Researchers develop safer way to make induced pluripotent
stem cells

Researchers at Johns Hopkins have found a better way to create induced pluripotent stem (iPS) cells - adult cells reprogrammed with the properties of embryonic stem cells-from a small blood sample. This new method, described last week in Cell Research, avoids creating DNA changes that could lead to tumour formation.

“These iPS cells are much safer than ones made with previous technologies because they don’t involve integrating foreign viruses that can potentially lead to uncontrolled, cancerous cell growth,” says Linzhao Cheng, PhD, an associate professor of medicine in the Division of Hematology and a member of the Johns Hopkins Institute of Cell Engineering. “This is important if iPS cells are to be used as therapies one day.”

Cheng says the higher-quality iPS cells will also be more reliable in research studies, “since we don’t have to worry about extra genetic changes associated with previous technologies interfering with study results.”

Johns Hopkins researchers created the safer iPS cells by transferring a circular piece of DNA into blood cells from anonymous donors to deliver the needed genetic components. The traditional way is to use viruses to carry DNA into a cell’s genome. Unlike the viral methods, the circular DNA the Hopkins team used is designed to stay separate from the host cell’s genome. After the iPS cells formed, the circular DNA delivered into the blood cells was gradually lost.

Using about a tablespoon of human adult blood or umbilical cord blood, the researchers grew the blood cells in the lab for eight to nine days. The researchers then transferred the circular DNA into the blood cells, where the introduced genes turned on to convert the blood cells to iPS cells within 14 days.

The research group verified conversion from mature blood cells to iPS cells by testing their ability to behave like stem cells and differentiate into other cell types, such as bone, muscle or neural cells. They also looked at the DNA from a dozen iPS cell lines to make sure there were no DNA rearrangements.

Cheng says the new method is also more efficient than the traditional use of skin cells to make iPS cells. “After a skin biopsy, it takes a full month to grow the skin cells before they are ready to be reprogrammed into iPS cells, unlike the blood cells that only need to grow for eight or nine days,” says Cheng. “The time it takes to reprogram the iPS cells from blood cells is also shortened to two weeks, compared to the month it takes when using cells.”

Cheng says, this easy method of generating integration-free human iPS cells from blood cells will accelerate their use in both research and future clinical applications”.

This study was funded by The Johns Hopkins University, a New York Stem Cell grant and grants from the US National Institutes of Health.

The Cheng lab:
Institute for Cell Engineering:

Genome code cracked for common paediatric brain cancer

Scientists at the Johns Hopkins Kimmel Cancer Center have deciphered the genetic code for medulloblastoma, the most common paediatric brain cancer and a leading killer of children with cancer, occurring in about 400 children per year in the U.S. The genetic “map” is believed to be the first reported of a paediatric cancer genome and is published in the online December issue of Science Express.

Notably, the findings show that children with medulloblastoma have five- to tenfold fewer cancer-linked alterations in their genomes compared with their adult counterparts. “These analyses clearly show that genetic changes in paediatric cancers are remarkably different from adult tumors. With fewer alterations, the hope is that it may be easier to use the information to develop new therapies for them,” says Victor Velculescu, M.D., Ph.D., associate professor of oncology at the Johns Hopkins gene pool Genetic research news from around the world Kimmel Cancer Center.

The Johns Hopkins team used automated tools to sequence hundreds of millions of individual chemicals called nucleotides, which pair together in a preprogrammed fashion to build DNA and, in turn, a genome. Combinations of these nucleotide letters form genes, which provide instructions that guide cell activity. Alterations in the nucleotides, called mutations, can create coding errors that transform a normal cell into a cancerous one. The scientists at Johns Hopkins have previously mapped genome sequences for pancreatic, adult brain, breast and colon cancers with similar methods.

