The ISSCR Issues Statement on Human Germ Line Genome Modification

The International Society for Stem Cell Research has released a statement calling for a moratorium on attempts to apply nuclear genome editing of the human germ line in clinical practice

Sunday, 22 March 2015

In a statement released on Thursday, the International Society for Stem Cell Research called for a moratorium on attempts at clinical application of nuclear genome editing of the human germ line to enable more extensive scientific analysis of the potential risks of genome editing and broader public discussion of the societal and ethical implications.

Technologies used to introduce changes into the DNA sequence of cells have advanced rapidly, making genome editing increasingly simple. Genome editing is feasible, not just in the somatic cells of an adult organism, but also in early embryos, as well as the gametes (sperm and egg) that carry the inheritable, germ line DNA. Research involving germ line nuclear genome editing has been performed to date in many organisms, including mice and monkeys, and applications to human embryos are possible.

The ISSCR statement raises significant ethical, societal and safety considerations related to the application of nuclear genome editing to the human germ line in clinical practice. Current genome editing technologies carry risks of unintended genome damage, in addition to unknown consequences. Moreover, consensus is lacking on what, if any, therapeutic applications of germ line genome modification might be permissible.

The statement calls for a moratorium on attempts to apply nuclear genome editing of the human germ line in clinical practice, as scientists currently lack an adequate understanding of the safety and potential long term risks of germ line genome modification. Moreover, the ISSCR asserts that a deeper and more rigorous deliberation on the ethical, legal and societal implications of any attempts at modifying the human germ line is essential if its clinical practice is ever to be sanctioned.

In calling for the above moratorium, the ISSCR is not taking a position on the clinical testing of mitochondrial replacement therapy, a form of germ line modification that entails replacing the mitochondria (found outside the nucleus) in the eggs of women at risk of transmitting certain devastating diseases to their children.

Source: International Society for Stem Cell Research

Contact: Michelle Quivey

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Newer Genome Editing Tool Shows Promise in Engineering Human Stem Cells

Johns Hopkins study could advance use of stem cells for treatment and disease research

Tuesday, 06 January 2015

A powerful “genome editing” technology known as CRISPR has been used by researchers since 2012 to trim, disrupt, replace or add to sequences of an organism’s DNA. Now, scientists at Johns Hopkins Medicine have shown that the system also precisely and efficiently alters human stem cells.

In a recent online report on the work in Molecular Therapy, the Johns Hopkins team says the findings could streamline and speed efforts to modify and tailor human-induced pluripotent stem cells (iPSCs) for use as treatments or in the development of model systems to study diseases and test drugs.

“Stem cell technology is quickly advancing, and we think that the days when we can use iPSCs for human therapy aren’t that far away,” says Zhaohui Ye, Ph.D., an instructor of medicine at the Johns Hopkins University School of Medicine.

“This is one of the first studies to detail the use of CRISPR in human iPSCs, showcasing its potential in these cells.”

CRISPR originated from a microbial immune system that contains DNA segments known as clustered regularly interspaced short palindromic repeats. The engineered editing system makes use of an enzyme that nicks together DNA with a piece of small RNA that guides the tool to where researchers want to introduce cuts or other changes in the genome.

Previous research has shown that CRISPR can generate genomic changes or mutations through these interventions far more efficiently than other gene editing techniques, such as TALEN, short for transcription activator-like effector nuclease.

Despite CRISPR’s advantages, a recent study suggested that it might also produce a large number of “off-target” effects in human cancer cell lines, specifically modification of genes that researchers didn’t mean to change.

To see if this unwanted effect occurred in other human cell types, Ye, Linzhao Cheng, Ph.D., a professor of medicine and oncology in the Johns Hopkins University School of Medicine; and their colleagues pitted CRISPR against TALEN in human iPSCs, adult cells reprogrammed to act like embryonic stem cells. Human iPSCs have already shown enormous promise for treating and studying disease.

The researchers compared the ability of both genome editing systems to either cut out pieces of known genes in iPSCs or cut out a piece of these genes and replace it with another. As model genes, the researchers used JAK2, a gene that when mutated causes a bone marrow disorder known as polycythemia vera; SERPINA1, a gene that when mutated causes alpha1-antitrypsin deficiency, an inherited disorder that may cause lung and liver disease; and AAVS1, a gene that’s been recently discovered to be a “safe harbour” in the human genome for inserting foreign genes.

Their comparison found that when simply cutting out portions of genes, the CRISPR system was significantly more efficient than TALEN in all three gene systems, inducing up to 100 times more cuts. However, when using these genome editing tools for replacing portions of the genes, such as the disease-causing mutations in JAK2 and SERPINA1 genes, CRISPR and TALEN showed about the same efficiency in patient-derived iPSCs, the researchers report.

Contrary to results of the human cancer cell line study, both CRISPR and TALEN had the same targeting specificity in human iPSCs, hitting only the genes they were designed to affect, the team says. The researchers also found that the CRISPR system has an advantage over TALEN: It can be designed to target only the mutation-containing gene without affecting the healthy gene in patients, where only one copy of a gene is affected.

The findings, together with a related study that was published earlier in a leading journal of stem cell research (Cell Stem Cell), offer reassurance that CRISPR will be a useful tool for editing the genes of human iPSCs with little risk of off-target effects, say Ye and Cheng.

“CRISPR-mediated genome editing opens the door to many genetic applications in biologically relevant cells that can lead to better understanding of and potential cures for human diseases,” says Cheng.

