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Future on Gene Therapy- Vicky Borquez (2A)

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Future on Gene Therapy- Vicky Borquez (2A)

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Within time Gene Therapy will be the cure for the diseases and sicknesses. As technology is still advancing there is still a lot of expectations with technology developing a more effective gene therapy.

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Abstract

Eventually, gene therapy will become a staple of 21st century medicine. But some experts say society will be better served if medical researchers proceed more slowly and prudently.

In its current manifestation, gene therapy is an elegant concept crudely executed. That's not an indictment - that's just the way it is for an extraordinarily complicated technology still in its infancy. After all, it has been only 5 years since the concept of gene therapy was convincingly demonstrated to provide, if not a cure, then at least a long-term therapeutic effect for X-linked severe combined immunodeficiency (X-SCID) disease.

(Here, gene therapy is defined as the introduction of genetic material via techniques of molecular biology into somatic cells [in contrast to germ cells] to treat or prevent disease.)

RELATIVELY BRIEF HISTORY

Many people still are under the impression that gene therapy's proof-of-concept was demonstrated as early as 1990. For example, on Jan. 26 of this year, the Los Angeles Times wrote that W. French Anderson, MD, was "dubbed 'the father of gene therapy' after a team he led in 1990 cured a hereditary disease of the immune system in a 4-year-old girl." That's not quite the way it happened.

Anderson did indeed gain renown for heading the team that in September 1990 carried out the first gene therapy clinical trial approved for use in a human. The goal of this phase 1 study was to define the safety issues involved. The 4-year-old girl had a genetic disease called adenosine deaminase (ADA) deficiency, which is caused by a defective gene for the enzyme ADA, resulting in SCID. Via a modified retrovirus, normal ADA genes were transferred to T lymphocytes that had been removed from the girl's body and grown in culture. The white cells then were returned to the patient. In January 1991, a 9-year-old girl underwent the same procedure.

Today both patients are alive and doing well, but conventional therapy (pegylated bovine ADA, or PEG-ADA) given before, during, and after their gene therapy confounded the results and makes any claim of "cure" based on the gene therapy problematic. One patient has an ADA level that is 25 percent of normal with the therapeutic gene present in 15 percent of her peripheral blood mononuclear cells. The other has an ADA level that is less than 5 percent of normal, and the presence of the therapeutic gene in the peripheral blood cells is negligible.

Anderson went on to collaborate in 12 of the first 20 gene therapy trials approved in the United States. In an article he wrote about the prospects for gene therapy, he stated, "Indeed, within 20 years, I expect that gene therapy will be used regularly to ameliorate -and even cure - many ailments." More recently, Time celebrated the 50th anniversary of Watson and Crick's 1953 discovery of the structure of DNA with a special issue about genetics, again featuring Anderson. Ever the optimist, Anderson looked 50 years ahead and told Time, "By 2053, there will be a gene-based treatment for essentially every disease. Cancer, heart disease, and other modern-day scourges will be vastly reduced."

Halfway through Anderson's 20-year window (but barely into his 50-year projection), you can count the number of clearly effective gene transfer therapies in nonexperimental clinical use with your nose. That's right - there's one. And you have to go halfway around the world to get it.

In October 2003, in China, Gendicine became the world's first gene therapy approved for commercial production. According to newspaper accounts, patients from around the world have been traveling there to receive this treatment for head and neck squamous cell carcinoma (HNSCC). The question is how long will Gendicine remain the world's only licensed gene therapy?

Probably, not very long - the Chinese approach is conceptually identical to one that was developed earlier in the United States but has been moving more slowly toward licensing - a pace that means gene therapy will not come into regular use anytime soon.

On the other hand, the number and variety of clinical trials of gene therapy are such that Anderson's 50-year projection could pan out. As of December 2004, 667 human gene transfer clinical protocols had been submitted for review by the National Institute of Health's Recombinant DNA Advisory Committee (RAC) and the U.S. Food and Drug Administration (through its Center for Biologics Evaluation and Research). Of these, 617 are for therapeutic purposes (see on page 56) - as opposed to marking and nontherapeutic purposes - but only a handful have advanced to phase 3. Most are phase 1 trials whose purpose is to demonstrate safety. Of the 59 protocols submitted in 2004, the majority originated in academia, but 37 percent had a corporate sponsor (sometimes working in collaboration with a nonprofit group).

MULTITUDE OF TARGETS

With all the hoopla surrounding the Human Genome Project, it's understandable that people would entertain high hopes for the advancement of gene therapy. The human genome is now known to contain some 25,000 genes, including about 22,000 protein-encoding genes that express about 100,000 proteins. But unresolved questions abound regarding what these genes and proteins actually do and how, when, where, and in what sequence. As answers emerge, gene therapy could evolve in ways that will provide numerous benefits to patients and without deleterious side effects.

Talking about gene therapy as though it were a single entity, though, isn't very helpful. As explained by David A. Sanders, PhD, associate professor of biological sciences at Purdue University, gene therapy falls into three groups:

Replacing a defective or mal-adaptive gene that's responsible for some monogenic disease (e.g., cystic fibrosis or sickle cell anemia)

Altering or killing an aberrant cell (e.g., infected by HIV or cancerous)

Inducing production of a therapeutic protein (e.g., treating hepatitis C by promoting secretion of interferon by other cells)

Initially, gene therapy focused on the first group, but most current research focuses on the other two. Whatever the application, numerous hurdles stand in the way of developing a successful gene therapy, such as:

Identifying an appropriate target for gene therapy

Getting a therapeutic transgene into the right cells (and only those cells) in the right amount

Delivering the transgene with a vector that doesn't trigger an immune response or, in the case of certain viral vectors, revert to a pathogenic form

Providing the appropriate regulatory elements for turning the gene on and off at the correct time

Keeping the transgene in the target cell long enough for it to do its job

Keeping the transgene from causing damage elsewhere (e.g., spurring development of neoplasms or autoimmune disease, which could happen if the transgene expresses a protein new to the patient's body

Aside from the 1,500 or so diseases known to be caused by a single defective gene, most involve multiple genes, so potential targets for gene therapy abound. Possible therapies aren't restricted to the naturally occurring genes and gene products in the human genome. Fusion genes and nonhuman genes also are being investigated. An example of a fusion gene is an investigational agent being developed by Targeted Genetics, based in Seattle, Wash. This agent fuses the gene for the Fc fragment of human immunoglobulin G with another gene for the soluble p75 receptor for tumor necrosis factor, producing the same molecule provided by etanercept (Enbrel). A phase 1 trial comprising patients who have rheumatoid arthritis was initiated in March 2004.

SKY-HIGH EXPECTATIONS

In the view of Theodore Friedmann, MD, who has been deeply involved in the study of gene therapy for three decades - essentially its entire modern history - hyperbole surrounding early claims had the effect of unrealistically heightening expectations that gene therapy would emerge quickly as a component of health care. Friedmann is a professor of pediatrics at the University of California-San Diego, and director of the UCSD Program in Gene Therapy. A medical ethicist and geneticist, he also has served on the RAC and was its chairman until last year.

That the initial human gene transfer protocol was hailed a success, Friedmann says, is a "perfect example of the confluence of exaggerated expectations and wishful thinking. Everyone wanted it to work." But, he adds, it was unfair to patients and the public that the heightened expectations generated by some of the scientists and their institutions, the media, and others served to raise false hope in many patients with many kinds of disease. "Hope is necessary, but knowingly making undeliverable promises and raising false hope is cruel," Friedmann says. "The delusion of a cure contributed to crashing disappointment later."

With their early expectations dashed, people concluded that gene therapy might be another biotech bust. Friedmann thinks the perception of failure was also fueled by widely publicized setbacks being portrayed as "disasters" - which he says were regarded as disasters only because expectations were so high to start with.

Friedmann sees gene therapy today at a point comparable to the early days of organ transplantation, when successes were scarce and failures frequent. Even the first clear success of gene therapy, he notes, has been muted by the emergence of three cases of leukemia (including one death) among the 18 children who were treated. The outcome of these cases is that gene transfer therapy is now reserved for patients who have had unsuccessful attempts at bone marrow transplantation or for whom this approach is not feasible.

