Skip to comments.Study first to show potential of light-activated gene therapy for knee injuries
Posted on 04/25/2006 2:08:02 PM PDT by Neville72
Could cellular defenses against sunlight be the key to effective gene therapy?
An early study has demonstrated for the first time that laser light can target gene therapy right up to the edge of damaged cartilage, while leaving nearby healthy tissue untouched, according to an article published in the April edition of the Journal of Bone and Joint Surgery. True repair of injuries to articular cartilage would enable millions of patients, currently consigned to worsening arthritis and joint replacement, to return to athletic exercise.
Study authors say that dramatic progress is being made toward a new form of light-activated gene therapy for cartilage repair that will be safe, fast, easy on patients and compatible with techniques used by most surgeons (e.g. arthroscopy). Beyond knee injuries, researchers believe the technology could one day guide precision gene therapy for cancer or heart disease, restore vision by repairing eye tissue and rebuild skin destroyed by burns.
As many as 10 percent of young, active patients with bleeding in the knee joint following injury have damaged articular cartilage, the sponge-like layer that protects joints from the punishing impact of running and jumping. Unlike a broken leg bone that mends itself, damaged cartilage does not. Over time, damaged cartilage erodes to become a leading cause of osteoarthritis, which causes joint inflammation and pain in 40 million Americans. Many eventually require total joint replacement surgery.
"For years researchers have been trying to turn on gene therapy precisely within areas of damaged tissue without harming surrounding healthy tissue," said Edward M. Schwarz, Ph.D., professor of Orthopaedics within the Center for Musculoskeletal Research at the University of Rochester Medical Center. "Our study shows that we can use our cellular defenses against, of all things, sunlight, to finally achieve safe, precise control over tissue repair."
Limits of Current Therapy
Present surgical treatments for damaged cartilage like lavage and debridement relieve pain and inflammation, but leave a hole in the cartilage. Other techniques are too expensive and invasive or apply to few injuries, according to the study authors. Given the limits of surgical repair, researchers have been attempting for years to use gene therapy to truly repair, or re-grow, articular cartilage.
The blueprint for the human body is encoded in genes, which store information that is converted into proteins which carry out bodily functions. Gene therapy works by inserting specially designed genes into cells that can, for instance, direct cells to divide, which makes a tissue grow. Animals like amphibians re-grow amputated limbs, including articular cartilage, but humans grow cartilage only once, during development, without help.
To deliver the genes into cells, researchers need an effective delivery vehicle, or vector. Viruses have evolved for millions of years to invade human cells and insert DNA into their prey. Researchers have harnessed their useful qualities while removing the harmful ones. Unfortunately, no viral vector to date has been able to turn on genes only in the damaged areas because they infect cells in nearby healthy tissue as well.
Sunlight: A Specific Solution
The solution to the problem of how to target some cells for gene therapy, while missing their neighbors, came from a strange source: our cellular defenses against sunlight. The sun gives off ultraviolet (UV) light, which can cause destructive changes (genetic mutations) when exposed to sensitive molecules like DNA. If not defended against, the changes in DNA caused by UV light would cause humans to constantly develop cancer, for instance, in exposed tissue. Thus, an SOS system evolved that calls for genetic repairs when UV light causes too many mutations. Specifically, UV light turns on signaling proteins called stress kinases, which activate DNA polymerase, the enzyme that re-builds DNA chains when damaged.
Current technologies can direct UV light with great precision. That, combined with the ability of UV light to turn on DNA polymerase, has granted researchers the ability to turn on gene therapy in one cell, but not its neighbors. In recent years, researchers have been working to develop a system where UV light pre-treats target tissue, so that only the cells exposed to light gain the ability to copy themselves and grow. What remained was to find the right combination of vector and light to make the therapy safe as well as effective.
Recombinant adeno-associated virus (rAAV) turned out to be the right vector because it has evolved to deliver into the cell only a single strand of deoxyribonucleic acids (DNA), not the usual two strands of molecules. A second strand of DNA must be built by DNA polymerase to form active, double-stranded DNA before genes, or a gene therapy, can take effect. Single-stranded delivery is the key rAAV's usefulness as part of light-activated gene therapy because, of the all the cells infected with a gene therapy, only those struck by UV light will turn on DNA polymerase. Only those cells will activate the therapeutic gene, divide and re-grow tissue.
