law. Funding should be the responsibility of those countries that support such research. Instead, EU funding should be given to somatic (adult) stem cell and umbilical stem cell research projects, which are accepted by all Member States. The European People’s Party and European Democrats (EPP-ED) Group in the Parliament, the largest political group within the Parliament, state in a press release that they initiated and garnered support for the letter.

 

The letter confirmed the European Parliament’s support for a resolution passed on 10 March 2005 on the trade in human egg cells that included a call for the EU to refuse to fund hESC research. The resolution stressed Parliament’s concern that women in developing countries could be enticed by financial incentives to donate their eggs, with such practices resulting in their exploitation. The resolution specifically cited a clinic in Romania that specialised in donating eggs to EU nationals, particularly UK citizens.   The resolution also noted that the UK had considered a £1000 payment to women donating their eggs. The Human Fertilisation and Embryology Authority, in their Sperm, Egg and Embryo Donation (SEED) Report, has recommended a limit of £250 for loss of earnings incurred. The resolution also asked the European Commission to refuse to fund embryo and hESC research; the open letter to Commissioner Barroso links these issues together, stating, “It is very clear that egg cells are needed for every embryo and that therefore there is a risk that women become instrumentalized as ‘suppliers of raw material.’”

 

Perhaps ironically, the UK, during their presidency of the Council of the EU (July-December 2005), will be responsible for redrafting the FP7 proposal, based on discussions held by the Competitiveness Council. UK Minister for Science and Innovation Lord David Sainsbury has said that the specific issues to be addressed will be research ethics including hESC research and the proposed European Research Council (ERC). Discussions on the next draft of FP7 will be held at the next meeting of the Competitiveness Council on 28-29 November 2005.

11 October 2005Japanese companies have announced that they have built desktop DNA machines that will allow doctors to analyse patients’ DNA as a prelude to prescribing [see D. Cyranoksi, Nature 437:796; BioNews 329]. Doctors will be able to analyse a single drop of a patient’s blood and, in one hour, according to claims, be able prescribe that patient the drug and dosage most appropriate for them. The machines will be tested first on patients who are being prescribed one of two drugs: an antibody called irinotecan and the anticoagulant warfarin. For some patients with mutations in their mitochondrial DNA, taking irinotecan can cause hearing loss. Likewise, some patients suffer from excessive bleeding when taking warfarin. These complications make these two drugs logical targets for personal testing prior to prescribing. The DNA analysis would tell doctors if a patient will react adversely to these drugs or whether they will be able to benefit from them.

 

Sir David Weatherall, who recently chaired a committee reporting on personalised medicine on behalf of the Royal Society, sounded a cautionary note. As reported by Nature, he said in the case of warfarin, “…metabolism…is related to at least two genes whose interaction is not understood. Other factors, such as the patient’s age or additional drugs being taken, also need to be considered.” The Royal Society report stated that these and other variables that affect a patient’s response to a drug (e.g. dosage, inherited factors, adherence to the drug regime, environmental factors) interact in complex ways and that more research is needed. Their report concluded that personalised medicine is still many decades from becoming a reality in mainstream clinical practice. Takaaki Sato, of the scientific equipment maker Shimadzu, disagrees. He believes that products such as theirs will be in use for day-to-day diagnosis and treatment in the near future. Initially, though, he agrees that their machine will be most useful for research purposes.

7 October 2005The Human Fertilisation and Embryology Authority (HFEA) has published its report from the Sperm, Egg and Embryo Donation (SEED) Review. Begun in November 2004, the SEED Review sought views from the public and stakeholders on a variety of issues related to the donation of gametes and embryos for fertility treatment and research. Results from the Review will help the HFEA decide whether to revise or maintain existing policies surrounding donor-assisted conception. In addition, the Review will enable the HFEA to consider what revisions will be needed to adequately transpose the European Directive on Tissues and Cells into UK law. The Directive imposes new standards for quality and safety that UK fertility clinics will have to meet. These requirements, with some exceptions, become mandatory in April 2006.