Source: Johns Hopkins Medicine

Contact: Marin Hedin

Reference:

Efficient and Allele-Specific Genome Editing of Disease Loci in Human iPSCs

Cory Smith, Leire Abalde-Atristain, Chaoxia He, Brett R Brodsky, Evan M Braunstein, Pooja Chaudhari, Yoon-Young Jang, Linzhao Cheng and Zhaohui Ye

Molecular Therapy, December 16, 2014; doi:10.1038/mt.2014.226

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Enhancing RNA Interference

Enhancing RNA Interference

Tuesday, 25 June 2013

Nanoparticles that deliver short strands of RNA offer a way to treat cancer and other diseases by shutting off malfunctioning genes. Although this approach has shown some promise, scientists are still not sure exactly what happens to the nanoparticles once they get inside their target cells.

A new study from MIT sheds light on the nanoparticles' fate and suggests new ways to maximize delivery of the RNA strands they are carrying, known as short interfering RNA (siRNA).

Lipid nanoparticles (carrying siRNA) are 

shown as they are transported inside cells 

using endocytic vesicles. Credit: Daria 

Alakhova and Gaurav Sahay. 

"We've been able to develop nanoparticles that can deliver payloads into cells, but we didn't really understand how they do it," says Daniel Anderson, the Samuel Goldblith Associate Professor of Chemical Engineering at MIT.

"Once you know how it works, there's potential that you can tinker with the system and make it work better."

Anderson, a member of MIT's Koch Institute for Integrative Cancer Research and MIT's Institute for Medical Engineering and Science, is the leader of a research team that set out to examine how the nanoparticles and their drug payloads are processed at a cellular and subcellular level. Their findings appear in the June 23 issue of Nature Biotechnology. Robert Langer, the David H. Koch Institute Professor at MIT, is also an author of the paper.

One RNA-delivery approach that has shown particular promise is packaging the strands with a lipid like material; similar particles are now in clinical development for liver cancer and other diseases.

Through a process called RNA interference, siRNA targets messenger RNA (mRNA), which carries genetic instructions from a cell's DNA to the rest of the cell. When siRNA binds to mRNA, the message carried by that mRNA is destroyed. Exploiting that process could allow scientists to turn off genes that allow cancer cells to grow unchecked.

Scientists already knew that siRNA-carrying nanoparticles enter cells through a process, called endocytosis, by which cells engulf large molecules. The MIT team found that once the nanoparticles enter cells they become trapped in bubbles known as endocytic vesicles. This prevents most of the siRNA from reaching its target mRNA, which is located in the cell's cytosol (the main body of the cell).

This happens even with the most effective siRNA delivery materials, suggesting that there is a lot of room to improve the delivery rate, Anderson says.

"We believe that these particles can be made more efficient. They're already very efficient, to the point where micrograms of drug per kilogram of animal can work, but these types of studies give us clues as to how to improve performance," Anderson says.

Molecular traffic jam

The researchers found that once cells absorb the lipid-RNA nanoparticles, they are broken down within about an hour and excreted from the cells.

They also identified a protein called Niemann Pick type C1 (NPC1) as one of the major factors in the nanoparticle-recycling process. Without this protein, the particles could not be excreted from the cells, giving the siRNA more time to reach its targets.

"In the absence of the NPC1, there's a traffic jam, and siRNA gets more time to escape from that traffic jam because there is a backlog," says Gaurav Sahay, an MIT postdoc and lead author of the Nature Biotechnology paper.

In studies of cells grown in the lab without NPC1, the researchers found that the level of gene silencing achieved with RNA interference was 10 to 15 times greater than that in normal cells.

Lack of NPC1 also causes a rare lysosomal storage disorder that is usually fatal in childhood. The findings suggest that patients with this disorder might benefit greatly from potential RNA interference therapy delivered by this type of nanoparticle, the researchers say. They are now planning to study the effects of knocking out the NPC1 gene on siRNA delivery in animals, with an eye toward testing possible siRNA treatments for the disorder.

The researchers are also looking for other factors involved in nanoparticle recycling that could make good targets for possibly slowing down or blocking the recycling process, which they believe could help make RNA interference drugs much more potent. Possible ways to do that could include giving a drug that interferes with nanoparticle recycling, or creating nanoparticle materials that can more effectively evade the recycling process.

Source: Massachusetts Institute of Technology 

Contact: Sarah McDonnell

Written by: Anne Trafton, MIT News Office

Reference:

Efficiency of siRNA delivery by lipid nanoparticles is limited by endocytic recycling

Gaurav Sahay, William Querbes, Christopher Alabi, Ahmed Eltoukhy, Sovan Sarkar, Christopher Zurenko, Emmanouil Karagiannis, Kevin Love, Delai Chen, Roberto Zoncu, Yosef Buganim, Avi Schroeder, Robert Langer & Daniel G Anderson

Nature Biotechnology (2013), doi:10.1038/nbt.2614

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Gene Therapy Reprograms Scar Tissue in Damaged Hearts into Healthy Heart Muscle

Gene Therapy Reprograms Scar Tissue in Damaged Hearts into Healthy Heart Muscle

Saturday, 05 January 2013

A cocktail of three specific genes can reprogram cells in the scars caused by heart attacks into functioning muscle cells, and the addition of a gene that stimulates the growth of blood vessels enhances that effect, said researchers from Weill Cornell Medical College, Baylor College of Medicine and Stony Brook University Medical Center in a report that appears online in the Journal of the American Heart Association.

"The idea of reprogramming scar tissue in the heart into functioning heart muscle was exciting," said Dr. Todd K. Rosengart, chair of the Michael E. DeBakey Department of Surgery at BCM and the report's corresponding author.

"The theory is that if you have a big heart attack, your doctor can just inject these three genes into the scar tissue during surgery and change it back into heart muscle. However, in these animal studies, we found that even the effect is enhanced when combined with the VEGF gene."

"This experiment is a proof of principle," said Dr. Ronald G. Crystal, chairman and professor of genetic medicine at Weill Cornell Medical College and a pioneer in gene therapy, who played an important role in the research.

"Now we need to go further to understand the activity of these genes and determine if they are effective in even larger hearts."