"These children received very effective treatment, but at a very high cost," Friedmann says, "and if additional cases of leukemia develop, we'll have a greater problem." But he adds, that doesn't mean that we should not push ahead with the field.

WHERE THE PATIENTS ARE

At first it was thought that gene therapy would focus on monogenic diseases - hereditary diseases such as SCID, hemophilia, or cystic fibrosis - that stem from a single defective gene. The thinking was that such diseases could be ameliorated, if not cured, by providing the patient with a properly functioning gene. Thus far, the initial expectations have not been fulfilled. Moreover, as gene therapy has evolved, it has drifted away from monogenic diseases and toward diseases like cancer.

This makes sense given that cancer is where the patients are and probably will be. Which is also where the money will be. According to the American Cancer Society, cancer has become the leading killer of Americans under the age of 85, surpassing cardiovascular disease. For demographic reasons, it is logical that CVD also would attract the attention of gene therapy researchers - and it has, along with incurable conditions such as Alzheimer's disease and Parkinson's disease.

Alameda, Calif.-based Avigen began a phase 1/2 clinical trial of AV201 for the treatment of severe Parkinson's at the end of 2004. This agent uses an adeno-associated virus (AAV) vector to deliver the gene for an enzyme, acetoacetate decarboxylase (AADC), directly into the striatum. The striatum is the section of the brain where movement is controlled via dopamine. As Parkinson's progresses, dopamine levels decrease. Patients are treated with levodopa, which is converted to dopamine by AADC. Eventually, the effectiveness of levodopa diminishes, presumably because the concentration of AADC declines. The idea behind AV201 is that with the enzyme restored, patients once again will respond to levodopa.

To take another example, some gene therapy research is exploring the role of angiogens - molecular mediators that promote the formation of blood vessels during normal cardiac and vascular development. One such angiogen is vascular endothelial growth factor (VEGF). The VEGF gene consists of eight exons that can be spliced in different ways (omitting one or more exons), leading to the synthesis of amino acid sequences of varying lengths (specifically, 121, 165, 189, and 206 amino acids). Using an adenovirus vector, attempts have been made to transfer cDNA for VEGF121 into skeletal muscle as a treatment for peripheral arterial disease and into myocardial tissue as a treatment for severe coronary artery disease.

It was once thought that the receptors for the VEGF group of proteins were restricted to endothelial cells, but they since have been found in cells of nonendothelial origin, including tumor cells. That finding points to a characteristic that gene therapy shares with pharmacotherapy: the molecular targets of therapy often are not restricted to cells in the tissue of interest. That puts a premium on developing vectors that deliver transgenes to specific cells and only those cells.

IMPROVING VIRAL VECTORS

Among the numerous vehicles for carrying therapeutic genes to target cells, vectors adapted from viruses stand out because of the ease with which viruses enter cells and then spill out their contents - the viral genes that induce the host to generate the components of new virions. When a therapeutic gene is inserted in place of most of the viral genome, the virion retains its ability to penetrate the target cells while delivering a presumably beneficial payload. The families that have been most often used as vectors are the adenoviruses and retroviruses, but AAV and lentivirus are among other viral vectors increasingly employed in gene therapy experiments. Lentiviruses actually are a genus in the retrovirus family, but they differ from other retroviruses in being able to integrate their genome into the chromosomes of nondividing cells (e.g., brain, peripheral nerves). Other retroviruses can transduce only dividing cells.

According to Sanders, one of Friedmann's major contributions, among many, to the development of gene therapy was showing that a recombinant pseudotyped virus could be created to serve as a vector for delivering genetic material to cells. The genome of a pseudotyped virus lacks the coding for one or more of its structural proteins, which confers a safety benefit and other advantages.

In contemporary gene therapy experiments, vesicular stomatitis virus G protein (VSV-G) pseudo-typed retroviruses and lentiviruses are commonly used but have several shortcomings, such as being toxic to cells producing virus in culture and targeting primary cells in culture or in vivo. In his own work, Sanders has been looking at other viruses that could be used to create pseudotyped viruses, and he says that he has found alphaviruses to be promising. Among the species in this insect-transmitted genus are the Ross River virus, Eastern equine encephalitis virus, Semliki Forest virus, and Venezuelan equine encephalitis virus.

The field of gene therapy is driven largely by medical professionals instead of scientists. Because patients are their primary concern, doctors might be predisposed to trying gene therapy experiments on severely ill patients - even if the science is still a bit ragged.

A protein biochemist by training, Sanders did postdoctoral work with Harvard's Richard Mulligan, PhD, who joins Friedman as another pioneer of gene therapy. Sanders is especially interested in the proteins found on the exterior of viruses, because the way these proteins match up with other proteins embedded in the cell membrane determines whether a virus can enter a given cell. Sanders has designed a new class of pseudo-typed viruses, constructing their shell from a variety of alphaviruses and their core from retroviruses and lentiviruses. Injected into the tail vein of a mouse, these pseudotyped viruses are delivered in quantity to the liver. And in the central nervous system, these vectors go specifically to glial cells but not to neurons, in contrast with VSV-G vectors, which enter neurons but not glial cells. Sanders says this property has important implications for the treatment of brain tumors, most of which are of glial origin.

In many cases, delivering a gene to the right cells is not sufficient. The gene also must be brought to the correct portion of the cell. That's because many cells are polarized, having one portion of their plasma membrane exposed to the outside world (the apical membrane) and another exposed to the blood stream (the basolateral membrane). The proteins embedded in the apical membrane differ from those in the basolateral membrane, and a tight junction prevents proteins from passing from one domain to the other. Because different proteins offer a foothold for different viruses, it's important to select a viral vector that's specific for the apical or basolateral membrane of polarized target cells, such as airway epithelial cells in the lung. Influenza viruses enter and leave these cells through the apical membrane, which is exposed to the outside. But a retrovirus like HIV or murine leukemia would have to enter through the basolateral membrane, which is exposed to the bloodstream in which these viruses find their major target (blood cells).

Sanders explains that a practical consequence of failure to appreciate the difference between apical and basolateral membranes is this: More than a decade ago, the gene responsible for cystic fibrosis was discovered. Great excitement ensued, and the success of gene therapy based on this discovery was eagerly anticipated. But success has been elusive, partly owing to difficulty in getting the therapeutic gene into the target cells. Sanders says that's because researchers used retroviral vectors that approached the epithelial cells from the wrong side (the basolateral side). He says a more promising approach may be to base a vector on a modified Ebola virus shell, which specifically targets airways epithelial cells and can enter through the apical membrane if it's aerosolized. This technique appears to improve gene delivery by two orders of magnitude compared to retroviral vectors, Sanders says.

WRONG DRIVER?

Setting aside the specifics of cystic fibrosis, the greater question focuses on why researchers would use the wrong vector in the first place. In Sanders' view, it's because the field of gene therapy is driven largely by medical professionals instead of scientists. The physician's paramount concern is to help a patient, and if the patient is severely ill, the physician may disregard loose ends or ambiguity in the science supporting a new technology.

"Medical doctors are interested in the individual patient's welfare at all costs," Sanders says. "I would demand that from my own physician or a physician for a member of my family. They are not necessarily the best evaluators of the societal and public health effects of their procedures. When confronted with seriously ill patients for whose condition there is no existing effective treatment they think, 'Let's go ahead and do the experiment anyway. Maybe it will work.'"

One frontrunner for the distinction of being the first gene therapy to win approval from the U.S. Food and Drug Administration is Advexin, made by Introgen, of Austin, Texas, and which resembles Gendicine. Both use an adenoviral vector to deliver the p53 tumor suppressor gene and can be used in combination with radiotherapy.

Advexin is being studied in two phase 3 trials that are enrolling patients with recurrent, unresectable head and neck squamous cell carcinoma. In one trial, Advexin monotherapy is being compared to methotrexate monotherapy, the primary outcome being the effect on survival time. The other compares combined chemotherapy plus Advexin versus the same chemotherapy without Advexin, with the primary endpoint being the time to progression.