 

The SEED Review makes several recommendations including:

• Gametes donated by an individual should not be used to produce children for more than 10 families in the UK
• Eggs collected from an individual during a single ‘cycle’ will not be shared with more than two other recipients
• Gamete donors may receive ‘benefits in kind’ for donating gametes to others requiring treatment, but these benefits should be limited to discounted treatment services for themselves
• Gametes procured from abroad should fulfil the same quality standards as apply in the UK; imports will only be approved where these standards can be met

The Review also makes recommendations regarding compensation for donors (see BBC news report 7/10/05). Donors will continue to be reimbursed for any reasonable out-of-pocket expenses incurred in relation to donating. In addition, donors may receive compensation for loss of earnings up to a limit of £250, with the daily maximum set at £55.19, the same for those serving on juries. The previous amount of £15, plus reasonable expenses, for a donation was seen as too little. However, giving too high an amount of compensation might inappropriately encourage donors. The HFEA notes “…that additional payment might encourage donors to disregard risks to the health or to withhold important medical information, or that it might attract donors with whose motives, when disclosed, could be difficult for offspring to come to terms.” The EU Tissue and Cells Directive states that donations must be voluntary and not seen as a way to make a profit. The limit of £250 is seen by the HFEA as just compensation for loss of income but not attractive enough to cause people to donate for other than altruistic motives.

The HFEA has also reported on the make-up of UK donors.  Sperm donors during 2004-2005 were primarily over the age of 30, with approximately 40% already having children of their own.  Ten years ago the majority of sperm donors were aged between 18-24.  The age distribution of egg donors has not changed significantly during the same time period.  Angela McNab, Chief Executive of the HFEA, said, "Understanding who donors are is a significant part of understanding how the area of donor-assisted conception works and how it can be made better...."

3 October 2005Preimplantation genetic diagnosis (PGD) is a technique whereby embryos created by in vitro fertilisation are screened for the presence of a disease associated genetic variant, and only embryos without the variant are implanted in the mother. The Human Fertilisation and Embryology Authority (HFEA) has announced that it will hold a public discussion on the use of preimplantation genetic diagnosis (PGD) for lower penetrance conditions – that is, conditions for which possession of a disease associated genetic variant does not necessarily mean that the disease will develop. For example, the penetrance of Huntingdon’s Disease is virtually 100%; whereas for the breast and ovarian cancer associated gene BRCA1 variants, penetrance is 50-80%, such that up to 50% of individuals with the gene variant will not go on to develop breast or ovarian cancer.The HFEA has already licensed the screening of embryos for a form of hereditary colorectal cancer, Familial Adenomatous Polyposis (FAP), which is not completely penetrant, but in this instance penetrance is both greater than 90% and is also typically early onset, with disease developing in young adults. In contrast, the lower penetrance conditions tend to affect people in later life (ages 40-80). The HFEA wishes to hear the views of stakeholder groups and the wider public on the possibility of licensing centres to screen embryos for lower penetrance conditions in the future. It will release a discussion document on the topic in October, and a public meeting is to be held in December of this year.

4 October 2005Recent draft guidance on the design of clinical trials involving gene transfer technology issued by the US Food and Drugs Agency (FDA) has called for long-term follow-up of trial participants, in order to properly monitor for long-term adverse effects of gene therapy treatments and mitigate their potential impact on participants. Gene transfer technology is defined as gene therapy products or cells or tissue that has been transduced with gene therapy products ex vivo. The document also provides a framework for risk-assessment to determine whether the trial involves high risks that merit long-term follow-up. Factors considered likely to increase the risk of delayed adverse events include persistence of the viral vector, integration of genetic material into the host genome, prolonged expression of the transgene, and altered expression of the host’s genes.

Special recommendations are made for trials involving the use of retroviral vectors, due to the occurrence in 2003 of therapy-associated leukaemia in three out of eleven children who received gene therapy for the rare genetic condition X-linked Severe Combined Immunodeficiency (X‑SCID) in a French trial. The leukaemia was caused by insertion of the retroviral vector used for the gene therapy. As well as increased levels of post-trial surveillance, it is recommended that consent forms for such trials include information about the risks of malignancies, including reference to the X-SCID trial.

3 October 2005Primary Inherited Aminoacidurias (PIA) are a group of rare genetic diseases involving defects in plasma membrane amino acid transporters which impair normal renal absorption of amino acids. A new EU-funded project aims to determine the genetic basis of these disorders, along with work intended to elucidate the mechanisms of disease and design therapeutic interventions. Eugindat (the European genomics initiative on disorders of plasma membrane amino acid transporters) will involve partners from 5 European countries; a key objective for the project is to establish a clinical and genetic description for PIA with a shared database of information on candidate gene studies that researchers can access. Other arms of the project will include a functional genomics work and the study of potential disease-associated polymorphisms in genetically isolated human populations. Project completion is scheduled for early 2007.