In addition, rAAV vectors used in human trials appear to be very safe. Other viral vectors used in early attempts at human gene therapy in some cases made permanent changes that caused violent immune reactions, and even cancer in a few cases, along with the therapeutic changes made. With safety more important than ever, it is almost impossible for rAAV to be dangerous because it does not contain any viral genes and is delivered using a harmless virus, Schwarz said. Lastly, rAAV appears to be the kind of virus that makes changes in the DNA of human cells for a few weeks, but then stops. This happens to be the perfect amount of time for a regenerative gene therapy, shutting down before too much re-growth occurs, according to researchers.
The Long and Short of UV Light
When discussing light-activated gene therapy, UV light used is categorized by its wavelength. The higher the intensity of light; the shorter its wavelength. An early attempt at light-activated gene therapy used short wavelength UV light (254 nm) because it can turn on DNA polymerase and direct tissue to re-grow. Unfortunately, it also destroys DNA in nearby healthy tissues. To overcome this obstacle, Schwarz and colleagues have been developing a system based on long-wavelength UV light with the goal of safely achieving site-specific therapy.
Long wavelength UV light is not absorbed by DNA, but still triggers the same natural SOS system. Instead of triggering the system by directly causing dangerous mutations like short wavelength UV light, the longer wavelength creates molecules called free radicals. Free radicals are highly reactive molecules that can themselves create mutations in DNA, but not nearly to the extent of short wavelength light. Free radicals, thus, turn on gene therapy before mutations are formed.
In addition, long wavelength UV light can be captured and transmitted through a cable, allowing for arthroscopic approaches, where short wavelength UV cannot. In arthroscopy, surgeons examine the injury with a fiber optic cable through the small incision, and in many cases make the repair through the same incision. Traditional surgery requires large incisions with long recovery times. This arthroscopic capability gives light-activated gene therapy the potential to become part of most orthopaedic medical practices in the country.
Thirdly, researchers developed a laser that can deliver the effective dose of long wavelength UV light within seconds. Using this laser light source means that the light-activated gene therapy could be completed in moments, not hours, for less pain and trouble on the part of patients.
The current study evaluated the ability of long-wavelength ultraviolet light to stimulate gene expression following infection by rAAV. Researchers evaluated the safety and efficacy of long-wavelength ultraviolet laser light to induce light-activated gene therapy in articular cartilage cells (chondrocytes). The study authors believe this is the first demonstration that site-directed gene delivery can safely and effectively treat articular defects in higher animal cartilage cells.
Given the safety concerns found with short wavelength, researchers were excited to find that the new long wavelength system is an order of magnitude more likely to turn on gene therapy as designed than to cause death by mutation (cytotoxity). Along with previous studies, the current research found rAAV to be highly efficient at turning on gene therapy in articular chondrocytes. Pretreatment with 6000 Joules per meter squared, a standard dose of UV light, led to a tenfold increase in the effect of gene therapy in target cells after one week. In addition, nearly half of cells exposed to the light expressed the inserted, therapeutic gene.
Schwarz is also president and founder of LAGeT, Inc. In 2003, LAGeT Inc. licensed the technology used in the study from the medical center, where he and his partners developed it. LAGeT is initially focusing on the development of therapies for musculoskeletal diseases, including osteoarthritis and spine-related repair, but recognizes its broader potential. One early project will attempt to use rAAV gene therapy to re-grow bone in patients who have lost bone to severe trauma or a bone tumor, and would otherwise lose the limb.
"While LAGeT's current focus is on musculoskeletal-related disorders, this approach could just as readily be used to deliver therapeutic genes to treat cancer and cardiovascular disease," Schwarz said. "We believe this area of research could represents a quantum leap from current treatments if successful because nothing out there yet brings about true regeneration of lost tissue."
Very exciting! Especially for people with grunged up knees like me.
Thanks for posting this.
Interesting, having had over 8 major orthopedic surgeries
including a TKR
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