Gene therapy methods

One of the most amazing genetic applications in medicine is gene therapy. Also known as somatic gene therapy and therapeutic gene therapy, this procedure involves inserting (or sometimes deleting) portions of the genes in diseased patients so that they can be cured and live healthier lives.

Bone marrow

Two methods exist for inserting genetic material into human chromosomes. The first, called the ex vivo technique, involves surgically removing cells from the affected tissue area, injecting or splicing the new DNA (the DNA that will correct the disease) into the cells and letting them divide in cultures. The new tissues are placed back into the affected area of the patient. Often, doctors need only culture the patient’s bone marrow because it produces the blood that will eventually travel throughout the body. This type of surgery, however, is especially painful, and patients usually have to undergo it twice--once to extract the marrow and then again to replace it--because the culturing time takes many hours to complete.The second method is called the in vivo technique and requires no surgery or even anesthesia. In this process, the therapeutic DNA is injected directly into the body cells, usually via one of two types of viruses. The most frequently used type is the very simple retrovirus. Dr. Richard Mulligan of MIT has synthetically created the perfect retrovirus: it has no reproduction sequence and exists solely to deliver therapeutic DNA during gene therapy. It has no viral DNA (DNA that would make the cell--and you-- sick) whatsoever and only carries the new DNA that has been spliced into it. After injecting the diseased cell with the new therapeutic DNA, it then dies. Using retroviruses is very safe and provides long-lasting effects. Unfortunately, the new DNA it injects will only help the new daughter cells and not those that already exist. The second type of virus used for the in vivo technique is called an adenovirus, the equivalent of the common cold virus. Although this virus will also die after injecting its spliced therapeutic DNA, it will be attacked by the immune system and the patient will suffer from a temporary sore throat and runny nose. The adenovirus works the same way the retrovirus does, but its effects are much more immediate--within 48 hours. Unlike the retrovirus, though, the new DNA’s effects wear off within weeks. Scientists like the fact that only a few millimeters of altered adenovirus solution is needed to cure the patient, whereas several liters of retrovirus are needed to obtain a much slower result.

Liver cell nucleus

There are other gene therapy techniques, although they aren’t as frequently used. One method involves inserting therapeutic DNA into cultured endothelium tissue (endothelium is the membrane that lines all of the blood vessels) and then grafting it into the patient. Another technique requires the patient to receive an electric shock while submerged in a bath of a therapeutic DNA solution. The shock opens the skin pores, allowing the DNA to enter. Still other options include skin grafts, connective tissue grafts, and injecting the liver with the therapeutic DNA

New Gene Therapy Technique To Cure Brain Disorders

To treat neurodegenerative diseases such as Alzheimer's, UCSF researchers have developed a new strategy for delivering gene therapy to the brain cells that stand to benefit most. Gene therapy to treat degenerative brain disorders is still regarded as a promising approach, and is again being tested in experimental clinical trials. It is difficult to get drugs that are large molecules, such as proteins, into the brain. Gene therapy puts genes into brain cells, so they can make their own therapeutic proteins. But then, there is still the problem of delivering the genes. Many drug developers continue to choose a common virus, called adeno-associated virus, as the gene delivery vehicle. But the brain poses special challenges for drug delivery. The favored approach has been to inject the gene therapy drug through several cannulae — kind of like very long, extremely thin straws — threaded through holes drilled in the skull and guided into place with fancy imaging equipment. Despite all that special effort, it remains difficult to control where the drug goes, and how far it goes toward reaching targeted cells.

Now a team at UCSF led by neuroscientist Dr. Krystof Bankiewicz has developed a way to get nerve cells themselves to help disperse gene therapy to targeted cells.

In research reported in the online edition of the Proceedings of the National Academy of Sciences, Bankiewicz and colleagues injected adeno-associated virus bearing either therapeutic or marker genes into specific cells within a brain structure called the thalamus, situated below the cerebral cortex. Neurons in the thalamus make specific connections to cells in the cortex through branching processes called axons.

Bankiewicz's research team found that gene therapy delivered to cells in the thalamus could be made to spread efficiently through axons to all cortical regions, where it made the gene-encoded proteins.