3 October 2005The Clinical Molecular Genetics Society (CMGS) performs an annual audit of activity in UK National Health Service laboratories; results from the 2003-4 audit are now available, and provide an interesting summary of activity in this area. The total number of postnatal tests performed was over 65,000, excluding the figures for aneuploidy and leukaemia testing, which contributed a further 5,000. Around 300 different disorders were tested for of which the most common were Fragile X and cystic fibrosis, representing 18% and 17% of the total number of tests, followed by familial breast cancer (BRCA gene mutations), hereditary haemochromatosis and the Factor V Leiden mutation. For prenatal testing (a total of 1260 reports, excluding those for aneuploidy), by far the most common disorder was cystic fibrosis (34% of the total), followed by sickle-cell anaemia (18%), Duchenne muscular dystrophy (8%) and beta-thalassaemia (7%). At least 82 different disorders were tested for prenatally; some of the rarest disorders were not reported individually.

The audit also reports the numbers of tests imported to and exported from participating laboratories, including internationally (around 12,000 and 8,400 respectively) and the disorders they covered, information on laboratory staffing, and also on average reporting times for certain tests. Of note, whereas routine requests for cystic fibrosis and Fragile X testing were met within three weeks, the much more complicated tests for mutations in the BRCA and HNPCC genes (involved in familial breast and colorectal cancers, respectively) took on average 23 and 27 weeks.

28 October 2005An article in Nature has announced the release of the first phase of the human haplotype map data. The International HapMap Consortium which consists of over 200 scientists from the US, UK, Canada, China, Japan and Nigeria are also presenting the results of the project at the American Society of Human Genetics conference, currently underway in Salt Lake City. This project results in a map of the common differences in the human genome and is a natural extension of the Human Genome Project.

Once the human genome sequence was finished, the logical follow-on project was to look for the indicators or markers that signify the differences between individuals, and their susceptibility to various diseases. Humans are genetically 99.9% identical to each other and the remaining 0.1% consists of the variation that accounts for important differences between individuals. There are thought to be around 10 million single nucleotide polymorphisms or SNPs in the human genome. In this project, over 1 million SNPs were genotyped in 269 individuals representing populations from Africa, the Far East and Western Europe, with 300,000 of these being ‘tagSNPs’. By using these tagging SNPs researchers were able to capture 90% of the information that would have been obtained by looking at all 10 million SNPs, since these are not all inherited independently. All the SNPs used in the tagSNP project were called common SNPs, because they all had a minor allele frequency of at least 5% and were spaced at approximately 1 every 5,000 bases. In addition to the 300,000 tagSNPs, the scientists also looked at ten 500 kilobase regions (the ENCODE regions) in detail and typed all SNPs identified in these regions irrespective of allele frequency in the full 269 samples, with an average spacing across the ten regions of 1 SNP typed every 279 bases. Data generated during the project were consequently analysed to look at the relationship or linkage disequilibrium (LD) between SNPs and across regions. The projected Phase II of the HapMap project aims to type an additional 4.6 million SNPs in the 269 samples and further SNPs and samples across the ENCODE regions.

The project is of considerable benefit to the scientific community, as the data produced during this project is publicly available and is already being utilised by other research groups. Additionally, the public SNP database, dbSNP, now contains 9.2 million reported SNPs; an increase of 6.6 million SNPs as a result of this venture. Scientists can now use bioinformatic tools to prioritise the SNPs thought to be putatively functional in order to narrow down the search for the causal variant of their particular disease of interest. However, other leading scientists have issued a note of caution, citing the fact that the project doesn’t look at rare variations (those with a minor allele frequency of less than 5%) that may be important in complex diseases. Another potential drawback may be that the data in the reported populations may not match well with that of a study population, therefore limiting its utility. Nonetheless it is agreed that the work published by the International HapMap Consortium does represent a considerable resource for other scientists, both in terms of information content and in terms of time and effort saving.