During a heart attack, blood supply is cut off to the heart, resulting in the death of heart muscle. The damage leaves behind a scar and a much weakened heart. Eventually, most people who have had serious heart attacks will develop heart failure.

Changing the scar into heart muscle would strengthen the heart. To accomplish this, during surgery, Rosengart and his colleagues transferred three forms of the vascular endothelial growth factor (VEGF) gene that enhances blood vessel growth or an inactive material (both attached to a gene vector) into the hearts of rats. Three weeks later, the rats received either Gata4, Mef 2c and Tbx5 (the cocktail of transcription factor genes called GMT) or an inactive material. (A transcription factor binds to specific DNA sequences and starts the process that translates the genetic information into a protein.)

The GMT genes alone reduced the amount of scar tissue by half compared to animals that did not receive the genes, and there were more heart muscle cells in the animals that were treated with GMT. The hearts of animals that received GMT alone also worked better as defined by ejection fraction than those who had not received genes. (Ejection fraction refers to the percentage of blood that is pumped out of a filled ventricle or pumping chamber of the heart.)

The hearts of the animals that had received both the GMT and the VEGF gene transfers had an ejection fraction four times greater than that of the animals that had received only the GMT transfer.

Rosengart emphasizes that more work needs to be completed to show that the effect of the VEGF is real, but it has real promise as part of a new treatment for heart attack that would minimize heart damage.

"We have shown both that GMT can effect change that enhances the activity of the heart and that the VEGF gene is effective in improving heart function even more," said Dr. Crystal.

The idea started with the notion of induced pluripotent stem cells – reprograming mature specialized cells into stem cells that are immature and can differentiate into different specific cells needed in the body. Dr. Shinya Yamanaka and Sir John B. Gurdon received the Nobel Prize in Medicine and Physiology for their work toward this goal this year.

However, use of induced pluripotent stem cells has the potential to cause tumors. To get around that, researchers in Dallas and San Francisco used the GMT cocktail to reprogram the scar cells into cardiomyocytes (cells that become heart muscle) in the living animals.

Now Rosengart and his colleagues have gone a step farther – encouraging the production of new blood vessels to provide circulation to the new cells.

Source: Weill Cornell Medical College 

Contact: Lauren Woods

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Researchers Print Live Cells with a Standard Inkjet Printer

Researchers Print Live Cells with a Standard Inkjet Printer
Monday, 19 March 2012

A fibroblast printed with the modified
inkjet printer. The interior of the cell
shows that the fluorescently tagged
actin monomers have been incorporated.

Credit: © The Journal of Visualized

Experiments. To watch the full video

article, please click here.

Researchers from Clemson University have found a way to create temporary holes in the membranes of live cells using a standard inkjet printer. The method will be published in JoVE, the Journal of Visualized Experiments, on March 16.

"We first had the idea for this method when we wanted to be able to visualize changes in the cytoskeleton arrangement due to applied forces on cells," said paper-author Dr. Delphine Dean.

She said other researchers have been using this method to print cells onto slides, but that they have only recently discovered that printing the cells causes the disruption in their membranes for a few hours. Creating temporary pores allow researchers to put molecules inside of cells that wouldn't otherwise fit, and study how the cells react.

"The authors have used an extremely innovative approach for bio-printing cells. Moreover, this approach can be used for applications other than cell printing," said JoVE Science Editor, Dr. Nandita Singh.

"Matrix proteins can be printed onto substrates with this technique for cell patterning. This JoVE publication will make this approach simple and approachable and enable other labs to replicate the procedure."

The printer is modified by removing the paper feed mechanism and adding a "stage" from which to feed the slides. The ink is replaced with a cell solution, and the cells are printed directly on to the slides.

Using this method, the researchers are able to process thousands of cells in a matter of minutes. Dr. Dean's team used the holes to introduce fluorescent molecules that illuminate the skeleton of the cell.

"We are actually interested in the cell mechanics of compressed cells. This method allows us to push on the cells and watch the response easily," said Dr. Dean.

"We are interested in cardiovascular cells, and how they respond to mechanical force."

Dr. Dean chose to submit her method to JoVE, the only peer reviewed, PubMed-indexed science journal to publish all of its content in both text and video format, because, according to her, "until you've seen it done, it's hard to understand the process."

Source: The Journal of Visualized Experiments

Contact: Katherine Scott

Reference:

Creating Transient Cell Membrane Pores Using a Standard Inkjet Printer
Owczarczak, A. B., Shuford, S. O., Wood, S. T., Deitch, S., Dean, D.
J. Vis. Exp. (61), e3681, DOI: 10.3791/3681 (2012)

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Embryonic Stem Cells Help Deliver 'Good Genes' in a Model of Inherited Blood Disorder

Embryonic Stem Cells Help Deliver 'Good Genes' in a Model of Inherited Blood Disorder
Thursday, 20 January 2011

Researchers at Nationwide Children's Hospital report a gene therapy strategy that improves the condition of a mouse model of an inherited blood disorder, Beta Thalassaemia. The gene correction involves using unfertilized eggs from afflicted mice to produce a batch of embryonic stem cell lines. Some of these stem cell lines do not inherit the disease gene and can thus be used for transplantation-based treatments of the same mice. Findings could hold promise for a new treatment strategy for autosomal dominant diseases like certain forms of Beta Thalassaemia, tuberous sclerosis or Huntington's disease.

Embryonic stem cells have the potential to produce unlimited quantities of any cell type and are therefore being explored as a new therapeutic option for many diseases. Unfertilized eggs can be cultured to form embryonic stem cells, so-called parthenogenetic embryonic stem cells.

"Parthenogenetic embryonic stem cells can differentiate into multiple tissue types as do stem cells from fertilized embryos," said K. John McLaughlin, PhD, principal investigator in the Center for Molecular and Human Genetics at The Research Institute at Nationwide Children's Hospital, Columbus, OH. Previously, the group demonstrated that blood cells derived from parthenogenetic cells could provide healthy, long-term blood replacement in mice.

