Safer Retinal Gene Delivery System Developed

PEG-POD (polyethylene glycol - peptide ocular delivery) is a non-viral approach to compacting DNA and delivering it to cells in the retina. The method offers a safer genetic approach to treating several degenerative disorders of eyesight, as well as some congenital ocular disorders.

Researchers at Tufts University School of Medicine and the Sackler School of Graduate Biomedical Sciences at Tufts have developed a new tool for gene therapy that significantly increases gene delivery to cells in the retina compared to other carriers and DNA alone, according to a study published in the January issue of The Journal of Gene Medicine. The tool, a peptide called PEG-POD, provides a vehicle for therapeutic genes and may help researchers develop therapies for degenerative eye disorders such as retinitis pigmentosa and age-related macular degeneration.

"For the first time, we have demonstrated an efficient way to transfer DNA into cells without using a virus, currently the most common means of DNA delivery. Many non-viral vectors for gene therapy have been developed but few, if any, work in post-mitotic tissues such as the retina and brain. Identifying effective carriers like PEG-POD brings us closer to gene therapy to protect the retinal cells from degeneration," said senior author Rajendra Kumar-Singh, PhD, associate professor of ophthalmology and adjunct associate professor of neuroscience at Tufts University School of Medicine (TUSM) and member of the genetics; neuroscience; and cell, molecular, and developmental biology program faculties at the Sackler School of Graduate Biomedical Sciences at Tufts.

Safe and effective delivery of therapeutic genes has been a major obstacle in gene therapy research. Deactivated viruses have frequently been used, but concerns about the safety of this method have left scientists seeking new ways to get therapeutic genes into cells. _RDMag

More on the basic method

Gene delivery systems will allow for the re-programming of cells that have gone wrong, and for cells that were programmed wrong from the very beginning. In other words, gene therapies will be for all humans regardless of age.

The idea is to make human life longer, healthier, more fulfilling, and more relevant to the universe at large. The way to the next level.

Wake Up Little Mousie -- Time to Dance

“Their relentless drive is not a mood disorder,” Jones said. “There is a strong affective and emotional component to the feeling that you always want to do something. They can’t imagine doing nothing.” _ImpactLab

Some people thrive on 4 - 6 hours of sleep a night, and find the energy to stay on the go -- day after day after day. How do they do it? Chances are, they do it because they like to be going. Why? The answer may be genetic.

...a genetic variation found in people who seem to need only about six hours’ sleep—compared to the often recommended 7½ to eight hours—was put into mice to create a colony of “insomniac” rodents. Like humans with the variation, which is called DEC2, mice who received the variant gene appeared to function normally even though they got less sleep than a control group that didn’t have the DEC2 variation. _ImpactLab

Great. As if the furry little vermin weren't active enough already. Instead of giving the "stay going longer" gene to mice, how about giving it to me?

The discovery arose after a 68-year-old woman contacted Jones’ collaborators to volunteer for sleep research, telling him she had an unusually early morning wake-up time. Both the woman and her daughter go to bed between 10 and 10:30 p.m. and wake up between 4 and 4:30 in the morning. Yet, their 18-hour day does not affect their energy level or ability to function.

“The mom is very energetic and extremely active,” Jones said. “In fact, it makes me feel tired to hear about the activities she does every day.”

The woman just returned from a 50-day cruise, dances several nights a week, and plays bridge every day. Intrigued by the woman’s ability to operate on less sleep, Jones contacted colleagues at UCSF, who examined the woman’s DNA and identified the DEC2 variation.

...The researchers precisely monitored when the mice were slumbering, and then interrupted their sleep cycle to see how it would affect them. Even with less sleep, the insomniac mice were more active than a group of control mice who didn’t have the DEC2 variation. The researchers determined this by monitoring how long both groups of mice spent running in wheels inside their cages, and the insomniac group spent an average 1 ½ more hours turning the wheels than the control group.

This heightened functioning raised the question of whether the insomniac mice slept deeper than the controls. But the Stanford group monitored their sleep and found it was no deeper than that of the control group.

The study begins to shed more light on two related aspects of sleep: the biological clock that lets people sleep in harmony with the cycle of day and night and the body’s sleep homeostat—a mechanism in a different part of the brain that tracks how long people are awake and asleep. Genes such as DEC2 are found in both the homeostat and biological clock. Yet, while some of those genes work in the homeostat, they do not appear to have a function in the biological clock. _IL

This type of gene therapy may be a useful treatment for depression. Depressed people tend to either sleep too much, or sleep too little with corresponding fatigue. Being able to sleep significantly less while feeling energetic and eager to carry out normal activities, sounds like the opposite of typical depression.