Comment: The immediate relevance of the publication of this work to human health is as yet unclear. There is no doubt that the report represents an advance in the ultimate goal of understanding human disease and facilitating personalised medicine, yet the direct benefits of this study are harder to perceive. If the genetic determinants of a complex disease are common, then this data may greatly advance the study of the disease, yet if the determinants are rarer, then Phase II of the HapMap project may be of greater utility than the current data release. The long term aim must be to understand the effect of these genome variations on public health.

26 October 2005

Embryonic stem cell lines are usually derived from the inner cell mass of the blastocyst, an early developmental stage of the mammalian embryo. This process leads to the destruction of the embryo and in the case of human embryonic stem (ES) cell research has led to a number of ethical objections from groups opposed to this elimination of a potential life.

Two novel methods for the production of embryonic stem cell lines, that avoid the destruction of viable embryos, have recently been proposed. The first method, reported in an advance online publication in Nature by Meissner and Jaenisch, uses a protocol termed 'altered nuclear transfer' (ANT) to create blastocysts which are incapable of developing into viable embryos but can still be used for the derivation of embryonic stem cell lines. During normal embryonic development, survival of the blastocyst after implantation in the uterus is dependent on the formation of the trophectodermic cell lineage. This lineage gives rise to the cells that form the foetal-maternal interface within the placenta and thus is essential for the development of the embryo. The inner cell mass (ICM) is the second cell lineage that is formed. It is responsible for all subsequent lineages in the embryo and when cultured in vitro gives rise to embryonic stem cell lines. The ANT method is based on the prevention of trophectoderm, and therefore placental, development in a process that leaves the ICM unaffected. The blastocysts that are created using this process have no potential to develop beyond this stage into a viable organism, but can still be used to derive embryonic stem cell lines.

The ANT method relies on genetically altering a somatic donor cell to prevent trophectoderm development, before transfer of its nucleus into a recipient denucleated or 'empty' egg cell. In this study the authors knocked-down expression of the Cdx2 gene in mice, in order to determine the effectiveness of the ANT approach. Cdx2 encodes a trophectoderm-specific transcription factor that is essential for the establishment and function of the trophectoderm lineage. Previous studies have shown that Cdx2-deficient blastocyts fail to maintain a blastocoel and lack epithelial integrity but do form an ICM and generate ES cells in culture. The authors used RNA interference (RNAi) to knock-down expression of the gene by introducing short hairpin (sh) RNAs against Cdx2 via a viral vector. This vector was used to infect mouse fibroblasts and expression of green fluorescent protein (GFP) was used as a marker to select cells to be used as donors for nuclear transfer. The researchers created a total of 526 new cells by nuclear transfer and of these 61 developed to the blastocyst stage. The Cdx2-deficient blastocysts were found to be morphologically abnormal and did not express Cdx2 when assessed by both immunohistochemistry and RT-PCR. None of the Cdx2-deficient blastocysts were able to implant and develop when transferred to the uteri of pseudo-pregnant mice, compared to 40% of a control sample.

The researchers then went on to assess whether ES cell lines could be derived from the Cdx2-deficient blastocysts. Following expansion in cultures ICMs taken from these cells generated ES cell lines with an efficiency that was comparable to that of nuclear transfer blastocysts derived from wild-type fibroblasts. The pluripotency of these lines was assessed by testing their ability to form chimeras when injected into diploid blastocysts. The GFP-labelled cells contributed to most tissues with the exception of the intestine, reinforcing previous reports that Cdx2 is necessary for development of the gastro-intestinal tract. In order to determine if full pluripotency could be restored to these cell lines, a plasmid was introduced which deleted the Cdx2 shRNA and thus restored normal Cdx2 expression and function. Nuclei from this new line were then transferred to recipient oocytes, and blastocysts which expressed wild-type levels of Cdx2 were generated. ES cell lines derived from these blastocysts were able to produce all somatic tissues, including intestinal cells, and these blastocysts were also able to implant when placed in pseudo-pregnant female mice. The researchers were thus able to generate abnormal blastocysts that were intrinsically unable to implant in the uterus and develop into a foetus, but which could be used for the derivation of ES cell lines which displayed reduced development potential compared to wild-type ES cells. However, full pluripotency could be restored by reversing the knock-down of Cdx2 expression.