"Advantages of parthenogenetic stem cells are not only that fertilization is not needed, but also that the recipient's immune system may potentially not view them as foreign, minimizing rejection problems. Furthermore, since parthenogenetic embryonic stem cells are derived from reproductive cells which contain only a single set of the genetic information instead of the double set present in body cells, they may not contain certain abnormal genes present in the other copy," said Dr. McLaughlin also one of the study authors.

A single copy of an abnormal gene inherited from one parent can cause so-called autosomal dominant diseases such as tuberous sclerosis or Huntington's disease. The affected person has one defective and one normal copy of the gene, but the abnormal gene overrides the normal gene, causing disease. In normal sexual reproduction, each parent provides one gene copy to offspring via their reproductive cells. Therefore, the reproductive cells of a patient with an autosomal dominant disease could either pass along a defective copy or a normal copy.

"As the donor patient has one defective gene copy and one normal, and only one copy is used for normal reproduction, we can select egg-cell-derived embryonic stem cells with two normal copies," said Dr. McLaughlin.

"These single-parent/patient-derived embryonic stem cells can theoretically be used for correction of a diverse number of diseases that occur when one copy of the gene is abnormal," said Dr. McLaughlin.

To test this theory, Dr. McLaughlin and colleagues from the University of Pennsylvania, University of North Carolina and University of Minnesota, examined whether parthenogenetic embryonic stem cells could be used for tissue repair in a mouse model of thalassaemia intermedia. Thalassaemia intermedia is an inherited blood disorder in which the body lacks sufficient normal haemoglobin, leading to excessive destruction of red blood cells and anaemia. They used a mouse model in which one defective gene copy causes anaemia.

Using approaches developed from a previous study done by this group, Nationwide Children's Research Fellow Sigrid Eckardt, PhD, derived embryonic stem cells from the unfertilized eggs of female mice with the disease, and identified those stem cell lines that contained only the "healthy" haemoglobin genes. These "genetically clean" embryonic stem cell lines were converted into cells that were transplanted into afflicted mice that were carriers of the disease-causing gene. Blood samples drawn five weeks after transplantation revealed that the delivered cells were present in the recipients' blood. Their red blood cells were also corrected to a size similar to normal mice and red blood cell count, hematocrit and haemoglobin levels became normal.

"Overall, we observed long-term improvement of thalassaemia in this model," said Dr. Eckardt.

"Our findings suggest that using reproductive cells to generate embryonic stem cells that are 'disease-free' may be a solution for genetic diseases involving large, complex or poorly identified deletions in the genome or that are not treatable by current gene therapy approaches."

Dr. McLaughlin says that this approach also contrasts with typical gene therapy approaches in that it requires no engineering of the genome, which is currently difficult to achieve in human embryonic and embryonic-like (IPS) stem cells.

Source: Nationwide Children's Hospital
Contact: Erin Pope
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Engineered Stem Cells May Limit Heart Attack Damage, Improve Function

Engineered Stem Cells May Limit Heart Attack Damage, Improve Function
Wednesday, 21 July 2010

Implanting tiny plastic scaffolds seeded with genetically engineered stem cells reduced organ damage and led to better cardiac function after a heart attack, according to an animal study presented at the American Heart Association's Basic Cardiovascular Sciences 2010 Scientific Sessions – Technological and Conceptual Advances in Cardiovascular Disease.

The study was designed to help determine what role cytokines – substances secreted by cells that have an effect on other cells – might play following a heart attack, said lead Matthias Siepe, M.D., lead author, assistant professor and staff surgeon at the Department of Cardiovascular Surgery, Medical University Center in Freiburg, Germany.

The researchers implanted five groups of 10 rats each with tiny polyurethane scaffolds seeded with different genetically engineered stem cells. Three groups received cells that overproduced one of three cytokines: hepatocyte growth-factor (HGF), stromal cell-derived factor 1 (SDF-1) or vascular endothelial growth factor (VEGF); one group received a gene called Akt1 associated with several cytokine pathways, and the fifth group received scaffolds seeded with unmodified stem cells, Siepe said. Five more groups were injected with the same types of modified and unmodified stem cells without the plastic scaffolding. An 11th group, the control group, received a sham operation, he said. A sham procedure is similar but omits a key therapeutic element of the treatment or procedure under investigation.

During six weeks of follow-up, the researchers observed significant improvements in blood pressure function in the rats implanted with scaffolds seeded with stem cells modified to overproduce Akt1, SDF-1 and HGF. There was no functional change in the group that received scaffolds containing VEGF-modified stem cells, he said.

In comparison, there was a decrease in blood pressure function in the control group that got the sham procedures. In addition, blood dynamics were stable in rats that received scaffolds with unmodified stem cells. In addition, two therapies – SDF-1 and Akt1 overproduction – seemed to limit cardiac damage from the heart attack, Siepe said.

Source: American Heart Association
Contact: Karen Astle
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Efficient Genetic Modification of Human Embryonic Stem Cells