The FDA has designated Advexin as an orphan drug, so if it's approved, Advexin could win seven years of marketing exclusivity. Advexin also is being studied as a treatment for many other cancers.

Looking at gene therapy from the perspective of public health, Sanders says he's opposed to doing gene therapy experiments too early. "Yes, we did learn some things from the death of Jesse Gelsinger [the 19-year-old who died during a gene therapy experiment at the University of Pennsylvania in 1999], but we didn't have to learn them in that way," he says.

As an example of how physicians' haste to help patients can lead researchers in the wrong direction, Sanders cites a key study from W. French Anderson that paved the way for the first human gene therapy experiments. With the concern that a viral vector might acquire the ability to replicate, it was important to demonstrate that replication did not occur if a retrovirus was used as a vector. Toward this end, Anderson and colleagues injected rhesus macaques (a proxy for human subjects) with a replication-competent murine leukemia retrovirus. Because no infection developed in the macaques, the researchers concluded that murine retrovirus probably would not pose an acute health risk in humans. The researchers attributed the lack of infection in the monkeys to neutralization of the retrovirus by complement.

Nonetheless, there was a problem, Sanders says. The presence of complement implies the presence of antibodies against the retrovirus. The question of where those antibodies came from should have given the researchers pause. When another team of researchers did a similar experiment at a later date, they used a recombinant retroviral vector that was contaminated with a replication-competent virus. And, whereas in the first experiment the virus was introduced in vivo, in the second the virus was mixed with cells in culture, and the cells were reintroduced into the monkeys. This time, the monkeys developed leukemia.

"There was a huge amount of retroviral replication," Sanders says. The results differed, he explains, because mice and most other mammals possess a sugar, galactose-alpha (1,3)-galactose, as a component of glycoproteins on cell surfaces, but Old World monkeys (and humans, along with the great apes) lack this sugar. Yet, they have ample antibodies against it - from 1 to 3 percent -because alpha-galactose is found on the coat of many viruses that infect these animals. When the antibodies recognize a virus sporting this sugar, the complement cascade is triggered. That's what happened in the first experiment, creating the illusion of safety. In the second experiment, with the transduction taking place in a cell culture instead of in vivo, there was no possibility of an immune response. As a result, the viruses produced in culture lacked the sugar and went undetected and replicated, causing leukemia when the cells were returned to the macaques.

ECOLOGY OF GENE THERAPY

Sanders likens gene therapy to the development of transgenic crops. As he sees it, the major issue with transgenic plants isn't their potential to affect human health, as many critics fear, but rather the possibility that genes introduced into crops in the hope of improving agriculture might find their way into other plants with detrimental results for the whole environment. He thinks similar ecological issues apply to gene therapy. If someone receives a treatment with a recombinant virus that has the potential to replicate, can untreated individuals also be receiving gene therapy? And if DNA enters the germ line as a result of gene therapy, the effects could be felt by future generations.

If society supports widespread gene therapy directed at disease prevention - in addition to treating existing disease -Sanders wonders, who will determine how and what resources should be allocated toward that end?

The societal implications of gene therapy are so profound that a cautious, step-by-step approach is warranted, Sanders says. "Gene therapy will become a component of 21st century medicine. There's no reason it can't work. But huge questions remain to be resolved. The history of mankind tells us that whenever you have new technology, you have problems. But by now we should be intelligent enough to anticipate the problems that might be associated with gene therapy."

What are some of these problems? For starters, should patients who have received gene therapy be allowed to donate blood? Probably not, in Sanders's opinion, given how little we know at this point about long-term outcomes in gene therapy. Then there's the important political question of whether the contemporary pharmaceutical industry should serve as the model for the development of gene therapy. Sanders observes that the big money generated by small molecules and biologics is for treating chronic diseases, not for preventing disease. If society supports widespread gene therapy directed at disease prevention in addition to treating existing disease, who will determine how and what resources should be allocated toward that end?

Given the genetic basis for most diseases, instead of contemplating the future of gene therapy, it might be equally interesting to wonder about the future of gene therapy in the context of drug therapy. Right now, whether the disease is cancer or CVD, gene therapy investigations for the most part are focused on developing new treatments for high-risk patients with severe illness - patients beyond the point where conventional treatment is effective. Eventually, however, conventional treatments and gene therapies will overlap. Thorny ethical and political issues will have to be addressed, but, over the long term, the future of drug therapy could be gene therapy.

Footnotes

¹ Since 1974, the RAC has reviewed technology involving recombinant DNA, including clinical trials involving human gene transfer if direct or indirect NIH funding is provided. Since 1997, the U.S. Food and Drug Administration has been the principal regulator and overseer of gene therapy trials, while the Recombinant Advisory DNA Committee has promoted public awareness and understanding of issues surrounding gene therapy. Yet, the RAC's function goes beyond education, taking on powerful and public scientific, ethics, and policy advisory oversight. The RAC sends its recommendations to the FDA and local committees, and while those RAC comments formally are recommendations only, they carry great influence on the final actions taken by the local review committees.

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Eventually, gene therapy will become a staple of 21st century medicine. But some experts say society will be better served if medical researchers proceed more slowly and prudently. In its current manifestation, gene therapy is an elegant concept crudely executed. That's not an indictment - that's just the way it is for an extraordinarily complicated technology still in its infancy.

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Gene therapy in the womb will not be tested in the near future. People are not willing to change something that they have no business changing. The question cannot be answered because people won't run experiments on their children

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A new gene therapy tested in human egg cells could lead to cures for mitochondrial diseases, though the technique hasn't been tested in human trials yet.

Credit: © Marina Dyakonova | Dreamstime.com

Efforts to develop a working gene therapy for certain inherited diseases have reached a milestone with a new method for altering a human egg cell.

If the technique, which was unveiled by Oregon Health & Science University and involves transplanting mitochondrial DNA, is ever approved for use in patients, a child's genetic makeup could be altered to cure certain genetic diseases before the baby is even born. Even so, the researchers acknowledged that realistically the treatment wouldn't likely be approved for testing in humans for a while.

The gene-tweaking technique, which is detailed online today (Oct. 24) in the journal Nature, is designed to treat diseases caused by genetic mutations in the cells' energy-making structures called mitochondria, said lead researcher Shoukhrat Mitalipov of OHSU School of Medicine.

Mitochondrial diseases can lead to diabetes, degeneration of nerves, or blindness, so the diseases themselves are often mistaken for other problems. Once the disease is identified, various supportive therapies are available, but generally there is no cure because the disease is caused by genetic mutations that are locked in. [ Top 10 Mysterious Diseases]

Swapping out DNA

Mitochondrial DNA (mtDNA) comes only from the mother and is contained in the cell's cytoplasm, between the nucleus and the membrane (unlike nuclear DNA, which sits in the cell's nucleus). In the new study, Mitalipov and colleagues took a donor egg cell and removed the nucleus. They replaced this nucleus with one from the mother's egg cell, resulting in a cell with the mother's DNA but the donor's mtDNA.

The cell was then fertilized, allowed to divide and become a blastocyst, or a small agglomeration of embryonic cells. From that, the researchers derived embryonic stem cells. The cells looked normal, just like the controls. [ 5 Amazing Stem Cell Discoveries]

This shows that the cells with the "new" mtDNA function just like ordinary egg cells, Mitalipov said in a press conference. "This shows the procedure is compatible with normal fertilization."

Since the mtDNA isn't in the nucleus, this type of gene therapy wouldn't change one's parents. (mtDNA does show up in some types of gene sequencing, but it is a very small number of genes - less than one in 10,000.)

Genetic diseases that result from problems with nuclear DNA wouldn't be affected by the therapy, so it isn't likely to be of any help for a disease such as cystic fibrosis or Down syndrome.

Ethics of gene therapy

The experiments on human cells build on work conducted in 2009 with macaque monkeys. In that case, the experiment involved two populations of healthy macaques, one from India and one from China. One group of macaques had their egg cells' nuclei transferred to donor cells from the other population. The result, Mitalipov said, was healthy macaque infants carrying the donor mtDNA. "The infants were normal even though they had foreign mtDNA," he said.