Comment: The methodology described in this paper attempts to develop a protocol for the derivation of ES cell lines that takes account of the moral and ethical views of those who object to the destruction of a potential life that this process normally involves. Those who oppose human ES cell research do so on the basis that the blastocyst that is destroyed when the ICM is removed and cultured in vitro to generate hES cell lines, could if implanted go on to develop into a foetus and eventually a new life. The authors of this study hope to overcome this objection by developing blastocysts that do not have this potential to develop into a new life, as they have been genetically engineered to lack expression of a key gene necessary for the implantation and development process. It remains to be seen if specifically creating an entity that will never have the potential to develop into a new life is morally or ethically more acceptable than destroying a blastocyst which does have this potential.

A number of scientific areas also need to be addressed before this technology could actually provide a viable alternative to the current use of wild-type human blastocysts. All the work performed by the report authors was carried out in mouse models. As the authors themselves concede, although CDX2 is known to be expressed in the human trophectoderm, it has not been established if its role in placental development is identical to that in the mouse. This will require further studies with human cells. The ANT protocol also adds steps to the regular single cell nuclear transfer technique that may have implications for the production and safety assessment of cell lines used in future therapies. Additionally the ANT technique did not generate fully pluripotent ES lines, as the loss of Cdx2 expression resulted in the inability to generate intestinal cells. Full pluripotency was only restored once Cdx2 expression had also been restored, but this resulted in the production of blastocysts that could implant in the uterus and therefore go on to normal embryonic development. Thus seemingly returning us to the original ethical dilemma about the destruction of viable embryos in this type of research.

The full paper can be found at: A. Meissnerand R. Jaenisch (2005) Generation of nuclear transfer-derived pluripotent ES cells from cloned Cdx2-deficient blastocysts. Nature advance online publication; doi: 10.1038/nature04257

A second alternative method for the derivation of embryonic stem cell lines has also been proposed. An advance online Nature publication by Chung et al., describes the creation of mouse embryonic stem cell lines from an eight-cell stage embryo, by removing a single blastomere and culturing this in vitro. This single-cell embryo biopsy model, is similar to that used in pre-implantation genetic diagnosis (PGD) of genetic diseases or defects, a process that does not affect the viability of the embryo tested (see previous items in PHGU newsletter).

The authors removed single blastomeres from eight-cell stage embryos in six different experiments. Each blastomere was co-cultured with GFP-expressing mouse ES cells, and after incubation for 24-48 hours a growing 'bud' of GFP-negative cells was observed in four of the cultures. These cell aggregates were expanded in cultures containing mouse feeder cells and the GFP-negative cells separated by hand under fluorescence microscopy. The cell lines produced expressed markers of pluripotency such as Oct-4, and exhibited a normal karyotype. They were also able to differentiate into cells of all three germ layers, and when used to create chimera, contributed to all organ systems and cell lineages. The embryos, which had been biopsied, were not impaired in their viability, and 49%, developed into live young compared to 51% of non-biopsied embryos in a control sample.

Comment: This study is the first report of the derivation of pluripotent ES cell lines from a single blastomere in vitro. Although only currently investigated in mice, the PGD method may provide a possible alternative for the production hES lines that does not result in the destruction of the embryo from which they are derived. However, a number of scientific hurdles to this still exist. Blastomeres removed from the early stage embryo did not generate ES cell lines when grown in cell culture medium and in the absence of mouse ES cells. It therefore appears that co-culture is critical to the development of new ES cell lines, and that this protocol requires the labour-intensive steps of separating out the various cell types before a homogeneous ES cell line can be established. The authors also acknowledge that further investigation is required to determine if this technique could be successfully performed using human embryos, as compared to mice. Even if this were to be established, it appears that potential sources of new cell lines would be limited to embryos that were being screened in approved IVF clinics. Many investigators are creating cell lines carrying specific defects or derived from patients with particular medical conditions (see previous newsletter item), and it does not appear that this potential source of embryonic material would have sufficient diversity to match these needs.

The full paper can be found at: Y Chung et al. (2005) Embryonic and extraembryonic stem cell lines derived from single mouse blastomeres.Nature advance online publication; doi: 10.1038/nature04277

18 October 2005A novel and technique for gene therapy has reportedly cured the genetic disease phenylketonuria (PKU) in mice. PKU is an autosomal recessive disease caused by a defective gene for the enzyme phenylalanine hydroxylase (PAH), which is a crucial for metabolism of the amino acid phenylalanine in the liver. In many countries including the UK, neonatal screening programmes test for PKU; affected children who are diagnosed early are restricted to a phenylalanine-free diet, which prevents the severe, irreversible mental retardation otherwise caused by the condition.