Efficient Genetic Modification of Human Embryonic Stem Cells Thursday, 07 January 2010 Biologists have developed an efficient way to genetically modify human embryonic stem cells. Their approach, which uses bacterial artificial chromosomes to swap in defective copies of genes, will make possible the rapid development of stem cell lines that can both serve as models for human genetic diseases and as testbeds on which to screen potential treatments, they say. "This will help to open up the whole human embryonic stem cell field. Otherwise, there's really few efficient ways you can study genetics with them," said Yang Xu, professor of biology at the University of California, San Diego who directed the research. Xu and co-authors Hoseok Song and Sun-Ku Chung, both postdoctoral fellows in Xu's research group, describe their technique in the January 8 issue of the journal Cell Stem Cell. Most attempts to alter the genetic makeup of the cells have proved too inefficient, Xu said. His group used bacterial artificial chromosomes, or BACs, to improve the yield. BACs are synthesized circles of human DNA, which bacteria will replicate just like their own native chromosomes. Commercially available BACs can be modified within bacterial cells to insert altered copies of specific genes. Once the modified BACs are introduced into human cells, they will sometimes pair up with a matching segment of a human chromosome and swap segments of DNA, a process called homologous recombination. The advantage of using BACS to alter the genetic code in human cells comes from the long flanking sequences on either side of the modified gene, which increases the chance that the BAC with line up with native DNA in position for a swap. Other genetic approaches have been limited by shorter segments of DNA. Using BACs, the team was able to substitute modified genes in 20 percent of treated cells. Standard methods of genetic modification typically achieve modification in fewer than one percent of cells, Xu said. His group successfully transferred a defective copy of the gene p53, which suppresses cancer, into a human embryonic stem cell line. By repeating the process in a second round, they developed a line of cells in which both copies of the genes were disrupted. They also report success with a different gene, ATM, which when mutated in humans causes Ataxia-telangiectasia, a disease characterized by a host of systemic defects including increased cancer risk, degeneration of specific types of brain cells and degraded telomeres, the protective caps at the end of each chromosome. Genetically engineered mice with two bad copies of the ATM gene share some of these traits with human patients, but not all. Neurons do not degenerate in ATM mice, for example, and the telomeres are long. "If you want to study accelerated shortening of telomeres, you can't do it in the mouse. You can only do it in human cells," Xu said. Those differences propelled Xu's group to develop human cell lines instead, with the hope that some of the processes that go wrong in human patients could be studied in the lab. Already, they have demonstrated that their ATM-deficient embryonic stem cell line has damaged telomeres. Other characteristics, such as the degeneration of specific types of neurons, will be the subject of future experiments, Xu said. The authors say their approach can easily be adapted to modify other human genes within other stem cells lines. For their initial work, Xu's group used a cell line that easily forms new colonies from single cells, but they also repeated the procedure in a cell line called H9, which has proved difficult to manipulate. Because H9 was among the few cells lines approved for use by researchers funded by the federal government before new lines began to be approved in mid-December 2009, many researchers already have considerable experience with coaxing the cells into differentiating into specific types of tissues, for example, which would make the ability to genetically modify them particularly valuable. Reference: Modeling Disease in Human ESCs Using an Efficient BAC-Based Homologous Recombination System Hoseok Song, Sun-Ku Chung, Yang Xu Cell Stem Cell, Volume 6, Issue 1, 80-89, 8 January 2010, 10.1016/j.stem.2009.11.016 ......... ZenMaster

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Gene Therapy and Stem Cells save Limb

Gene Therapy and Stem Cells save Limb Wednesday, 09 December 2009 Blood vessel blockage, a common condition in old age or diabetes, leads to low blood flow and results in low oxygen, which can kill cells and tissues. Such blockages can require amputation resulting in loss of limbs. Now, using mice as their model, researchers at Johns Hopkins have developed therapies that increase blood flow, improve movement and decrease tissue death and the need for amputation. The findings, published online last week in the early edition of the Proceedings of the National Academy of Sciences, hold promise for developing clinical therapies. "In a young, healthy individual, hypoxia — low oxygen levels — triggers the body to make factors that help coordinate the growth of new blood vessels but this process doesn't work as well as we age," says Gregg Semenza, M.D., Ph.D., professor of paediatrics and genetic medicine and director of the vascular biology program at the Johns Hopkins Institute for Cell Engineering. "Now, with the help of gene therapy and stem cells we can help reactivate the body's response to hypoxia and save limbs." Previously, Semenza's team generated a virus that carries the gene encoding an active form of the HIF-1 protein, which turns on genes necessary for building new blood vessels. When injected into the hind legs of otherwise healthy mice and rabbits that had been treated to reduce blood flow, the HIF-1 virus treatment partially restored blood flow. People with diabetes have a 40 times higher risk of losing a limb to amputation, says Semenza. To find out if HIF-1 gene therapy could improve blood flow in a diabetic animal, the team then tested the same virus in diabetic and non-diabetic mice that had blood flow cut off to one hind leg. Twenty-one days after treatment, the HIF-1 virus-treated mice had 85 percent recovery of blood flow compared with 24 percent in the mock-treated mice. In addition, treated, diabetic mice had much less tissue damage compared to the untreated diabetic mice. These results were reported in the Nov. 3 issue of the Proceedings of the National Academy of Sciences. In the current study, the team asked if the same gene therapy treatment could improve reduced blood flow associated with advanced age. Comparing 13-month-old mice to 3-month-old mice, blocking the femoral artery in the hind leg causes all older mice to lose their legs while only about a third of younger mice have to lose their legs. The research team treated young and old mice with the HIF-1 virus and examined blood flow and tissue health. They found that while treatment improved young mice, it did not make a noticeable difference in the older mice. However, it was known that when HIF-1 normally activates signals in the body to build new vessels, one of the many types of cells recruited to the site of new vessel growth is a population of stem cells from the bone marrow, which are called bone marrow-derived angiogenic cells. Therefore, the team isolated these cells from mice and grew them under special conditions that would turn on HIF-1 in these cells. When the researchers treated the mice with both the HIF-1 virus and simultaneously injected bone marrow-derived angiogenic cells, treated, older mice were less likely to lose their legs compared to their untreated counterparts. Further study of these mice showed that activating HIF-1 in the cells appeared to turn on a number of genes that help these cells not only home to the ischemic limb, but to stay there once they arrive. To figure out how the cells stay where they're needed, the research team built a tiny micro-fluidic chamber and tested the cells' ability to stay stuck with fluid flowing around them at rates mimicking the flow of blood through vessels in the body. They found that cells under low oxygen conditions were better able to stay stuck only if those same cells had HIF-1 turned on. "Our results are promising because they show that a combination of gene and cell therapy can improve the outcome in the case of critical limb ischemia associated with aging or diabetes," says Semenza. "And that's critical for bringing such treatment to the clinic." ......... ZenMaster