Mitalipov also experimented on frozen macaque egg cells and showed the technique works with them as well, though the success rate was lower.

If this treatment were used on humans, it would work only in future children, since it involves changing the genes of an embryo before development. That opens up a lot of ethical questions about parents who want to alter their child's genes - even if it is for health reasons.

"Your mitochondrial DNA really is a part of your identity," said Gerard D'Souza, assistant professor of pharmaceutics at the Massachusetts College of Pharmacy and Health Sciences, who wasn't involved in the current study. He noted that entire studies have traced ancestry via mitochondrial DNA, suggesting people think of it as a piece of who they are.

D'Souza added that Mitalipov's approach was a big departure from a lot of current thinking about gene therapy. For gene therapy to work, the genes (or the agents to repair them) have to be delivered to many cells at once. That's why viruses are often used as a vector, or carrier. "Rather than deliver DNA to multiple cells, he just let one cell become the individual," he said.

For his part, Mitalipov said he and his team are in discussions with the Food and Drug Administration about how to set up a clinical trial of the technique in humans. During the press briefing, Mitalipov said the technique is safe thus far, and the experiment passed muster with the university's institutional review board. The experiments were funded privately.

Still, it likely will be some time before a human trial is approved.

Among the ethical questions raised by the technique concern the hope that it would offer to people at risk for genetic disease. "People try all sorts of treatments," D'Souza said. "They realize nothing works, and only then do they check if it's a [mitochondrial genetic] disorder."

Charles Mohan, CEO of the United Mitochondrial Disease Foundation, said that if the new treatment becomes available to parents, it will allow them to at least make decisions about the health of their children in the first place. Right now, for anyone who carries the genes for mitochondrial disease, it's a roll of the genetic dice - even assuming one knows they have the genes in the first place. Mohan's daughter died from a mitochondrial disease at 15, and did not show symptoms until she was 10. Meanwhile, his son is now an adult, and healthy.

"It provides an alternative," he said. "If we had known earlier, what decisions would we have made?" Whatever the controversies about genetically engineering humans, a cure like this would at least mean that it's possible to make such decisions.

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Could Genetic Diseases Be Cured in the Womb?

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Efforts to develop a working gene therapy for certain inherited diseases have reached a milestone with a new method for altering a human egg cell. If the technique, which was unveiled by Oregon Health & Science University and involves transplanting mitochondrial DNA, is ever approved for use in patients, a child's genetic makeup could be altered to cure certain genetic diseases before the baby is even born.

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Ethical concerns on somatic Gene Therapy - Vicky Borquez (2A)

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Ethical concerns on somatic Gene Therapy - Vicky Borquez (2A)

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Gene Therapy is creating controversy in religious views due to questioning God's creations and power. Over time people became more accepting of gene therapy because it has the cures of a lot of disorder or diseases. However, with modern technology scientist have found a way to alter your genes causing your DNA to change. Concerning religions due to challenging God's creations.

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Adenosine deaminase occurs in the  lymphocytes, which are immune system cells.   Deoxyadenosine is created when DNA is broke down, and is very toxiz to lymphocytes.

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The ADA gene provides instructions for producing the enzyme adenosine deaminase. This enzyme is produced in all cells, but the highest levels of adenosine deaminase occur in immune system cells called lymphocytes, which develop in lymphoid tissues. These lymphoid tissues include the thymus, which is a gland located behind the breastbone, and lymph nodes, which are found throughout the body.

# data[].attributes.attachment_link.readable_text
A new gene therapy tested in human egg cells could lead to cures for mitochondrial diseases, though the technique hasn't been tested in human trials yet.

Credit: © Marina Dyakonova | Dreamstime.com

Efforts to develop a working gene therapy for certain inherited diseases have reached a milestone with a new method for altering a human egg cell.

If the technique, which was unveiled by Oregon Health & Science University and involves transplanting mitochondrial DNA, is ever approved for use in patients, a child's genetic makeup could be altered to cure certain genetic diseases before the baby is even born. Even so, the researchers acknowledged that realistically the treatment wouldn't likely be approved for testing in humans for a while.

The gene-tweaking technique, which is detailed online today (Oct. 24) in the journal Nature, is designed to treat diseases caused by genetic mutations in the cells' energy-making structures called mitochondria, said lead researcher Shoukhrat Mitalipov of OHSU School of Medicine.

Mitochondrial diseases can lead to diabetes, degeneration of nerves, or blindness, so the diseases themselves are often mistaken for other problems. Once the disease is identified, various supportive therapies are available, but generally there is no cure because the disease is caused by genetic mutations that are locked in. [ Top 10 Mysterious Diseases]

Swapping out DNA

Mitochondrial DNA (mtDNA) comes only from the mother and is contained in the cell's cytoplasm, between the nucleus and the membrane (unlike nuclear DNA, which sits in the cell's nucleus). In the new study, Mitalipov and colleagues took a donor egg cell and removed the nucleus. They replaced this nucleus with one from the mother's egg cell, resulting in a cell with the mother's DNA but the donor's mtDNA.

The cell was then fertilized, allowed to divide and become a blastocyst, or a small agglomeration of embryonic cells. From that, the researchers derived embryonic stem cells. The cells looked normal, just like the controls. [ 5 Amazing Stem Cell Discoveries]

This shows that the cells with the "new" mtDNA function just like ordinary egg cells, Mitalipov said in a press conference. "This shows the procedure is compatible with normal fertilization."

Since the mtDNA isn't in the nucleus, this type of gene therapy wouldn't change one's parents. (mtDNA does show up in some types of gene sequencing, but it is a very small number of genes - less than one in 10,000.)

Genetic diseases that result from problems with nuclear DNA wouldn't be affected by the therapy, so it isn't likely to be of any help for a disease such as cystic fibrosis or Down syndrome.

Ethics of gene therapy

The experiments on human cells build on work conducted in 2009 with macaque monkeys. In that case, the experiment involved two populations of healthy macaques, one from India and one from China. One group of macaques had their egg cells' nuclei transferred to donor cells from the other population. The result, Mitalipov said, was healthy macaque infants carrying the donor mtDNA. "The infants were normal even though they had foreign mtDNA," he said.

Mitalipov also experimented on frozen macaque egg cells and showed the technique works with them as well, though the success rate was lower.

If this treatment were used on humans, it would work only in future children, since it involves changing the genes of an embryo before development. That opens up a lot of ethical questions about parents who want to alter their child's genes - even if it is for health reasons.

"Your mitochondrial DNA really is a part of your identity," said Gerard D'Souza, assistant professor of pharmaceutics at the Massachusetts College of Pharmacy and Health Sciences, who wasn't involved in the current study. He noted that entire studies have traced ancestry via mitochondrial DNA, suggesting people think of it as a piece of who they are.

D'Souza added that Mitalipov's approach was a big departure from a lot of current thinking about gene therapy. For gene therapy to work, the genes (or the agents to repair them) have to be delivered to many cells at once. That's why viruses are often used as a vector, or carrier. "Rather than deliver DNA to multiple cells, he just let one cell become the individual," he said.

For his part, Mitalipov said he and his team are in discussions with the Food and Drug Administration about how to set up a clinical trial of the technique in humans. During the press briefing, Mitalipov said the technique is safe thus far, and the experiment passed muster with the university's institutional review board. The experiments were funded privately.

Still, it likely will be some time before a human trial is approved.

Among the ethical questions raised by the technique concern the hope that it would offer to people at risk for genetic disease. "People try all sorts of treatments," D'Souza said. "They realize nothing works, and only then do they check if it's a [mitochondrial genetic] disorder."

Charles Mohan, CEO of the United Mitochondrial Disease Foundation, said that if the new treatment becomes available to parents, it will allow them to at least make decisions about the health of their children in the first place. Right now, for anyone who carries the genes for mitochondrial disease, it's a roll of the genetic dice - even assuming one knows they have the genes in the first place. Mohan's daughter died from a mitochondrial disease at 15, and did not show symptoms until she was 10. Meanwhile, his son is now an adult, and healthy.