For the study, scientists sequenced nearly all protein-encoding genes in 22 samples of paediatric medulloblastoma and compared these sequences with normal DNA from each patient to identify tumorspecific changes or mutations. Each tumor sample had an average of 11 mutations. There were 225 mutations in all. Then, the investigators searched through a second set of 66 medulloblastomas, including some samples from adults, to find how these mutations altered the proteins made by the genes.

The team found that most of the mutations congregate within a few gene families or pathways. The most prevalent pathway ordered the way long strands of DNA, that make up chromosomes, are twisted and shaped into dense packets that open and close depending on when genes need to be activated. Such a process is regulated by chemicals that operate outside of genes, termed “epigenetic” by scientists. Within the epigenetic pathway, two commonly mutated genes were both involved in how molecules called histones wrap around DNA.

Mutations in MLL2 and MLL3 were identified in 16% of the entire set of 88 medulloblastoma samples. Add to this three other epigenetic alterations found by the scientists in the genome scan, and the total set accounts for 20% of mutations in all the brain cancer samples. Second to epigenetic pathways were gene mutations in pathways such as Hedgehog and Wnt that control tissue and organ development in humans and other animals. Both pathways have previously been linked to childhood medulloblastoma.

Modified immune cells used to successfully treat soft tissue tumour

Results of an intermediate stage clinical trial of several dozen people provides evidence that a method that has worked for treating patients with metastatic melanoma can also work for patients with metastatic synovial cell sarcoma, one of the most common soft tissue tumours in adolescents and young adults. This study is the first to use genetically modified immune cells, in a technique known as adoptive therapy, to cause cancer regression in patients with a solid cancer as opposed to melanoma. This approach represents a method for obtaining immune cells from any cancer patient and converting them into ones that can recognise cancer cells expressing the target antigen, NY-ESO-1, according to researchers at the National Cancer Institute. The study appeared in the 31 January 2011, issue of the Journal of Clinical Oncology.

NY-ESO-1 is a protein found in up to 50% of melanomas and cancers of the breast, prostate, oesophagus, lung, and ovary, and in 80% of synovial sarcomas. “Since NY-ESO-1 is expressed in a substantial number of cancers, beside melanoma and synovial sarcoma, it is an attractive target for immune-based therapies against these cancers as well,” said lead investigator Steven Rosenberg, MD, PhD, chief of the Surgery Branch in NCI’s Center for Cancer Research.

This work builds upon previously published results in patients with metastatic melanoma. Those studies showed that metastatic melanoma patients could be treated by infusion with their own genetically modified T cells, or white blood cells, that had receptors on their surfaces that recognised an antigen on the melanoma cells.

In this study, 17 patients with synovial cell sarcoma or metastatic melanoma, whose tumours expressed NY-ESO-1, received therapy with their own immune cells engineered to express a T cell receptor capable of recognising the NYESO- 1 antigen. To perform this treatment, the investigators isolated lymphocytes from each patient’s blood and modified these cells by inserting the gene encoding the anti-tumour T cell receptor into them. These genetically modified cells were then able to recognise and destroy NY-ESO-1- expressing cancer cells. The results showed tumour regression in four of the six patients with synovial cell sarcoma and in five of the 11 melanoma patients. A partial response that lasted 18 months was observed in one of the synovial cell sarcoma patients, while two of the melanoma patients demonstrated complete ongoing regression responses that lasted 20 months or longer, which for patients with these diseases, is significant.

“Now that we have shown that a patient’s own cells genetically engineered to express a receptor against the NY-ESO- 1 antigen can mediate tumour regression, we will be optimising this treatment and extending it to the treatment of patients with other common cancers,” said Rosenberg.

● Reference: Robbins PF, et al., Tumor regression in patients with metastatic synovial sarcoma and melanoma using engineered lymphocytes reactive with NYESO- 1, Journal of Clinical Oncology, DOI: 10.1200/JCO.2010.32.2537, 31 January 2011.