Time will tell. And no doubt, there are other similar genes that interact with the body and brain's need for regular sleep.

I am not sure how well such persons could adapt to polyphasic sleep -- a method of breaking up a night's sleep into short, multiple naps throughout the day. There is a lot of research to be done into ways humans can free themselves from the chains of compulsion that rule so much of our lives.

By the way, optimal functioning on less sleep is another method of life extension. I want to work in as many effective methods as possible.

Forget Stem Cells! Switch Cell Types by Simply Changing the Messenger RNA

"What's new about this approach is that we didn't have to make the host cell pluripotent, that is the ability to develop into any of three major tissue types, we can directly convert from one cell type to another, without the intermediate step," explains Eberwine. _Physorg

This sounds much too easy to be true, so it probably won't be that simple. But something interesting is happening when you can turn a neuron into an astrocyte simply by injecting astrocytic mRNA into the neuron.

By simply flooding one cell type, a nerve cell, with the an abundance of a specific type of messenger RNA (mRNA) from another cell type, the investigators changed a neuron into an astrocyte-like cell, a star-shaped brain cell that helps to maintain the blood-brain barrier, regulates the chemical environment around cells, responds to injury, and releases regulatory substances.

James Eberwine, PhD, Elmer Holmes Bobst Professor of Pharmacology, Junhyong Kim, PhD, Edmund J. and Louise W. Kahn Term Endowed Professor of Biology and first author Jai-Yoon Sul, PhD, Assistant Professor of Pharmacology, and colleagues report their findings online this week in the Proceedings of the National Academy of Sciences. This approach offers the possibility for a new type of cell-based therapy for neurodegenerative and other diseases.

"In some ways, this is akin to what a virus does," explains Eberwine, "When a virus infects a cell it affects the host cell genome and the RNAs that it can make." By putting the RNA of one cell type, in the correct amounts, into another cell type, we were able to change its function."

"This research overturns the notion that all cells are permanently hardwired with little ability to change their physiology," notes Sul. _PO

This is just the beginning, of course. With better tools for manipulating the molecules of life inside living tissues and cells, the learning shifts into warp speed.

Soon, artificially created viruses will be performing these tasks like tiny nanobots, driving cell and tissue development in animals, plants, large-scale tissue vats, etc. It is time for biology to start getting a little respect.

Live Twice as Long: Burn the Candle at Both Ends

At least it seems to work that way for PEPCK-C genetically augmented mice. An updated study on these brave new mice reinforces earlier findings: these genetically modified mice live longer, are stronger, more assertive, and bear young at a previously unprecedented old age.

Two founder lines generated by this procedure were bred together, creating a line of mice that have 9.0 units/g skeletal muscle of PEPCK-C, as compared to 0.080 units/g in muscle from control animals. The mice were more active than controls in their cages and could run for up to 5 km, at a speed of 20 m/min without stopping (control mice run for 0.2 km at the same speed). Male PEPCK-Cmus mice are extremely aggressive, as well as hyperactive. During strenuous exercise, they use fatty acids as a fuel more efficiently than do controls and produce far less lactate than do control animals, perhaps due to the greatly increased number of mitochondria in their skeletal muscle. PEPCK-Cmus mice also store up to five-times more triglyceride in their skeletal muscle, but have only marginal amounts of triglyceride in their adipose tissue depots, despite eating 60% more than controls. The concentration of leptin and insulin the blood of 8–12 months of PEPCK-Cmus mice is far lower than noted in the blood of control animals of the same age. These mice live longer than controls and the females remain reproductively active for as long as 35 months. __Biochimie__via__Ouroborus

Remember, these mice were specially bred, twice over. They are the cross-bred offspring of two lines of mice which were genetically engineered mice for heightened PEPCK-C expression. There is no pill that you can take to achieve the same result.

But there are lessons to be learned from these super-mice. Humans live long enough so that genetic modifications can be made to them long after they are born--but long before they are due to die.

Think of what Olympic or professional athletes could do with higher levels of skeletal muscle PEPCK-C, and more muscle mitochondria. Who will be the first humans to volunteer for PEPCK-C gene therapy? Where will the human research be done first? China? Russia?

The total package would have to include higher intelligence, longer life, greater strength and speed, less need for sleep, higher resistance to infections and disease, and heightened executive function. But we have to start somewhere.