"It provides an alternative," he said. "If we had known earlier, what decisions would we have made?" Whatever the controversies about genetically engineering humans, a cure like this would at least mean that it's possible to make such decisions.

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Could Genetic Diseases Be Cured in the Womb?

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Efforts to develop a working gene therapy for certain inherited diseases have reached a milestone with a new method for altering a human egg cell. If the technique, which was unveiled by Oregon Health & Science University and involves transplanting mitochondrial DNA, is ever approved for use in patients, a child's genetic makeup could be altered to cure certain genetic diseases before the baby is even born.

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Different techniques 3) Killing of Specific cells 

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Different techniques 3) Killing of Specific cells 

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Similar to gene inhibition
insert dna to kill the cell completely

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eliminate the activity of a gene that "encourages" bad cells to reproduce
ex; cancer

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There are 10 different types of severe combined immunodeficiency, they have all be reported at birth, and they all have to do with missing T cells. Children born with SCID are all at risk of severe infections.

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Two life-threatening conditions. One remarkable wish.

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DIfferent techniques 1)  Gene Augmentation therapy AG

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DIfferent techniques 1)  Gene Augmentation therapy AG

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used to treat diseases caused by a mutation - stops it from functioning (protein)
adds DNA containing a functional version of the lost gene back into the cell
only works if the effects of the disease are reversible or there isnt a lot of damage

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Gene therapy is when DNA is introduced into a patient to treat a genetic disease. The new DNA usually contains a functioning gene to correct the effects of a disease-causing mutation.

Gene therapy uses sections of DNA (usually genes) to treat or prevent disease.

The DNA is carefully selected to correct the effect of a mutated gene that is causing disease.

The technique was first developed in 1972 but has, so far, had limited success in treating human diseases.

Gene therapy may be a promising treatment option for some genetic diseases, including muscular dystrophy and cystic fibrosis.

There are two different types of gene therapy depending on which types of cells are treated:

Somatic gene therapy: transfer of a section of DNA to any cell of the body that doesn't produce sperm or eggs. Effects of gene therapy will not be passed onto the patient's children.

Germline gene therapy: transfer of a section of DNA to cells that produce eggs or sperm. Effects of gene therapy will be passed onto the patient's children and subsequent generations.

Gene therapy techniques

There are several techniques for carrying out gene therapy. These include:
Gene augmentation therapy

This is used to treat diseases caused by a mutation that stops a gene from producing a functioning product, such as a protein.

This therapy adds DNA containing a functional version of the lost gene back into the cell.

The new gene produces a functioning product at sufficient levels to replace the protein that was originally missing.

This is only successful if the effects of the disease are reversible or have not resulted in lasting damage to the body.

For example, this can be used to treat loss of function disorders such as cystic fibrosis by introducing a functional copy of the gene to correct the disease (see illustration below).

Gene inhibition therapy

Suitable for the treatment of infectious diseases, cancer and inherited disease caused by inappropriate gene activity.

The aim is to introduce a gene whose product either:

inhibits the expression of another gene

interferes with the activity of the product of another gene.

The basis of this therapy is to eliminate the activity of a gene that encourages the growth of disease-related cells.

For example, cancer is sometimes the result of the over-activation of an oncogene (gene which stimulates cell growth). So, by eliminating the activity of that oncogene through gene inhibition therapy, it is possible to prevent further cell growth and stop the cancer in its tracks.

Killing of specific cells

Suitable for diseases such as cancer that can be treated by destroying certain groups of cells.

The aim is to insert DNA into a diseased cell that causes that cell to die.

This can be achieved in one of two ways:

the inserted DNA contains a "suicide" gene that produces a highly toxic product which kills the diseased cell

the inserted DNA causes expression of a protein that marks the cells so that the diseased cells are attacked by the body's natural immune system.

It is essential with this method that the inserted DNA is targeted appropriately to avoid the death of cells that are functioning normally.

How is DNA transfer done?

A section of DNA/gene containing instructions for making a useful protein is packaged within a vector, usually a virus, bacterium or plasmid.

The vector acts as a vehicle to carry the new DNA into the cells of a patient with a genetic disease.

Once inside the cells of the patient, the DNA/gene is expressed by the cell's normal machinery leading to production of the therapeutic protein and treatment of the patient's disease.

Challenges of gene therapy

Delivering the gene to the right place and switching it on:

it is crucial that the new gene reaches the right cell

delivering a gene into the wrong cell would be inefficient and could also cause health problems for the patient

even once the right cell has been targeted the gene has to be turned on

cells sometimes obstruct this process by shutting down genes that are showing unusual activity.

Avoiding the immune response:

The role of the immune system is to fight off intruders.

Sometimes new genes introduced by gene therapy are considered potentially-harmful intruders.

This can spark an immune response in the patient, that could be harmful to them.

Scientists therefore have the challenge of finding a way to deliver genes without the immune system 'noticing'.

This is usually by using vectors that are less likely to trigger an immune response.

Making sure the new gene doesn't disrupt the function of other genes:

Ideally, a new gene introduced by gene therapy will integrate itself into the genome of the patient and continue working for the rest of their lives.

There is a risk that the new gene will insert itself into the path of another gene, disrupting its activity.

This could have damaging effects, for example, if it interferes with an important gene involved in regulating cell division, it could result in cancer.

The cost of gene therapy:

Many genetic disorders that can be targeted with gene therapy are extremely rare.

Gene therapy therefore often requires an individual, case-by-case approach. This may be effective, but may also be very expensive.

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Gene therapy is when DNA is introduced into a patient to treat a genetic disease. The new DNA usually contains a functioning gene to correct the effects of a disease-causing mutation. Gene therapy uses sections of DNA (usually genes) to treat or prevent disease.

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The concept of Gene Therapy first came into medicine in the 1960's and 1970's. The first person to be treated with Gene Therapy was a 4 year old girl that was treated in the NIH Clinical Center in 1990. (AH)

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Cord blood has been studied extensively for the treatment of hematological diseases such as cancer and sickle cell anemia, but is now in phase II trials for the alleviation of autism. Dr. Ann Smith, director of Smart Cells who provided the cord blood for the trial, discusses the research and it's potential.

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Cord blood has been studied extensively for the treatment of hematological diseases such as cancer and sickle cell anemia, but is now in phase II trials for the alleviation of autism. Dr. Ann Smith, director of Smart Cells who provided the cord blood for the trial, discusses the research and it's potential.

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Somatic :transfer of a section of DNA to any cell of the body that doesn’t produce sperm or eggs

Germline : transfer of a section of DNA to cells that produce eggs or sperm

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Gene therapy - An Introduction to Genetic Analysis - NCBI Bookshelf

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The general approach of gene therapy is nothing more than an extension of the technique for clone selection by functional complementation (Chapter 12). The functions absent in the recipient as a result of a defective gene are introduced on a vector that inserts into one of the recipient's chromosomes and thereby generates a transgenic animal that has been genetically "cured."

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Severe combined immune deficiency (SCID), and adenosine deaminase (ADA)  were some of the very first accomplishments of gene therapy; both inherited immune disorders. Gene therapy is also helping many different blindness.

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Researchers have been working for decades to bring gene therapy to the clinic, yet very few patients have received any effective gene-therapy treatments. But that doesn't mean gene therapy is an impossible dream. Even though gene therapy has been slow to reach patients, its future is very encouraging. Decades of research have taught us a lot about designing safe and effective vectors, targeting different types of cells, and managing and minimizing immune responses in patients. We've also learned a lot about the disease genes themselves. Today, many clinical trials are underway, where researchers are carefully testing treatments to ensure that any gene therapy brought into the clinic is both safe and effective.

Below are some gene therapy success stories. Successes represent a variety of approaches-different vectors, different target cell populations, and both in vivo and ex vivo approaches-to treating a variety of disorders.