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Cure for Colour Blindness in Monkeys

Gene therapy used to treat adult vision disorders involving cone cells Wednesday, 16 September 2009 Researchers from the University of Washington and the University of Florida used gene therapy to cure two squirrel monkeys of colour blindness — the most common genetic disorder in people. Writing online Wednesday in the journal Nature, scientists cast a rosy light on the potential for gene therapy to treat adult vision disorders involving cone cells — the most important cells for vision in people. "We've added red sensitivity to cone cells in animals that are born with a condition that is exactly like human colour blindness," said William W. Hauswirth, Ph.D., a professor of ophthalmic molecular genetics at the UF College of Medicine and a member of the UF Genetics Institute and the Powell Gene Therapy Center. "Although colour blindness is only moderately life-altering, we've shown we can cure a cone disease in a primate, and that it can be done very safely. That's extremely encouraging for the development of therapies for human cone diseases that really are blinding." The finding is also likely to intrigue millions of people around the world who are colour-blind, including about 3.5 million people in the United States, more than 13 million in India and more than 16 million in China. The problem mostly affects men, leaving about 8 percent of Caucasian men in the United States incapable of discerning red and green hues that are important for everyday things like recognizing traffic lights. "People who are colour-blind feel that they are missing out," said Jay Neitz, Ph.D., a professor of ophthalmology at the University of Washington. "If we could find a way to do this with complete safety in human eyes, as we did with monkeys, I think there would be a lot of people who would want it. Beyond that, we hope this technology will be useful in correcting lots of different vision disorders." The discovery comes about 10 years after Neitz and his wife Maureen Neitz, Ph.D., a professor of ophthalmology at the University of Washington and senior author of the study, began training two squirrel monkeys named Dalton and Sam. In addition to teaching the animals, the Neitz research group worked with the makers of a standard vision-testing technique called the Cambridge Colour Test to perfect a way the monkeys could "tell" them which colours they were seeing. The tests are similar to ones given to elementary children the world over, in which students are asked to identify a specific pattern of coloured dots among a field of dots that vary in size, colour and intensity. The researchers devised a computer touch screen the monkeys could use to trace the colour patterns. When the animals chose correctly, they received a reward of grape juice. Likewise, decades were spent by Hauswirth and colleagues at the University of Florida, to develop the gene-transfer technique that uses a harmless adeno-associated virus to deliver corrective genes to produce a desired protein. In this case, researchers wanted to produce a substance called long-wavelength opsin in the retinas of the monkeys. This particular form of opsin is a colourless protein that works in the retina to make pigments that are sensitive to red and green. "We used human DNA’s, so we won't have to switch to human genes as we move toward clinical treatments," said Hauswirth, who is also involved in a clinical trial with human patients to test gene therapy for the treatment of Leber congenital amaurosis, a form of blindness that strikes children. About five weeks after the treatment, the monkeys began to acquire colour vision, almost as if it occurred overnight. "Nothing happened for the first 20 weeks," Neitz said. "But we knew right away when it began to work. It was if they woke up and saw these new colours. The treated animals unquestionably responded to colours that had been invisible to them." It took more than a year and a half to test the monkeys' ability to discern 16 hues, with some of the hues varying as much as 11-fold in intensity. Dalton is named for John Dalton, an English chemist who realized he was colour-blind and published the first paper about the condition in 1798. "We've had Dalton and Sam for 10 years. They are like our children," Neitz said. "This species are friendly, docile monkeys that we just love. We think it is useful to continue to follow them — it's been two years now that they've been seeing in colour, and continuing to check their vision and allowing them to play with the computer is part of their enrichment." With the discovery, the researchers are the first to address a vision disorder in primates in which all photoreceptors is intact and healthy, providing a hint of gene therapy's full potential to restore vision. About 1 in 30,000 Americans have a hereditary form of blindness called achromatopsia, which causes nearly complete colour blindness and extremely poor central vision. "Those patients would be targets for almost exactly the same treatment," Hauswirth said. Even in common types of blindness such as age-related macular degeneration and diabetic retinopathy, vision could potentially be rescued by targeting cone cells, he said. "The major thrust of the study is you can ameliorate if not cure colour blindness with gene therapy," said Gerald H. Jacobs, Ph.D., a research professor of psychology at the University of California, Santa Barbara, who was not involved in the research. "There are still questions about safety, but in these monkeys at least, there were no untoward effects. Those who are motivated to ameliorate their colour defect might take some hope from the findings.” "This is also another example of how utterly plastic the visual system is to change," Jacobs said. "The nervous system can extract information from alterations to photo-pigments and make use of it almost instantaneously." Reference: Gene therapy for red–green colour blindness in adult primates Katherine Mancuso, William W. Hauswirth, Qiuhong Li, Thomas B. Connor, James A. Kuchenbecker, Matthew C. Mauck, Jay Neitz & Maureen Neitz Nature advance online publication 16 September 2009, doi:10.1038/nature08401 ......... ZenMaster

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Mitochondrial Gene Replacement in Primate Offspring and Embryonic Stem Cells

Breakthrough could help break the chain of several maternally-based diseases passed from generation to generation 
Wednesday, 26 August 2009 

 Researchers at Oregon Health & Science University's Oregon National Primate Research Center (ONPRC) believe they have developed one of the first forms of genetic therapy – a therapy aimed at preventing serious diseases in unborn children. Specifically, the therapy would combat inherited diseases passed on from mothers to their children through mutated DNA in cell mitochondria. The research is published in the Aug. 26 advance online edition of the journal Nature. 