Jumping genes

An ambitious hunt by Johns Hopkins scientists for actively “jumping genes” in humans has yielded compelling new evidence that the genome, anything but static, contains numerous mobile elements that may help to explain why people have such a variety of physical traits and disease risks.

Using bioinformatics to compare the standard assembly of genetic elements as outlined in the reference human genome to raw whole-genome data from 310 individuals recently made available by the 1000 Genomes Project, the team revealed 1,016 new insertions of RIPs, or retrotransposon insertion polymorphisms, thereby expanding the catalog of insertions that are present in some individuals and absent in others. Their results appeared online in Genome Research.

Retrotransposons are travelling bits of DNA that replicate by copying and pasting themselves at new locations in the genome. Having duplicated themselves and accumulated over evolutionary history, transposable elements now make up about half of the human genome. However, only a tiny subfamily of these insertions known as LINE-1 (L1) is still active in humans. Line 1 insertions are able to mobilise not only themselves but also other pieces of DNA.

“In any individual, only between 80 to 100 retrotransposons are actively copying and inserting into new sites,” says Haig Kazazian, M.D., professor of human genetics, McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine. “We're not only discovering where they are and who has which ones, but also finding out that they insert with a remarkable frequency: On the order of one in every 50 individuals has a brand-new insertion that wasn't in their parents.”

“Our genome contains around half a million interspersed L1 sequences that have accumulated over evolutionary history, along with over a million more repeats, most of which were mobilised by L1 elements,” explains Adam D. Ewing, Ph.D., a postdoctoral fellow in Kazazian's lab. “Since the vast majority of these are ancestral and therefore common to all humans and even some of our primate relatives, we can ignore them and focus on L1s that contain human-specific characters in their sequences. Those are the actively mobilised elements responsible for considerable genomic diversity among human individuals.”

The high frequency of these L1 insertions gives us a better idea about the extent of human diversity, according to Kazazian, whose 22-year focus on retrotransposons seeks to reveal how they alter the expression of human genes.

Just as the structural variants known as single nucleotide polymorphisms (or SNPs, pronounced “snips”) serve as markers for various diseases, the hope is that RIPs – which are up to 6,000 times bigger than SNPs, and therefore may have a stronger effect on gene expression – will correlate with disease phenotypes.

“In that same way that someone had to go out and find the SNPs, this study was about finding RIPs that remain active and continue to produce new insertions,” Kazazian says. “Now we have the background necessary to begin studies that may correlate these L1 insertions with everything from autism to cancer.”

Genome study reveals links to abnormal cardiac rhythms behind sudden death

A study among almost 50,000 people worldwide has identified DNA sequence variations linked with the heart’s electrical rhythm in several surprising regions among 22 locations across the human genome. The variants were found by an international consortium, including Johns Hopkins researchers, funded by the National Institute of Health and reported in the Nature Genetics advance online publication.

Among the notable discoveries were variations in two side-by-side genes that regulate electrically charged particles to produce signals that start contraction of the heart and register as pulsing waves seen on heart monitors. One of the genes, named SCN5A, was known to be involved in controlling how signals start from specialised muscle cells and travel across the heart to cause its rhythmic contractions. Its neighbour, SCN10A, previously was an unsuspected player in cardiac electrical activity.

The study’s genome-wide hunt focused on the QRS interval, a measure of electrical depolarisation in the main lower pumping chambers of the heart (ventricles), easily detectable with a simple EKG machine. A prolonged QRS interval suggests a diseased ventricular conduction system and has been associated with increased risk for sudden cardiac death, among other heart disorders.

Fifteen genome-wide association or socalled GWAS studies among healthy American and European populations who had EKGs were combined for large-scale meta-analysis. Each GWAS reported what effects were observed at which locations in a scan of 2.5 million single nucleotide polymorphisms (SNPs) throughout the genome. Pronounced snips, SNPs are sites where a single letter in the DNA code is variable. Study subjects whose EKGs showed prolonged QRS intervals had SNPs in common with each other.