Sebastian Misztal was a patient in a hemophilia gene therapy trial in 2011. Following the treatment, Misztal no longer had spontaneous bleeding episodes.
Credit: UCLH/UCL NIHR Biomedical Research Centre

Several inherited immune deficiencies have been treated successfully with gene therapy. Most commonly, blood stem cells are removed from patients, and retroviruses are used to deliver working copies of the defective genes. After the genes have been delivered, the stem cells are returned to the patient. Because the cells are treated outside the patient's body, the virus will infect and transfer the gene to only the desired target cells.

Severe Combined Immune Deficiency (SCID) was one of the first genetic disorders to be treated successfully with gene therapy, proving that the approach could work. However, the first clinical trials ended when the viral vector triggered leukemia (a type of blood cancer) in some patients. Since then, researchers have begun trials with new, safer viral vectors that are much less likely to cause cancer.

Adenosine deaminase (ADA) deficiency is another inherited immune disorder that has been successfully treated with gene therapy. In multiple small trials, patients' blood stem cells were removed, treated with a retroviral vector to deliver a functional copy of the ADA gene, and then returned to the patients. For the majority of patients in these trials, immune function improved to the point that they no longer needed injections of ADA enzyme. Importantly, none of them developed leukemia.

Gene therapies are being developed to treat several different types of inherited blindness-especially degenerative forms, where patients gradually lose the light-sensing cells in their eyes. Encouraging results from animal models (especially mouse, rat, and dog) show that gene therapy has the potential to slow or even reverse vision loss.

The eye turns out to be a convenient compartment for gene therapy. The retina, on the inside of the eye, is both easy to access and partially protected from the immune system. And viruses can't move from the eye to other places in the body. Most gene-therapy vectors used in the eye are based on AAV (adeno-associated virus).

In one small trial of patients with a form of degenerative blindness called LCA (Leber congenital amaurosis), gene therapy greatly improved vision for at least a few years. However, the treatment did not stop the retina from continuing to degenerate. In another trial, 6 out of 9 patients with the degenerative disease choroideremia had improved vision after a virus was used to deliver a functional REP1 gene.

Credit: Jean Bennett, MD, PhD, Perelman School of Medicine, University of Pennsylvania; Manzar Ashtari, Ph.D., of The Children's Hospital of Philadelphia, Science Translational Medicine.

People with hemophilia are missing proteins that help their blood form clots. Those with the most-severe forms of the disease can lose large amounts of blood through internal bleeding or even a minor cut.

In a small trial, researchers successfully used an adeno-associated viral vector to deliver a gene for Factor IX, the missing clotting protein, to liver cells. After treatment, most of the patients made at least some Factor IX, and they had fewer bleeding incidents.

Patients with beta-Thalassemia have a defect in the beta-globin gene, which codes for an oxygen-carrying protein in red blood cells. Because of the defective gene, patients don't have enough red blood cells to carry oxygen to all the body's tissues. Many who have this disorder depend on blood transfusions for survival.

In 2007, a patient received gene therapy for severe beta-Thalassemia. Blood stem cells were taken from his bone marrow and treated with a retrovirus to transfer a working copy of the beta-globin gene. The modified stem cells were returned to his body, where they gave rise to healthy red blood cells. Seven years after the procedure, he was still doing well without blood transfusions.

A similar approach could be used to treat patients with sickle cell disease.

In 2012, Glybera became the first viral gene-therapy treatment to be approved in Europe. The treatment uses an adeno-associated virus to deliver a working copy of the LPL (lipoprotein lipase) gene to muscle cells. The LPL gene codes for a protein that helps break down fats in the blood, preventing fat concentrations from rising to toxic levels.

Several promising gene-therapy treatments are under development for cancer. One, a modified version of the herpes simplex 1 virus (which normally causes cold sores) has been shown to be effective against melanoma (a skin cancer) that has spread throughout the body. The treatment, called T-VEC, uses a virus that has been modified so that it will (1) not cause cold sores; (2) kill only cancer cells, not healthy ones; and (3) make signals that attract the patient's own immune cells, helping them learn to recognize and fight cancer cells throughout the body. The virus is injected directly into the patient's tumors. It replicates (makes more of itself) inside the cancer cells until they burst, releasing more viruses that can infect additional cancer cells.

A completely different approach was used in a trial to treat 59 patients with leukemia, a type of blood cancer. The patients' own immune cells were removed and treated with a virus that genetically altered them to recognize a protein that sits on the surface of the cancer cells. After the immune cells were returned to the patients, 26 experienced complete remission.

Patients with Parkinson's disease gradually lose cells in the brain that produce the signaling molecule dopamine. As the disease advances, patients lose the ability to control their movements.

A small group of patients with advanced Parkinson's disease were treated with a retroviral vector to introduce three genes into cells in a small area of the brain. These genes gave cells that don't normally make dopamine the ability to do so. After treatment, all of the patients in the trial had improved muscle control.

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Researchers have been working for decades to bring gene therapy to the clinic, yet very few patients have received any effective gene-therapy treatments. But that doesn't mean gene therapy is an impossible dream. Even though gene therapy has been slow to reach patients, its future is very encouraging.

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CRISPR includes two different molecules in order to allow it to work. The two molecules are an enzyme called Cas9 as well as an organism's RNA. The RNA that is found in CRISPR is used to bind the sequence within the DNA. The enzyme Cas9 that is found in CRISPR plays a similar if not identical role in the DNA of the organism. (AH)

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CRISPR-Cas9 is a genome editing tool that is creating a buzz in the science world. It is faster, cheaper and more accurate than previous techniques of editing DNA and has a wide range of potential applications.

What is CRISPR-Cas9?

CRISPR-Cas9 is a unique technology that enables geneticists and medical researchers to edit parts of the genome by removing, adding or altering sections of the DNA sequence.

It is currently the simplest, most versatile and precise method of genetic manipulation and is therefore causing a buzz in the science world.

How does it work?

The CRISPR-Cas9 system consists of two key molecules that introduce a change (mutation) into the DNA. These are:

an enzyme called Cas9. This acts as a pair of 'molecular scissors' that can cut the two strands of DNA at a specific location in the genome so that bits of DNA can then be added or removed.

a piece of RNA called guide RNA (gRNA). This consists of a small piece of pre-designed RNA sequence (about 20 bases long) located within a longer RNA scaffold. The scaffold part binds to DNA and the pre-designed sequence 'guides' Cas9 to the right part of the genome. This makes sure that the Cas9 enzyme cuts at the right point in the genome.

The guide RNA is designed to find and bind to a specific sequence in the DNA. The guide RNA has RNA bases that are complementary to those of the target DNA sequence in the genome. This means that, at least in theory, the guide RNA will only bind to the target sequence and no other regions of the genome.

The Cas9 follows the guide RNA to the same location in the DNA sequence and makes a cut across both strands of the DNA.

At this stage the cell recognises that the DNA is damaged and tries to repair it.

Scientists can use the DNA repair machinery to introduce changes to one or more genes in the genome of a cell of interest.

Some bacteria have a similar, built-in, gene editing system to the CRISPR-Cas9 system that they use to respond to invading pathogens like viruses, much like an immune system.

Using CRISPR the bacteria snip out parts of the virus DNA and keep a bit of it behind to help them recognise and defend against the virus next time it attacks.

Scientists adapted this system so that it could be used in other cells from animals, including mice and humans.

What other techniques are there for altering genes?

Over the years scientists have learned about genetics and gene function by studying the effects of changes in DNA.

If you can create a change in a gene, either in a cell line or a whole organism, it is possible to then study the effect of that change to understand what the function of that gene is.

For a long time geneticists used chemicals or radiation to cause mutations. However, they had no way of controlling where in the genome the mutation would occur.

For several years scientists have been using 'gene targeting' to introduce changes in specific places in the genome, by removing or adding either whole genes or single bases.

Traditional gene targeting has been very valuable for studying genes and genetics, however it takes a long time to create a mutation and is fairly expensive.

Several 'gene editing' technologies have recently been developed to improve gene targeting methods, including CRISPR-Cas systems, transcription activator-like effector nucleases (TALENs) and zinc-finger nucleases (ZFNs).

The CRISPR-Cas9 system currently stands out as the fastest, cheapest and most reliable system for 'editing' genes.

What are the applications and implications?