Repairing (and Building) Muscle With Stem Cells

Your muscles have stem cells too, in case of injury. But muscle stem cells can not only heal muscle injuries, they can build new muscle--if they receive the proper stimulation.

One of the key players in regeneration following injury are muscle stem cells - so called satellite cells. A critical regulator of this process is calcineurin. It is activated by injury and controls the activity of other proteins involved in stem cell differentiation and the overall response to damage.

Nadia Rosenthal, head of EMBL's Mouse Biology Unit, and her team have now found a naturally occurring version of calcineurin, called CnAß1 that is permanently active - it does not depend on whether the person is injured or not.

...The study results To test the effects of permanent CnAß1 expression Enrique Lara-Pezzi from Rosenthal's lab overexpessed CnAß1 in muscle cells, and observed increased proliferation of muscle stem cells. Switching off the protein had the opposite effect; stem cells stopped dividing and differentiated into muscle cells instead. When CnAß1 was overexpressed in the muscles of transgenic mice, the animals were resistant to the destructive effects of muscle injury and regenerated the damage more efficiently.

..."Supplementary CnAß1 also reduces the formation of scars in damaged muscle, helps speed up the resolution of inflammation and protects muscle cells from atrophy [wasting] under starvation," said Rosenthal. "These effects make CnAß1 a promising candidate for new therapeutic approaches against muscle wasting."

Source

For conventional researchers, the injury-healing effect of CnAB1 (calcineurin) is the primary objective. Researchers looking into extreme performance will go further, to find ways to first proliferate more stem cells, then differentiate them into skeletal muscle (and cardiac muscle in the heart) to augment strength and performance. This is inevitable. A pulsed expression of CnAB1 would be one approach--with research needed to identify optimal timing for various types of desired athletic performance.

Silica Nano-spheres Proving Useful in Plant Genetic Research

Iowa State researchers have developed yet another use for their porous silicon nanospheres. They are using the nanoparticles to introduce both new genes and the chemicals that trigger the gene's expression, at the same time, into plant cells.

A team of Iowa State University plant scientists and materials chemists have successfully used nanotechnology to penetrate plant cell walls and simultaneously deliver a gene and a chemical that triggers its expression with controlled precision. Their breakthrough brings nanotechnology to plant biology and agricultural biotechnology, creating a powerful new tool for targeted delivery into plant cells.

The research, "Mesoporous Silica Nanoparticles Deliver DNA and Chemicals into Plants," is a highlighted article in the May issue of Nature Nanotechnology.

...."With the mesoporous nanoparticles, we can deliver two biogenic species at the same time," Wang said. "We can bring in a gene and induce it in a controlled manner at the same time and at the same location. That's never been done before."

The controlled release will improve the ability to study gene function in plants. And in the future, scientists could use the new technology to deliver imaging agents or chemicals inside cell walls. This would provide plant biologists with a window into intracellular events.

...."The team found a chemical we could use that made the nanoparticle look yummy to the plant cells so they would swallow the particles," Torney said.

It worked. The nanoparticles were swallowed by the plant protoplasts, which are a type of spherical plant cells without cell walls.

Most plant transformation, however, occurs with the use of a gene gun, not through endocytosis. In order to use the gene gun to introduce the nanoparticles to walled plant cells, the chemists made another clever modification on the particle surface. They synthesized even smaller gold particles to cap the nanoparticles. These "golden gates" not only prevented chemical leakage, but also added weight to the nanoparticles, enabling their delivery into the plant cell with the standard gene gun.

The biologists successfully used the technology to introduce DNA and chemicals to Arabidopsis, tobacco and corn plants.

Source

The Iowa State researchers are becoming quite clever in the use of their silicon nano-spheres. This is research worth following.

One Pill to Treat 1800 Genetic Diseases?

Within the next three years, PTC Therapeutics plans to offer PTC 124 to treat over 1800 genetic diseases.

As well as offering hope of a first effective treatment for two conditions that are at present incurable, the drug has excited scientists because research suggests it should also work against more than 1,800 other genetic illnesses.

PTC124 targets a particular type of mutation that can cause very different symptoms according to the gene that is disrupted. This makes it potentially useful against a range of inherited disorders.

The same drug could be given to patients with Duchenne muscular dystrophy, the most serious form of the muscle-wasting condition, cystic fibrosis, which mainly affects the lungs, and haemophilia, in which the blood does not clot. It can be taken orally, and safety trials have not revealed any major side effects.