“The size of the study really gave us the power to identify many genes not previously suspected to play a role in heart conduction,” says Dan Arking, Ph.D., assistant professor in the McKusick- Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine.

To further investigate one of the 22 regions deemed significant, team members used mouse hearts to find where in the heart the ion channel protein products of the SCN10A gene form, locating an abundance of them in the fibres that conduct electrical signals. Next, the researchers treated mice with a drug that blocked the function of the ion channels produced by the SCN10A gene, and used EKGs to show that the QRS duration was extended in these animals.

“These genes potentially could be very useful for identifying individuals for whom adverse drug reactions may cause life threatening cardiac events,” Arking says.

Nano scale gene switch

In a proof of principal study in mice, scientists at Johns Hopkins and the Virginia Commonwealth University (VCU) have shown that a set of genetic instructions encased in a nanoparticle can be used as an “ignition switch” to rev up gene activity that aids cancer detection and treatment.

The switch, called a promoter, is a set of chemical letters that interacts with DNA to turn on gene activity. In this case, the scientists used a promoter called PEG-Prom, cloned by VCU researcher Paul Fisher, Ph.D. PEG-Prom is activated only when inside cancer cells, not in normal ones.

“With current imaging devices like CT and PET, we can tell if something is wrong in a patient, but we don’t have definitive tools to distinguish cancer from inflammation or infection,” says Martin Pomper, M.D., Ph.D., professor of radiology at Johns Hopkins. “It generally takes at least one month after giving patients certain cancer treatments before existing imaging tools can measure the patient’s response to the therapy.”

To differentiate cancer cells from normal cells, Johns Hopkins scientists connected PEG-Prom to either a gene that produces firefly luciferase, the substance that make fireflies glow, or a gene called HSV1tk, which initiates a chemical reaction with radioactive labels inside the cell that can be detected by imaging devices. Once inside a cancer cell, the PEG-Prom switch is turned on, and it activates either the luciferase or HSV1tk gene.

Then, they stuffed the PEG-Prom/gene combination into tiny spheres – about 50,000 times smaller than the head of a pin – and intravenously injected the nanoparticles into mice with either metastatic breast cancer or melanoma.

The findings, reported in the December 12 online edition of Nature Medicine, reveal a 30-fold difference in identifying cancer cells containing luciferase and normal cells that did not contain the substance. Similar results were observed in cancer cells filled with the radioactive labels and normal ones that were not.

In addition to diagnostic and monitoring tools, the technique could be designed to deliver therapies to the heart of cancer cells. One approach, Pomper says is to use radioactive isotopes to make cancer cells radioactive from the inside, instead of delivering radiation to the patient externally. Such a technique would still be limited to identifying tumours that are two millimetres or larger, amounting to millions of cells, because current imaging devices cannot detect anything smaller. He also says that certain doses of nanoparticles could be toxic, so his team is conducting tests to find the best nanoparticle.

Pancreatic cancer gene code cracked

Scientists at Johns Hopkins have deciphered the genetic code for a type of pancreatic cancer, called neuroendocrine or islet cell tumours. The work, described online in the January 20, 2011 issue of Science Express, shows that patients whose tumours have certain coding ‘mistakes’ live twice as long as those without them.

“One of the most significant things we learned is that each patient with this kind of rare cancer has a unique genetic code that predicts how aggressive the disease is and how sensitive it is to specific treatments,” says Nickolas Papadopoulos, Ph.D., associate professor at the Johns Hopkins Kimmel Cancer Center and director of translational genetics at Hopkins’ Ludwig Center. “What this tells us is that it may be more useful to classify cancers by gene type rather than only by organ or cell type.”