CRISPR-Cas9 has a lot of potential as a tool for treating a range of medical conditions that have a genetic component, including cancer, hepatitis B or even high cholesterol.

Many of the proposed applications involve editing the genomes of somatic (non-reproductive) cells but there has been a lot of interest in and debate about the potential to edit germline (reproductive) cells.

Because any changes made in germline cells will be passed on from generation to generation it has important ethical implications.

Carrying out gene editing in germline cells is currently illegal in the UK and most other countries.

By contrast, the use of CRISPR-Cas9 and other gene editing technologies in somatic cells is uncontroversial. Indeed they have already been used to treat human disease on a small number of exceptional and/or life-threatening cases.

What's the future of CRISPR-Cas9?

It is likely to be many years before CRISPR-Cas9 is used routinely in humans.

Much research is still focusing on its use in animal models or isolated human cells, with the aim to eventually use the technology to routinely treat diseases in humans.

There is a lot of work focusing on eliminating 'off-target' effects, where the CRISPR-Cas9 system cuts at a different gene to the one that was intended to be edited.

Better targeting of CRISPR-Cas9

In most cases the guide RNA consists of a specific sequence of 20 bases. These are complementary to the target sequence in the gene to be edited. However, not all 20 bases need to match for the guide RNA to be able to bind.

The problem with this is that a sequence with, for example, 19 of the 20 complementary bases may exist somewhere completely different in the genome. This means there is potential for the guide RNA to bind there instead of or as well as at the target sequence.

The Cas9 enzyme will then cut at the wrong site and end up introducing a mutation in the wrong location. While this mutation may not matter at all to the individual, it could affect a crucial gene or another important part of the genome.

Scientists are keen to find a way to ensure that the CRISPR-Cas9 binds and cuts accurately. Two ways this may be achieved are through:

the design of better, more specific guide RNAs using our knowledge of the DNA sequence of the genome and the 'off-target' behaviour of different versions of the Cas9-gRNA complex.

the use of a Cas9 enzyme that will only cut a single strand of the target DNA rather than the double strand. This means that two Cas9 enzymes and two guide RNAs have to be in the same place for the cut to be made. This reduces the probability of the cut being made in the wrong place.

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CRISPR-Cas9 is a genome editing tool that is creating a buzz in the science world. It is faster, cheaper and more accurate than previous techniques of editing DNA and has a wide range of potential applications. CRISPR-Cas9 is a unique technology that enables geneticists and medical researchers to edit parts of the genome by removing, adding or altering sections of the DNA sequence.

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Legislation in the US- if involving humans must go through (OHRP) Office for Human Research Protections)+  Institutional Review Boards (IRB) and get approved
- Discuss physical/ethical/mental issues
The U.S. Food and Drug Administration (FDA) ensures that drugs, medical devices and biological products are safe and effective before they are used
- if approved then something goes wrong have the right to revoke approval

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In the United States, the Department of Health and Human Services (DHHS) has been charged with oversight of clinical trials. Two organizations within DHHS, the Office for Human Research Protections (OHRP) and the U.S. Food and Drug Administration (FDA), have specific authority described in the Code of Federal Regulations (CFR). All investigators, must comply with these regulations when conducting clinical gene therapy trials. The OHRP mandates that all research involving human subjects undergo Institutional Review Boards (IRB) review and approval. An IRB is charged with evaluating research risk to subjects and must approve research protocols and informed consent documents prior to beginning a study.
Another DHHS agency, the National Institutes of Health (NIH), oversees the conduct of federally funded clinical trials through a series of guidelines that add additional requirements to those specified in the CFR.

NIH - OBA - RAC
The NIH established the Recombinant DNA Advisory Committee (RAC) on October 7, 1974 in response to public concerns regarding the safety of manipulating genetic material through the use of recombinant DNA techniques. Although the RAC's responsibilities have evolved over time with scientific understanding and developments in this technology, it continues to serve the NIH as a critically important forum for open, public deliberation on the panoply of scientific, ethical, and legal issues raised by recombinant DNA technology and its basic and clinical research applications. A major responsibility of the RAC at present is to review human gene transfer research on behalf of the NIH. Human gene transfer trials conducted at, or sponsored by, institutions receiving NIH funding for recombinant DNA research are registered with the Office of Biotechnology Activities (OBA) and reviewed by the RAC. The OBA is responsible for the NIH system of oversight of recombinant DNA research. Clinical gene therapy protocols that raise novel or particularly important scientific, safety or ethical considerations are discussed by the RAC at one of its quarterly public meetings. RAC proceedings and reports are posted to the OBA website to enhance their accessibility to the scientific and lay publics.
The NIH also consults the RAC for advice concerning various advances in recombinant DNA technology, as well as ethical and safety considerations associated with novel or possibly risky forms of recombinant DNA research.

FDA - CBER
The U.S. Food and Drug Administration (FDA) is the primary government agency charged with protecting the health of U.S. citizens by ensuring that drugs, medical devices and biological products are safe and effective before they are used by doctors, nurses, other health care professionals, hospitals and consumers.
FDA's Center for Biologics Evaluation and Research (CBER) regulates human gene therapies, which fall under the legal definition of a "biologic." Manufacturers of gene therapy products must test their products extensively and meet FDA requirements for safety, purity and potency before they can be sold in the United States. A manufacturer who is considering selling a gene therapy product in the United States first must tell FDA of its intentions, and then must test the product in a laboratory and then in research animals. When a manufacturer is ready to study the gene therapy product in humans, it must obtain a special permission exemption from FDA before starting. This exemption is called an investigational new drug application or (IND). In the IND, the manufacturer explains how it intends to conduct the study, what possible risks may be involved and what steps it will take to protect patients, and provides data in support of the study. As part of the IND process, the manufacturer also must get approval from a committee of scientific and medical advisors and consumers (called an Institutional Review Board), which focuses on protecting persons who may participate in the study. Researchers also must inform the persons who may be part of the study about the study's potential risks and benefits, and obtain their consent.
When FDA's scientists receive an IND application for gene therapy, they review it carefully before permitting the manufacturer or researcher to begin the study. FDA may ask the study sponsor to do more laboratory tests and include more safeguards to ensure the safety of patients, such as giving patients smaller doses. As discussed in the next section, if unexpected problems arise, FDA may tell the manufacturer to change the study or stop it altogether.
FDA has not yet approved for sale any human gene therapy product.

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In the United States, the Department of Health and Human Services (DHHS) has been charged with oversight of clinical trials. Two organizations within DHHS, the Office for Human Research Protections (OHRP) and the U.S. Food and Drug Administration (FDA), have specific authority described in the Co...

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CRISPR is an acronym in which stands for Clustered Regularly Interspaced Short Palindromic Repeat. The name CRISPR refers to the unique sequences of DNA found in a genome. (AH)

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Have you heard? A revolution has seized the scientific community. Within only a few years, research labs worldwide have adopted a new technology that facilitates making specific changes in the DNA of humans, other animals, and plants. Compared to previous techniques for modifying DNA, this new approach is much faster and easier. This technology is referred to as "CRISPR," and it has changed not only the way basic research is conducted, but also the way we can now think about treating diseases [1,2].

What is CRISPR

CRISPR is an acronym for Clustered Regularly Interspaced Short Palindromic Repeat. This name refers to the unique organization of short, partially palindromic repeated DNA sequences found in the genomes of bacteria and other microorganisms. While seemingly innocuous, CRISPR sequences are a crucial component of the immune systems [3] of these simple life forms. The immune system is responsible for protecting an organism's health and well-being. Just like us, bacterial cells can be invaded by viruses, which are small, infectious agents. If a viral infection threatens a bacterial cell, the CRISPR immune system can thwart the attack by destroying the genome of the invading virus [4]. The genome of the virus includes genetic material that is necessary for the virus to continue replicating. Thus, by destroying the viral genome, the CRISPR immune system protects bacteria from ongoing viral infection.