“There are literally thousands of genetic diseases that could benefit from this approach,” Lee Sweeney, of the University of Pennsylvania, who is leading the research, said. “What’s unique about this drug is it doesn’t just target one mutation that causes disease, but a whole class of mutations.”

....PTC124 works by binding to a part of the cell called the ribosome, which translates genetic code into protein, and allows it to ignore nonsense mutations. The gene can be read straight through and a normal protein is produced.

The beauty of the drug is that it should be useful with any disease caused by a nonsense mutation, no matter what its outward effects. The error is not corrected, but ignored. Patients would have to take the pill throughout their lives.

PTC124, which is made by PTC Therapeutics, has been staggeringly successful in animal models. A study published today in Nature shows that in mice with a nonsense mutation that causes Duchenne muscular dystrophy, the drug starts dystrophin production and restores their muscles to health.

The drug has passed safety trials in humans, and the results of phase-two trials on cystic fibrosis and Duchenne muscular dystrophy will be published shortly.

Source

This ribosome targeting approach may eventually palliate between 5% - 15% of genetic disorders. Other approaches to gene therapy would target different parts of the gene expression network. This drug reveals how many possible approaches to treating genetic conditions there must be.

Using viral and non-viral vectors to replace faulty DNA in the nucleus is the most straightforward approach. But the potential for gene therapy exists from nuclear DNA to transcription factors and enzymes to RNA in coding and noncoding forms, to proteomics, to glycomics, etc.

Pharmaceutical companies exist to make a profit. If crusading politicians are allowed to remove the profitability from the pharma business, the number of approaches taken in the laboratory to solve these disease problems will drop significantly. In other words, we will be stuck with "local optima," when better possibilities existed but were not pursued for lack of backing.

In a market, profit-driven economy, multiple approaches are taken. In a state-planned economy, singular approaches are taken. Watch your politicians closely to determine which type of economy they favour.

Special Delivery of DNA to Cells: Interesting Method

David M. Lynn and fellow engineers at UW Madison have developed an ultrathin film for DNA packaging and delivery, for use in gene therapy.

When placed in or near a body tissue, the films are designed to degrade and release the DNA. Large strands of DNA cannot normally penetrate cells, so Lynn constructs his films with special polymers designed to bundle the genes into small tight packages that cells can import. Once inside, the genes instruct the cells to make proteins.

Lynn and his colleagues create the films one layer at a time using a dip-coating method, dunking first in one solution, then another. The individual layers are so thin it would take roughly 10,000 of them to equal the thickness of a single sheet of paper.

....The researchers alternate layers of DNA with layers of a polymer that is stable when dry but that degrades when exposed to water. Because the polymers carry a positive electric charge that is attractive to DNA, each polymer layer also "primes" the surface to accept the next layer of DNA. While electrostatic forces between the layers keep the film stable in dry, room-temperature conditions, the polymers break down easily in a wet biological environment - like the inside of a patient's body.

Lynn's laboratory has engineered a whole toolbox of different polymers to fine-tune the DNA delivery properties of their films. Using the layering method, they can control the amount of DNA by adding more layers, or can even layer multiple ingredients in a specific order. Tweaking the polymer structure slightly can change how quickly the films erode and thus how long cells are exposed to the gene therapy.

Source

Given the past morbidity and mortality of using viral vectors for gene therapy, it is important to find safe and effective alternatives. Although this method lacks much of the gene insertion machinery naturally present in viruses, it represent a very safe beginning for an alternative approach.

Gene Therapy: Cancer, Heart Disease, Life Extension

Since before Crick and Watson, people have been dreaming about making changes to the human genome. First of all, watching a child die from an inherited disorder is heart-wrenching. Most parents and physicians would do almost anything to change that child's destiny. Then, the knowledge that many adults get sick and die prematurely, lose their strength and their minds prematurely, leads many physicians and scientists to think about changing the genes that cause that early decline. Finally, the knowledge that the degenerative changes of most common disease, including cancer and heart disease--and of aging itself--are moderated by genetic processes, led many scientists to think of adjusting the genetic compliment routinely.

With the discovery of restriction enzymes and practical methods of gene sequencing, the race to map the genes was on. It was important to understand the normal human genome before scientists could identify the genes that led to disease. Once all the disease genes were identified, it was thought that perhaps substituting healthy genes for the disease genes might cure the underlying problem.

But the story was not that simple. Early attempts to introduce genes into human subjects met with unforeseen obstacles. And when it was discovered that there were only 25,000 to 30,000 human genes--instead of the expected 100,000--it began to dawn on scientists that there was more to the story than just the genes. Epigenetic factors play into gene expression, which complicated the plot significantly.