"We believe this discovery in nonhuman primates can rapidly be translated into human therapies aimed at preventing inherited disorders passed from mothers to their children through the mitochondrial DNA, such as certain forms of cancer, diabetes, infertility, myopathies and neurodegenerative diseases," explained Shoukhrat Mitalipov, Ph.D.. Dr. Mitalipov is an associate scientist in the Division of Reproductive Sciences at ONPRC, the Oregon Stem Cell Center and the departments of Obstetrics and Gynecology and Molecular & Medical Genetics of Oregon Health & Science University (OHSU). 

"Currently there are 150 known diseases caused by mutations of the mitochondrial DNA, and approximately 1 out of every 200 children is born with mitochondrial mutations." 

 Mitochondria are structures that are found in all cells that provide energy for cell growth and metabolism, which is why they are often called the cell's "power plant." The structures produce energy to power each individual cell. Mitochondria also carry their own genetic material. When an egg cell is fertilized by a sperm cell during reproduction, the embryo almost exclusively inherits the maternal mitochondria present in the egg. This means that any disease-causing genetic mutations that a mother carries in her mitochondrial DNA can be passed on to her offspring. 

The method developed by OHSU researchers transfers the mother's chromosomes to a donated egg that has had its chromosomes removed, but which has healthy mitochondria, thereby preventing the disease from being passed on to one's offspring. 

How the OHSU researchers' method works 
Scientists collected groups of unfertilized eggs from two female rhesus macaque monkeys (monkeys A and B). They then removed the chromosomes, which contain the genes found in the cell nucleus, from the eggs of monkey B, and then transplanted the nuclear genes from the eggs of monkey A into the eggs of monkey B. Then the eggs from monkey B, which now contained their own mitochondria but monkey A's nuclear genes, were fertilized. The fertilized eggs developed into embryos that were implanted in surrogate monkeys. The initial implantation of two embryos resulted in the birth of healthy twin monkeys, nicknamed "Mito" and "Tracker" (in reference to the procedure used for imaging of mitochondria). These monkeys are the world's first animals derived by spindle transfer. 

Follow-up testing showed that there was little to no trace of cross-animal mitochondrial transfer using this procedure. This demonstrates that the researchers were successful in isolating nuclear genetic material from mitochondrial genetic material during the transfer process. 

"In theory, this research has demonstrated that it is possible to use this therapy in mothers carrying mitochondrial DNA diseases so that we can prevent those diseases from being passed on to their offspring," added Mitalipov. 

"We believe that with the proper governmental approvals, our work can rapidly be translated into clinical trials for humans, and, eventually, approved therapies." "This breakthrough is an excellent example of how OHSU's research findings can often be rapidly translated into health therapies that benefit residents of our state and the country as a whole," said Dr. Joe Robertson, M.D., M.B.A., president of OHSU. 

"Recent findings suggest that mitochondrial disorders play a role in at least some proportion of many human disorders," said Duane Alexander, M.D, director of the Eunice Kennedy Shriver National Institute of Child Health and Human Development, which provided funding for the study. 

"Pending further research, the findings hold the potential of allowing a couple to have a child who is biologically their own, but is free of any conditions associated with defects in maternal mitochondria." 

Using the technique, the researchers created fertilized eggs and achieved three successful pregnancies in rhesus monkeys, which have resulted in four healthy newborns. Recent advances in the transfer of hereditary material and in microscopy facilitated the achievement, they wrote. The researchers said that the technique did not appear to pose any risk of chromosomal damage. Analysis of 5-6-day-old embryos (blastocysts) resulting from the fertilized eggs, and of embryonic stem cell lines established from them, did not uncover any evidence of damage to the chromosomes. Analysis of cells from the infant monkeys born after the procedure failed to detect any mitochondrial DNA from the mother. 

Reference: 
Mitochondrial gene replacement in primate offspring and embryonic stem cells 
Masahito Tachibana, Michelle Sparman, Hathaitip Sritanaudomchai, Hong Ma, Lisa Clepper, Joy Woodward, Ying Li, Cathy Ramsey, Olena Kolotushkina & Shoukhrat Mitalipov 
Nature advance online publication 26 August 2009, doi:10.1038/nature08368

See also: 
DNA swap could avoid inherited diseases 
David Cyranoski 
Nature News 26 August 2009, doi:10.1038/news.2009.860 
......... 

ZenMaster

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Technique Enables Efficient Gene Splicing in Human Embryonic Stem Cells