How does it work?
Figure 1 ~ The steps of CRISPR-mediated immunity. CRISPRs are regions in the bacterial genome that help defend against invading viruses. These regions are composed of short DNA repeats (black diamonds) and spacers (colored boxes). When a previously unseen virus infects a bacterium, a new spacer derived from the virus is incorporated amongst existing spacers. The CRISPR sequence is transcribed and processed to generate short CRISPR RNA molecules. The CRISPR RNA associates with and guides bacterial molecular machinery to a matching target sequence in the invading virus. The molecular machinery cuts up and destroys the invading viral genome. Figure adapted from Molecular Cell 54, April 24, 2014 [5].
Interspersed between the short DNA repeats of bacterial CRISPRs are similarly short variable sequences called spacers (FIGURE 1). These spacers are derived from DNA of viruses that have previously attacked the host bacterium [3]. Hence, spacers serve as a 'genetic memory' of previous infections. If another infection by the same virus should occur, the CRISPR defense system will cut up any viral DNA sequence matching the spacer sequence and thus protect the bacterium from viral attack. If a previously unseen virus attacks, a new spacer is made and added to the chain of spacers and repeats.

The CRISPR immune system works to protect bacteria from repeated viral attack via three basic steps [5]:

Step 1) Adaptation - DNA from an invading virus is processed into short segments that are inserted into the CRISPR sequence as new spacers.

Step 2) Production of CRISPR RNA - CRISPR repeats and spacers in the bacterial DNA undergo transcription, the process of copying DNA into RNA (ribonucleic acid). Unlike the double-chain helix structure of DNA, the resulting RNA is a single-chain molecule. This RNA chain is cut into short pieces called CRISPR RNAs.

Step 3) Targeting - CRISPR RNAs guide bacterial molecular machinery to destroy the viral material. Because CRISPR RNA sequences are copied from the viral DNA sequences acquired during adaptation, they are exact matches to the viral genome and thus serve as excellent guides.

The specificity of CRISPR-based immunity in recognizing and destroying invading viruses is not just useful for bacteria. Creative applications of this primitive yet elegant defense system have emerged in disciplines as diverse as industry, basic research, and medicine.

What are some applications of the CRISPR system?
In Industry
The inherent functions of the CRISPR system are advantageous for industrial processes that utilize bacterial cultures. CRISPR-based immunity can be employed to make these cultures more resistant to viral attack, which would otherwise impede productivity. In fact, the original discovery of CRISPR immunity came from researchers at Danisco, a company in the food production industry [2,3]. Danisco scientists were studying a bacterium called Streptococcus thermophilus, which is used to make yogurts and cheeses. Certain viruses can infect this bacterium and damage the quality or quantity of the food. It was discovered that CRISPR sequences equipped S. thermophilus with immunity against such viral attack. Expanding beyond S. thermophilus to other useful bacteria, manufacturers can apply the same principles to improve culture sustainability and lifespan.

Beyond applications encompassing bacterial immune defenses, scientists have learned how to harness CRISPR technology in the lab [6] to make precise changes in the genes of organisms as diverse as fruit flies, fish, mice, plants and even human cells. Genes are defined by their specific sequences, which provide instructions on how to build and maintain an organism's cells. A change in the sequence of even one gene can significantly affect the biology of the cell and in turn may affect the health of an organism. CRISPR techniques allow scientists to modify specific genes while sparing all others, thus clarifying the association between a given gene and its consequence to the organism.

Rather than relying on bacteria to generate CRISPR RNAs, scientists first design and synthesize short RNA molecules that match a specific DNA sequence-for example, in a human cell. Then, like in the targeting step of the bacterial system, this 'guide RNA' shuttles molecular machinery to the intended DNA target. Once localized to the DNA region of interest, the molecular machinery can silence a gene or even change the sequence of a gene (Figure 2)! This type of gene editing can be likened to editing a sentence with a word processor to delete words or correct spelling mistakes. One important application of such technology is to facilitate making animal models with precise genetic changes to study the progress and treatment of human diseases.
Figure 2 ~ Gene silencing and editing with CRISPR. Guide RNA designed to match the DNA region of interest directs molecular machinery to cut both strands of the targeted DNA. During gene silencing, the cell attempts to repair the broken DNA, but often does so with errors that disrupt the gene-effectively silencing it. For gene editing, a repair template with a specified change in sequence is added to the cell and incorporated into the DNA during the repair process. The targeted DNA is now altered to carry this new sequence. In Medicine
With early successes in the lab, many are looking toward medical applications of CRISPR technology. One application is for the treatment of genetic diseases. The first evidence that CRISPR can be used to correct a mutant gene and reverse disease symptoms in a living animal was published earlier this year [7]. By replacing the mutant form of a gene with its correct sequence in adult mice, researchers demonstrated a cure for a rare liver disorder that could be achieved with a single treatment. In addition to treating heritable diseases, CRISPR can be used in the realm of infectious diseases, possibly providing a way to make more specific antibiotics that target only disease-causing bacterial strains while sparing beneficial bacteria [8]. A recent SITN Waves article discusses how this technique was also used to make white blood cells resistant to HIV infection [9].

The Future of CRISPR

Of course, any new technology takes some time to understand and perfect. It will be important to verify that a particular guide RNA is specific for its target gene, so that the CRISPR system does not mistakenly attack other genes. It will also be important to find a way to deliver CRISPR therapies into the body before they can become widely used in medicine. Although a lot remains to be discovered, there is no doubt that CRISPR has become a valuable tool in research. In fact, there is enough excitement in the field to warrant the launch of several Biotech start-ups that hope to use CRISPR-inspired technology to treat human diseases [8].
Ekaterina Pak is a Ph.D. student in the Biological and Biomedical Sciences program at Harvard Medical School.
References:

1. Palca, J. A CRISPR way to fix faulty genes. (26 June 2014) NPR < http://www.npr.org/blogs/health/2014/06/26/325213397/a-crispr-way-to-fix-faulty-genes> [29 June 2014]

2. Pennisi, E. The CRISPR Craze. (2013) Science, 341 (6148): 833-836.

3. Barrangou, R., Fremaux, C., Deveau, H., Richards, M., Boyaval, P., Moineau, S., Romero, D.A., and Horvath, P. (2007). CRISPR provides acquired resistance against viruses in prokaryotes. Science 315, 1709-1712.

4. Brouns, S.J., Jore, M.M., Lundgren, M., Westra, E.R., Slijkhuis, R.J., Snijders, A.P., Dickman, M.J., Makarova, K.S., Koonin, E.V., and van der Oost, J. (2008). Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321, 960-964.

5. Barrangou, R. and Marraffini, L. CRISPR-Cas Systems: Prokaryotes Upgrade to Adaptive Immunity (2014). Molecular Cell 54, 234-244.

6. Jinkek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.(2012) 337(6096):816-21.

7. CRISPR reverses disease symptoms in living animals for first time. (31 March 2014). Genetic Engineering and Biotechnology News. < http://www.genengnews.com/gen-news-highlights/crispr-reverses-disease-symptoms-in-living-animals-for-first-time/81249682/> [27 July 2014]

8. Pollack, A. A powerful new way to edit DNA. (3 March 2014). NYTimes < http://www.nytimes.com/2014/03/04/health/a-powerful-new-way-to-edit-dna.html?_r=0> [16 July 2014]

9. Gene editing technique allows for HIV resistance? < http://sitn.hms.harvard.edu/flash/waves/2014/gene-editing-technique-allows-for-hiv-resistance/> [13 June 2014]

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CRISPR: A game-changing genetic engineering technique - Science in the News

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Have you heard? A revolution has seized the scientific community. Within only a few years, research labs worldwide have adopted a new technology that facilitates making specific changes in the DNA of humans, other animals, and plants. Compared to previous techniques for modifying DNA, this new approach is much faster and easier.

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What is gene therapy and how does it work? BS

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What is gene therapy and how does it work? BS

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Gene therapy is the idea of treating, curing, and preventing a disease or illness. The way gene therapy works is that scientist package healthy DNA into a device called a vector. This device is then released into a tissue that is affected by the illness. The vector travels to the infected area and releases it's DNA package. Once the DNA is placed, it tells the body to produce the proteins that will treat the infected area.