"For the first time, specific regions of the cortex can be supplied with therapeutic agents by targeting defined regions of the thalamus," Bankiewicz says. "Critical and widespread cortical regions can be reached through each individual cannula, using this new approach."

The technique is called convection-enhanced delivery. The fluid containing the gene therapy is injected under pressure, delivered in pulses. The pulsation acts a pump to deliver the therapy over greater distances.

"This procedure can now be performed under the direct guidance and control of interventional MRI, to assure precise delivery of the viral vector," Bankiewicz says.

"Translational experiments now are in progress to evaluate the potential of this unique gene delivery technology for the treatment of cortical dementias such as Alzheimer's disease and lysosomal storage disorders in children."

How Is the Gene for Cystic Fibrosis Inherited?

Cystic fibrosis is inherited in an autosomal recessive pattern, which means that two copies of the cystic fibrosis gene in each cell are altered. In most cases, the parents of an individual with an autosomal recessive disorder are carriers of one copy of the altered gene, but do not show signs and symptoms of the disorder.

When two cystic fibrosis carriers have a baby, the baby has a:

• One in four chance of inheriting two abnormal CFTR genes and having cystic fibrosis.
  
  
• One in four chance of inheriting two normal CFTR genes and not having cystic fibrosis or being a carrier.
  
  
• Two in four chance of inheriting one normal CFTR gene and one abnormal CFTR gene. The baby will not have cystic fibrosis, but will be a carrier like its parents.

Gene therapy for pancreatic cancer

Gene transfer technology has the potential to revolutionize cancer treatment. Developments in molecular biology, genetics, genomics, stem cell technology, virology, bioengineering, and immunology are accelerating the pace of innovation and movement from the laboratory bench to the clinical arena. Pancreatic adenocarcinoma, with its particularly poor prognosis and lack of effective traditional therapy for most patients, is an area where gene transfer and immunotherapy have a maximal opportunity to demonstrate efficacy. In this review, we have discussed current preclinical and clinical investigation of gene transfer technology for pancreatic cancer. We have emphasized that the many strategies under investigation for cancer gene therapy can be classified into two major categories. The first category of therapies rely on the transduction of cells other than tumor cells, or the limited transduction of tumor tissue. These therapies, which do not require efficient gene transfer, generally lead to systemic biological effects (e.g., systemic antitumor immunity, inhibition of tumor angiogenesis, etc) and therefore the effects of limited gene transfer are biologically "amplified." The second category of gene transfer strategies requires the delivery of therapeutic genetic material to all or most tumor cells. While these elegant approaches are based on state-of-the-art advances in our understanding of the molecular biology of cancer, they suffer from the current inadequacies of gene transfer technology. At least in the short term, it is very likely that success in pancreatic cancer gene therapy will involve therapies that require only the limited transduction of cells. The time-worn surgical maxim, "Do what's easy first," certainly applies here.

Cystic Fibrosis Gene Therapy

With gene therapy, treatment targets the cause of cystic fibrosis rather than just treating the symptoms. Although the first gene therapy experiments have involved lung cells, scientists hope that these technologies will be adapted to treat other organs affected by cystic fibrosis.

Is There a "Cystic Fibrosis Gene?"

The cause of cystic fibrosis (CF) is a defect in the cystic fibrosis transmembrane conductance regulator (CFTR) gene.

This gene for cystic fibrosis makes a protein that controls the movement of salt and water in and out of your cells. In people with cystic fibrosis, the gene does not work effectively. As a result, cells that line the passageways of the lungs, pancreas, and other organs produce abnormally thick, sticky mucus. This mucus obstructs the airways and glands, which causes the characteristic signs and symptoms of cystic fibrosis.

Other factors may influence the course of cystic fibrosis. For example, changes in genes other than CFTR might help explain why some people with the disease are more severely affected than others. However, most of these genetic changes have not been identified.

Gene therapy: A cure for congenital blindness

Congenital Blindness is blindness that occurs when a child is born. There are many forms of congenital blindness. One specific form of congenital blindness is Leber’s Congenital Amaurosis.

Leber’s Congenital Amaurosis (LCA) is a form of blindness that is usually found at a very young age. It is extremely rare and occurs in 3 in 100,000 newborns.