Reporting in the advance online edition of the journal Proceedings of the National Academy of Sciences of the United States of America (PNAS), US scientists outline a procedure whereby a viral (bacteriophage) based system was used to direct integration of a functional mouse PAH gene to a specific region of the mouse genome [Chen L and Woo SLC (2005) PNAS Early Edition October 17, 10.1073/pnas.0503877102]. The PKU mouse model is a fairly accurate recreation of the human disorder, and has been used before for gene therapy studies by different groups.

In this new paper the authors, who have previously reported transient correction of the PKU mouse phenotype by adenovirus-mediated PAH gene delivery, use a phiBT1 phage system to deliver the gene. This system comprises the gene of interest in a DNA construct flanked by attachment sites, and delivered with another construct containing the gene for the viral integrase protein that catalyses insertion of DNA sequences between the attachment sites via recombination with complementary attachment sites in target DNA. It was tested in mouse cells in vitro, and reporter gene expression observed in 27% of samples.

The researchers next identified a total of eight sites in the mouse genome that could function as pseudo-attachment sites for the phiBT1 phage system, by integrating reporter genes to mouse cells and then sequencing the sites of integration. These sites showed up to 51% sequence similarity with the bacterial attachment sites. Groups of nude mice (a strain lacking competent immune systems) were injected with components of the phiBT1 system, including a reporter gene; integration occurred in positive but not negative controls, and more than 95% of gene integration took place at a single site, mpsP3, and a further 4% at two additional sites.

Having established that it was feasible to achieve site-specific integration of DNA sequences into mammalian genomes using the phage system, the researchers then looked at PKU mice to see whether delivery of the PAH gene in this manner could alleviate disease symptoms. Twelve PKU mice were injected in the tail vein with an integrating DNA construct containing the mouse PAH gene; six of these mice also received the integrase gene construct, whilst the other six received an integrase-negative construct (ie. one which would not support integration of the PAH gene to the mouse genome). Injections were repeated in weeks 4 and 12, and blood samples were taken biweekly from the mice and analysed for phenylalanine.

Serum phenylalanine levels dropped significantly in both groups of mice following injection, but in the integrase-negative control group it returned to pre-treatment level (1600–1800mM) within two weeks of each injection. However, the integrase-positive mice maintained the decreased level of 650–850mM following the initial injection; this dropped further to 300-500mM after the second injection and remained stable until the injections were repeated again in week 12, after which serum phenylalanine to was reduced to 90-120mM, a measure within the normal range for healthy mice. This level remained stable until week 20, when the mouse livers were examined for PAH activity. Untreated PKU mice show

13 October 2005Metastatic potential as a heritable trait. Threadgill DW (2005). Nat. Genet. 37, 1026-1027. News and Views piece accompanying new report of a genetic modifier of cancer metastasis efficiency in mice, outlining the model whereby metastatic potential is considered as a heritable trait influenced by host genetic polymorphisms.

Stem cells, asymmetric division and cancer. Clevers H (2005). Nat. Genet. 37, 1027-1028 News and Views piece.

The latest issue of Nature Medicine has two pieces commenting on the American College of Medical Genetics (ACMG) guidelines on newborn screening, released in March this year, which recommended a uniform screening programme for all US states:

Newborn screening grows up. Nat. Med. 11, 1013 (2005). Editorial broadly in favour of the move towards uniform screening in the US, although the author notes that screening programmes are only one aspect of improved care and that education of healthcare providers will be critical.

News Feature: Screen savers. Nat. Med. 11, 1020-1021 (2005). News article looking at potential benefits of the expanded newborn screening panel, and reporting on opposition to the inclusion of some conditions for which the medical benefits of diagnosis are not certain in the panel.

Perspectives on the properties of stem cells. McCulloch EA and Till JE (2005) Nat. Med. 11, 1026-1028. Autobiographical and technical commentary article.

Tools for genomics. Southern E (2005) Nat. Med. 11, 1029-1034. Autobiographical and technical commentary article.