Technique Enables Efficient Gene Splicing in Human Embryonic Stem Cells Thursday, 13 August 2009 A novel technique allows researchers to efficiently and precisely modify or introduce genes into the genomes of human embryonic stem cells (ESCs) and induced pluripotent stem (iPS) cells, according to Whitehead scientists. The method uses proteins called zinc finger nucleases and is described in the August 13 issue of Nature Biotechnology. For years, scientists have easily swapped genes in and out of mouse ESC or iPS cell genomes, but have had a notoriously difficult time disrupting or inserting genes into their human equivalents. "It's not clear where this hurdle of genetic manipulation lies; it could be purely technical, but it could also be an inherent difference between human and mouse cells," says Dirk Hockemeyer. Hockemeyer and Frank Soldner are first authors on the article and postdoctoral researchers in Whitehead Member Rudolf Jaenisch’s lab. "Other people have genetically manipulated these human cells, but the process has been extremely laborious and extremely time consuming. Using the zinc finger nucleases makes the process very easy," says Hockemeyer. Earlier methods are so inefficient that fewer than 15 genes have been swapped into human ESCs since that cell type was discovered 10 years ago. By comparison, hundreds of genes have been introduced into the genomes of mouse ESCs. According to Jaenisch, this method could open a new phase in human genetics. "This is a proof of principle that zinc finger nucleases can be used to swap out many, many additional genes in human ESCs and iPS cells," says Jaenisch, who is also a professor of biology at MIT. "Now human ESC and iPS cell genetics can catch up to mouse genetics, which has had a 20-year head start." The inability to alter human ESC and iPS cells' genomes has hindered researchers from routinely creating specific cell types for modelling genetic diseases (e.g., the brain cells affected by Parkinson’s disease) and studying how embryonic stem cells mature into adult cells. (iPS cells are adult cells that have been reprogrammed to an embryonic-stem-cell-like state, so they have similar properties of ESCs: the ability to self-propagate and the ability to mature into any of an adult's approximately 220 cell types. iPS cells have the added benefit of possessing the same genes as the patient who donated the adult cells, thereby accurately reflecting that patient's specific genetic profile.) To substitute a gene in ESCs and iPS cells, Hockemeyer and Soldner adapted a recently developed technique to cut out one gene from the human ESCs and iPS cells and substitute it with another by putting two zinc finger nucleases and the replacement gene into the ESCs and iPS cells. Each zinc finger nuclease recognizes a particular sequence in a cell's DNA and then cuts through both strands of DNA at that site. The cell's DNA repair machinery recognizes that the DNA has been cut and tries to fix it using the replacement gene resulting in the desired alteration of the original gene. In addition to working so efficiently, the method can be tailored to precisely swap nearly any gene in the genome. "We can produce zinc finger nucleases that out of about three billion DNA base pairs can identify one specific site," says Soldner. "We also spent quite a bit of energy to see if the zinc finger nucleases cut somewhere other than the intended target site, and it was very unlikely." Reference: Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases Dirk Hockemeyer, Frank Soldner, Caroline Beard, Qing Gao, Maisam Mitalipova, Russell C. DeKelver, George E. Katibah, Ranier Amora, Elizabeth A. Boydston, Bryan Zeitler, Xiangdong Meng, Jeffrey C. Miller, Lei Zhang, Edward J. Rebar, Philip D. Gregory, Fyodor D. Urnov and Rudolf Jaenisch Nature Biotechnology, Published online: 13 August 2009, doi:10.1038/nbt.1562 ......... ZenMaster

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Researchers Edit Genes in Human Stem Cells

Researchers Edit Genes in Human Stem Cells Thursday, 18 June 2009 Researchers at the Johns Hopkins School of Medicine have successfully edited the genome of human- induced pluripotent stem cells, making possible the future development of patient-specific stem cell therapies. Reporting this week in Cell Stem Cell, the team altered a gene responsible for causing the rare blood disease paroxysmal nocturnal haemoglobinuria, or PNH, establishing for the first time a useful system to learn more about the disease. "To date, only about six genes have been successfully targeted or edited in human stem cells out of countless people and attempts — that's just not efficient enough if we want to move disease research and therapy forward," says Linzhao Cheng, Ph.D., an associate professor of gynaecology and obstetrics and member of the Johns Hopkins Institute of Cell Engineering. "We've been able to improve gene targeting and editing in human embryonic stem cells more than 200 fold." Cheng's lab and collaborators at Johns Hopkins study PNH, a condition where "friendly fire" kills patients' own blood cells and the body can't replenish the lost blood cells due to loss of normal blood stem cells. PNH is an acquired disease that occurs only in adults, according to Cheng. "It's a tough condition to study because we need to study it in blood stem cells and they're difficult to grow in the lab. So for years we've been trying to develop another cell system to better understand and perhaps fix what's going on in PNH." To establish a system for research, they used human embryonic stem cells which can be expanded unlimitedly in the laboratory, but they also had to create a mutation as found in a PNH patient. To target and remove the function of the one specific gene known to cause PNH, the research team improved on the standard approach of gene targeting, which can remove a functional gene or replace a dysfunctional gene. The gene targeting technology, first used successfully for mouse embryonic stem cells, won a Nobel Prize in Physiology or Medicine in 2007. Gene targeting exploits a cell's own ability to repair broken DNA. When DNA breaks from exposure to mutagens or other agents like DNA-cutting enzymes, DNA-repairing enzymes in the cell find and re-join the two exposed DNA ends. However, if another piece of DNA with exposed ends is floating around, it effectively can be spliced into the broken DNA during repair, and replace the defective copy. The team's technological improvement includes the use of custom-designed molecular scissors that are made by collaborators at Harvard University and University of Texas Southwestern Medical Center. These engineered DNA cutting enzymes make a precise break at specific locations in a cell's DNA — in this case in the gene that causes PNH. They added the molecular scissors and a fragment of DNA containing a gene that confers selection of rare targeted clones in both human embryonic stem cells and induced pluripotent stem cells. The latter, also known as iPS cells, are very similar to embryonic stem cells in biological properties, but generated by using adult tissues such as skin. Of all the cells surviving selection, they picked and grew eight iPS cell lines to study further, and five of those contained a targeted insertion at the gene site. Further examination showed that the cells contained the correct number of chromosomes, no longer contained any trace of the molecular scissors and had characteristics as cells from PNH patients that lack a group of cell surface molecules. "I commend my team, especially Dr. Jizhong Zou who spent three years with the help of many collaborators on this challenging project," says Cheng. "We're very excited about this accomplishment; it will enable better studies for other blood diseases. But there's still much to do before we can really use human iPS cells in clinical therapies." Cheng's team will continue to improve on techniques and begin applying these techniques to iPS cells from patients. Reference: Gene Targeting of a Disease-Related Gene in Human Induced Pluripotent Stem and Embryonic Stem Cells Jizhong Zou, Morgan L. Maeder, Prashant Mali, Shondra M. Pruett-Miller, Stacey Thibodeau-Beganny, Bin-Kuan Chou, Guibin Chen, Zhaohui Ye, In-Hyun Park, George Q. Daley, Matthew H. Porteus, J. Keith Joung, and Linzhao Cheng Cell Stem Cell, 18 June 2009, doi:10.1016/j.stem.2009.05.023 ......... ZenMaster

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