Pancreatic neuroendocrine cancers account for about five percent of all pancreatic cancers. Some of these tumours produce hormones that have noticeable effects on the body, including variations in blood sugar levels, weight gain, and skin rashes while others have no such hormone ‘signal.’ In contrast, hormone-free tumours grow silently in the pancreas, and “many are difficult to distinguish from other pancreatic cancer types,” according to Ralph Hruban, M.D., professor of pathology and oncology, and director of the Sol Goldman Pancreatic Cancer Research Center at Johns Hopkins.

For the new study, the team investigated non-hormonal pancreatic neuroendocrine tumours in 68 men and women. Patients whose tumours had mutations in three genes –MEN-1, DAXX and ATRX – lived at least 10 years after diagnosis, while more than 60% of patients whose tumours lacked these mutations died within five years of diagnosis.

The Johns Hopkins team used automated tools to create a genetic map that provides clues to how tumours develop, grow and spread. Within the code are individual chemicals called nucleotides, which pair together in a pre-programmed fashion to build DNA and, in turn, a genome. Combinations of these nucleotide letters form genes, which provide instructions that guide cell activity. Changes in the nucleotide pairs, called mutations, can create coding errors that transform a normal cell into a cancerous one.

In the first set of experiments, the Johns Hopkins scientists sequenced nearly all protein-encoding genes in 10 of the 68 samples of pancreatic neuroendocrine tumours and compared these sequences with normal DNA from each patient to identify tumour-specific changes or mutations. In another set of experiments, the investigators searched through the remaining 58 pancreatic neuroendocrine tumours to determine how often these mutated genes appeared.

The most prevalent mutation, in the MEN-1 gene, occurred in more than 44% of all 68 tumours. MEN-1, which has been previously linked to many cancers, creates proteins that regulate how long strands of DNA are twisted and shaped into dense packets that open and close depending on when genes need to be activated. Such a process is regulated by proteins and chemicals that operate outside of genes, termed ‘epigenetic’ by scientists.

Two other commonly mutated genes, DAXX and ATRX, which had not previously been linked to cancer, also have epigenetic effects on how DNA is read. Of the samples studied, mutations in DAXX and ATRX were found in 25% and 17.6%, respectively. The proteins made by these two genes interact with specific portions of DNA to alter how its chemical letters are read.

“This is a great example of the potential for personalised cancer therapy,” says Hruban. “Patients who are most likely to benefit from a drug can be identified and treated, while patients whose tumours lack changes in the mTOR pathway could be spared the side effects of drugs that may not be effective in their tumours.”

Prototype patented for rapid genome sequencing

Scientists from Imperial College London are developing technology that could ultimately sequence a person’s genome in mere minutes, at a fraction of the cost of current commercial techniques. The researchers have patented an early prototype technology that they believe could lead to an ultrafast commercial DNA sequencing tool within ten years. Their work is described in a study published in the journal Nano Letters and it is supported by the Wellcome Trust Translational Award and the Corrigan Foundation.

The research suggests that scientists could eventually sequence an entire genome in a single lab procedure, whereas at present it can only be sequenced after being broken into pieces in a highly complex and time-consuming process. Fast and inexpensive genome sequencing could allow ordinary people to unlock the secrets of their own DNA, revealing their personal susceptibility to diseases such as Alzheimer’s, diabetes and cancer. Medical professionals are already using genome sequencing to understand populationwide health issues and research ways to tailor individualised treatments or preventions.

Dr Joshua Edel, one of the authors on the study from the Department of Chemistry at Imperial College London, says: “Compared with current technology, this device could lead to much cheaper sequencing: just a few dollars, compared with $1m to sequence an entire genome in 2007. We haven’t tried it on a whole genome yet but our initial experiments suggest that you could theoretically do a complete scan of the 3,165 million bases in the human genome within minutes, providing huge benefits for medical tests or DNA profiles for police and security work. It should be significantly faster and more reliable, and would be easy to scale up to create a device with the capacity to read up to 10 million bases per second, versus the typical 10 bases per second you get with the present day single molecule real-time techniques.”  

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