A diagram of how gene therapy works

Though LCA does not currently have a cure, experts have been successfully using gene therapy to allow patients with the disease to gain vision. Gene therapy is a process in which a working gene is inserted into a patient to repair a malfunctioning gene.

In 2007, a team of experts at the University of Pennsylvania and Children’s Hospital of Philadelphia, conducted gene therapy on twelve patients with LCA. The above diagram shows how the gene therapy worked.

1. An eye syringe containing a vector, or genetically engineered virus, is injected into the patient’s photoreceptor cells.
2. The vector releases the healthy gene, in this case, the RPE65 gene.
3. After gene therapy was performed on twelve patients, vision was almost completely restored to their eyes. The restored vision has been permanent from the time of the experiment (started in October 2007).

What are the ethical issues surrounding gene therapy?

 

Because gene therapy involves making changes to the body’s set of basic instructions, it raises many unique ethical concerns. The ethical questions surrounding gene therapy include:

• How can “good” and “bad” uses of gene therapy be distinguished?
• Who decides which traits are normal and which constitute a disability or disorder?
• Will the high costs of gene therapy make it available only to the wealthy?
• Could the widespread use of gene therapy make society less accepting of people who are different?
• Should people be allowed to use gene therapy to enhance basic human traits such as height, intelligence, or athletic ability?

Current gene therapy research has focused on treating individuals by targeting the therapy to body cells such as bone marrow or blood cells. This type of gene therapy cannot be passed on to a person’s children. Gene therapy could be targeted to egg and sperm cells (germ cells), however, which would allow the inserted gene to be passed on to future generations. This approach is known as germline gene therapy.

The idea of germline gene therapy is controversial. While it could spare future generations in a family from having a particular genetic disorder, it might affect the development of a fetus in unexpected ways or have long-term side effects that are not yet known. Because people who would be affected by germline gene therapy are not yet born, they can’t choose whether to have the treatment. Because of these ethical concerns, the U.S. Government does not allow federal funds to be used for research on germline gene therapy in people.

How does gene therapy work?

Gene therapy is designed to introduce genetic material into cells to compensate for abnormal genes or to make a beneficial protein. If a mutated gene causes a necessary protein to be faulty or missing, gene therapy may be able to introduce a normal copy of the gene to restore the function of the protein.

A gene that is inserted directly into a cell usually does not function. Instead, a carrier called a vector is genetically engineered to deliver the gene. Certain viruses are often used as vectors because they can deliver the new gene by infecting the cell. The viruses are modified so they can’t cause disease when used in people. Some types of virus, such as retroviruses, integrate their genetic material (including the new gene) into a chromosome in the human cell. 

Other viruses, such as adenoviruses, introduce their DNA into the nucleus of the cell, but the DNA is not integrated into a chromosome.

The vector can be injected or given intravenously (by IV) directly into a specific tissue in the body, where it is taken up by individual cells. Alternately, a sample of the patient’s cells can be removed and exposed to the vector in a laboratory setting. The cells containing the vector are then returned to the patient. If the treatment is successful, the new gene delivered by the vector will make a functioning protein.

Researchers must overcome many technical challenges before gene therapy will be a practical approach to treating disease. For example, scientists must find better ways to deliver genes and target them to particular cells. They must also ensure that new genes are precisely controlled by the body.

What is gene therapy?

Genes, which are carried on chromosomes, are the basic physical and functional units of heredity. Genes are specific sequences of bases that encode instructions on how to make proteins. Although genes get a lot of attention, it’s the proteins that perform most life functions and even make up the majority of cellular structures. When genes are altered so that the encoded proteins are unable to carry out their normal functions, genetic disorders can result.

Gene therapy is a technique for correcting defective genes responsible for disease development. Researchers may use one of several approaches for correcting faulty genes:

• A normal gene may be inserted into a nonspecific location within the genome to replace a nonfunctional gene. This approach is most common.
  
• An abnormal gene could be swapped for a normal gene through homologous recombination.
  
• The abnormal gene could be repaired through selective reverse mutation, which returns the gene to its normal function.
  
• The regulation (the degree to which a gene is turned on or off) of a particular gene could be altered