Genetic fingerprinting. Jeffreys AJ (2005) Nat. Med. 11, 1035-1039. Autobiographical and technical commentary article.

Finding genetic modifiers of cystic fibrosis. Haston CK and Hudson TJ (2005) N. Engl. J. Med. 353, 1509-1511. Editorial piece accompanying research article, and considering the problems in identifying modifier genes.

Mind the (biomedical funding) gap. Klausner A (2005) Nat. Biotech. 23, 1217-1218. Commentary.

6 October 2005Repeat Instability: mechanisms of dynamic mutations. Pearson CE, Edamura KT and Cleary JD (2005) Nature Reviews Genetics 6, 729-742. Review on the genetic mechanisms underlying a range of neurological disorders associated with DNA repeat expansion mutations.

Diseases of unstable repeat expansion: mechanisms and common principles. Gatchel JR and Zoghbi HY (2005) Nature Reviews Genetics 6, 743-755. Review on these diseases and the associations between disordered gene expression and disease pathogenesis.

Therapeutics development for triplet repeat expansion diseases. Di Prospero NA and Fischbeck KH (2005) Nature Reviews Genetics 6, 756-767. Review of research into disease mechanisms and interventions.

The molecular genetics of Huntington disease – a history. Bates GP (2005) Nature Reviews Genetics 6, 766-773. Summary of progress since mapping of the HD gene in 1983 and cloning in 1993 to current studies into the molecular pathogenesis using genetic models.

The new cytogenetics: blurring the boundaries with molecular biology. Speicher MR and Carter NP (2005) Nature Reviews Genetics 6, 782-792. Review looking at the impact of technological advances such as high resolution array-comparative genomic hybridisation on research and diagnostic cytogenetics.

Applications of behavioural genetics: outpacing the science? Rothstein MA (2005) Nature Reviews Genetics 6, 793-798. 'Science and Society' article on the potential applications and abuses of research in behavioural genetics, and the necessary safeguards to prevent inappropriate use.

Genetic association studies. Cordell HJ and Clayton DG (2005) Lancet 366, 1121-1131. Third in a series of summary pieces on aspects of genetic epidemiology.

Shaking the tree: mapping complex disease genes with linkage disequilibrium. Plamer LJ and Cardon LR (2005) Lancet 366, 1223-1234. Fourth in a series of summary pieces on aspects of genetic epidemiology.

A new grammar for drug discovery. Fishman MC and Porter JA (2005) nature 437, 491-493. News and Views feature on approaches to realising the potential of the genome in drug discovery by focusing on conserved molecular signalling pathways, including the use of genetic disorders in highlighting key features of such pathways.

Targeting specific cell types with silencing RNA. Williams BR (2005) N. Engl. J. Med. 353, 1410-1411. Commentary on technique originally reported in Nature Biotechnology.

6 October 2005A recent paper in Science reports on a new mouse model for trisomy 21 (Down’s Syndrome, or DS), one of the most common forms of human aneuploidy in which there is an additional third copy of human chromosome 21 [O’Doherty A et al. (2005) Science 309, 2033-2037]. Murine models have previously been used in the study of the molecular genetics and pathology of trisomy 21, but have had various limitations. The mouse chromosome 16 carries around two-thirds of the homologous (equivalent) genes to those present on human chromosome 21 (Hsa21), so hitherto the best murine disease models have been partial trisomy 16 strains engineered to have additional copies of chromosomal regions corresponding to some of those present in human trisomy 21. The new murine model is a trans-species chimera, having a normal mouse genome with the addition of most of the human chromosome 21; this ‘trans-chromosomic’ mouse is therefore trisomic only for human chromosome 21 genes, since it carries both these and the homologous mouse genes on murine chromosomes 10, 16 and 17. The authors propose that this model is better than previous ones because it more closely models the situation in humans, minimises disruption of mouse gene sequences and also comprises not only most of the human chromosome 21 genes but also non-coding sequences including regulatory regions.