Protein interaction, gene regulator proteins, non-coding RNA, and glycomics--among other things--influence gene expression and the ultimate fate of the cell. Of course, in humans, cells exist within tissues, tissues within organs, and organs within the human organism. Complex interactions occur at every level.

To introduce new genes into a cell with defective genes, you must have a vector. Gene vectors are typically viral, since viruses make a living by introducing their genes into an animal cell for its own replication. There are also non-viral vectors that can be used to introduce genes into cells. Viruses have a billion+ year advantage as gene vectors, but nonviral methods are improving despite the challenges.

Besides introducing genes into cells, modern gene therapy also involves silencing of genes by various means. Sometimes it involves activating dormant genes that are already present. And sometimes it means delivering the entire cell--genes, nucleus, cytoplasm, and all.

Human Gene Therapy journal has made an entire issue freely available on the internet. The latest news on gene therapy is available through various sources. A recent article in The Scientist discussed future directions of gene therapy.

The SENS approach to life extension involves interventions against seven causes of aging: Cell depletion, Unwanted Cells, Chromosomal Mutations, Mitochondrial Mutations, Protein Crosslinks, Extracellular Junk, and Intracellular Junk. None of them are insurmountable, and much of the new knowledge from molecular biology, stem cell biology, cell biology, and other areas of biotechnology, are applicable to these challenges.

The very proliferation of new knowledge in all of these areas present a difficulty. Bioinformatics has evolved to keep track of the impossible quantity of data being generated, but what is actually needed is a super-human intelligence to make sense of it all, and to prioritise new research. In lieu of that, we will have to muddle through. Bit by bit, we seem to be succeeding. It would go much faster with some next-level supervision, but such is life.

Designer Genes: Wear With Pride

Medical News Today provides an intriguing report on the development of an advanced computer program from Johns Hopkins that should speed up the design and manufacture of artificial genes for genomics and proteomics research.

The program, called GeneDesign, guides the design of blueprints for DNA segments to the exacting specifications required for studying gene function and genetically engineering cells. The blueprints are then used by companies or other investigators to synthesize the gene.

....GeneDesign automates the process of determining which base pairs -- the building blocks of DNA -- should be linked together in a particular order to make a gene, according to Jef Boeke, Ph.D., professor of molecular biology and genetics and director of the High Throughput Biology Center at The Johns Hopkins University School of Medicine. A gene codes for a specific protein, and the order of the hundreds or thousands of base pairs making up that gene determines the order of the amino acid building blocks making up that protein. Boeke is senior author of the paper.

"GeneDesign not only guides the user in designing the gene, but also automatically diagnoses design flaws in the sequence of bases making up the gene," said Boeke.

....GeneDesign consists of six modules that can be used individually or in series to automate the tasks required to design and manipulate synthetic DNA sequences. The program allows the user to start with either the sequence of the amino acid making up the protein or the bases making up the gene that codes for that protein. Then the user moves through a series of steps that guide the design of the gene and vector that will carry the gene into the cell. Users can follow the main "Design a Gene" path or use the modules individually as needed. Vectors are mobile pieces of DNA that are used to carry artificial genes into cells.

A major advantage to GeneDesign is the ability to choose specific codons that work especially well in specific organisms, Boeke said. A codon is a trio of bases in a gene that codes for a specific amino acid building block. Most amino acids are represented by more than one codon. For example, the codons GCU, GCC, GCA, GCG can each code for the amino acid alanine.

....Another advantage of the GeneDesign is ease of creating restriction sites -- places along the DNA where the gene can be cut, said Sarah M. Richardson, a Ph.D. candidate in the Department of Genetic Medicine at Hopkins and first author of the paper. Scientists use molecular scissors called restriction enzymes to make these cuts, which allow them to do the cutting and pasting needed to put artificial genes into vectors.

"GeneDesign guides the choice of the series of base pairs where the restriction enzymes cut the DNA," Richardson said. "That lets investigators use different restriction enzymes to make cuts exactly where they want to.

Here is the link to the website for GeneDesign Beta2.0, which includes instructions for using the program's modules, and the program manual.

This is just one more tool in a long line of exciting new tools for advancing knowledge in the big three fields of the future: molecular biology (genomics, proteomics, RNA etc), nanotechnology, and information science/technology. A powerful synergy between these fields has developed which steepens the knowledge acquisition curve even more.