Female mouse embryonic stem (ES) cell lines containing human Hsa21 fragments were created using a technique called irradiation microcell-mediated chromosome transfer; the ES cell lines were examined for human chromosomal content using fluorescence in situ hybridisation (FISH). Five cell lines containing a single, freely-segregating Hsa21 fragment (comprising around 90% of the full chromosome 21), but no other human chromosomal fragments, were identified. ES cells from these lines were injected to mouse blastocysts to create chimeric embryos; the adult female chimeras were mated with normal male mice. Germline transmission of the Hsa21 fragment was achieved from one chimeric mouse to create a trans-chromosomic mouse strain called Tc1. The Tc1 mouse colony reportedly shows stable transmission of the Hsa21 fragment to more than 40% of progeny mice, and both male and female mice carrying the human genes have been aged up to 20 months and seem healthy.

The authors then analysed the Tc1 mice for human gene expression, using Hsa21 probe sequences; 51 out of 205 sequences (representing 39 out of a total of 139 genes) tested for showed increased expression in Tc1 mice compared with control mice. For comparison, the paper reports that only 9 out of 22,078 non-Hsa21 sequences showed higher expression in the Tc1 mice, leading them to conclude that there were significant levels of human chromosome 21 gene expression in the Tc1 mice. This was found to include expression of a number of specific genes previously implicated in aspects of the DS phenotype, such as the SOD1, BACE2 and APP genes.

Finally, the paper reports analysis of phenotypic features of the Tc1 trans-chromosomic mice similar to those commonly observed in humans with trisomy 21. They observed minor changes in craniofacial morphology and a slightly decreased density of neurons in the cerebellum, proposed to parallel the slightly decreased cerebellar volume seen in affected humans. Seven out of eleven Tc1 mice were found to have significant cardiac defects, primarily affecting the ventricular septum, which were similar to the most common congenital heart defects observed in DS humans. In order to assess the mice for traits equivalent to the mental retardation observed in humans, they were tested for aspects of learning and memory, and found to have impaired long-term memory and a tendency towards hyperactivity compared with control animals.

Some of the phenotypic features of the Tc1 mice have been observed in other DS mouse models. The authors propose that this new DS model is likely to be superior to these mouse partial trisomy models, at least in some aspects, because they more closely parallel the human condition, although they caution that further work is needed to establish how well. They also observe that the new technique for creating trans-chromosomic mice may serve for modelling of other human aneuploidies. Author Dr Victor Tybulewicz commented: "Aneuploidies are seen in at least 5% of all pregnancies and are therefore a big cause of human illness, death and miscarriage. This technology will provide a crucial genetic tool in understanding this complex human syndrome" (see MRC press release). The other project leader, Professor Elizabeth Fisher, said: "This is a technical step forward in stem cell technology. It opens up a lot of possibilities", adding that they hoped it would not only be of use in the study of DS but also "help unravel the picture of individual genes responsible for complex conditions, such as diabetes, and to create artificial chromosomes for gene therapy" (see BBC news report).

Comment: This paper represents an interesting application of stem cell technology in the creation of potentially superior animal models of disease, by permitting the incorporation of large human chromosomal fragments into the germ-line. Such models are crucial in the study of disease processes and in developing therapeutic interventions, but are often limited due to the differences between species. How far this particular mouse model will be of use for dissecting the precise genetic causes of the features of Down’s Syndrome remains to be seen, but in the longer term the basic technology seems likely to be of value in the study of genetic influences on health.

3 October 2005The Use of Racial, Ethnic, and Ancestral Categories in Human Genetics Research. NHGRI Race, Ethnicity, and Genetics Working Group (2005) Am. J. Hum. Genet. 77, 519-532. Review looking at concepts of race and ethnicity, and how the use of such categorisation in genetics can imply that differences between groups are solely biologically (as opposed to socially or environmentally) mediated. The authors call for careful investigation of traits associated with different ethnic groups as an essential component of disease research.

Reforming research ethics committees. BMJ (2005) 331, 587-588. Editorial commenting on the UK Department of Health report on the operation of NHS research ethics committees.

Genetic linkage studies. Teare MD and Barrett JH (2005) Lancet 366, 1036-1044. Second in a series of summary pieces on aspects of genetic epidemiology.

Massively parallel sequencing. Rogers Y and Venter JC (2005) Nature 437, 326-327. News and views article on a novel form of DNA sequencing system reported in the same issue, which is around 100 times faster than current advanced sequencing methods, looking at advantages and limitations.

Beta-Thalassemia. Rund D and Rachmilewitz E (2005) N. Engl. J. Med. 353, 1135-1146. Review article including a section on progress in prenatal diagnosis.