Scientists test gene therapy for AIDS, sickle cell cures
By Chukwuma Muanya
FOLLOWING the apparent success of a case in which German doctors cured a man of Human Immuno-deficiency Virus (HIV)/Acquired Immune Deficiency Syndrome (AIDS) using a bone marrow transplant, American researchers have developed a mouse model that allows pre-clinical testing of their new gene therapy- protocol that offers real hope as cure for the deadly virus.
A University of California (UC) Davis, United States, stem cell researcher, Prof. Gerhard Bauer, has been working for more than 10 years on a similar cure for AIDS based on replacing the devastated immune system of an HIV-infected patient with stem cells that have been engineered to resist human immunodeficiency syndrome.
Also, using a harmless virus to insert a corrective gene into mouse blood cells, scientists at St. Jude Children's Research Hospital, United States, have alleviated sickle cell disease pathology. In their studies, the researchers found that the treated mice showed essentially no difference from normal mice.
Although the scientists caution that applying the gene therapy to humans presents significant technical obstacles, they believe that the new therapy will become an important treatment for the disease. The experiments were reported in the advanced online issue of the journal Molecular Therapy.
According to a study pre-published online in Blood, the official journal of the American Society of Hematology, stem cells derived from bone marrow may serve as a novel therapeutic option to treat a disease called epidermolysis bullosa (EB), a disorder characterized by extraordinarily fragile skin.
Epidermolysis bullosa is a disorder characterised by extraordinarily fragile skin and blistering on touch, akin to third degree burns. While the disease is often lethal in the neonatal period, more severe forms of the disease, such as recessive dystrophic EB (referred to as RDEB), can lead to years of painful blistering and mutilating scarring. The condition is caused by significantly reduced collagen type 7 protein (col7) production, a key component of the anchoring fibrils that connect the cutaneous membranes to the dermis of the skin and mucosal tissues in the gastrointestinal tract. A lack of these fibrils means the dermal-epidermal connection is very sensitive, and any action, which can include simple functions such as walking or eating, and the touch of clothing, creates friction between the skin layers that creates blisters and painful sores.
Children with RDEB, who are often referred to as "butterfly children" because their skin is said to be as sensitive as butterfly wings, develop painful skin and mucosal blistering, mutilating scarring, alopecia (hair loss), and other erosions shortly after birth. As a result of the extreme fragility of the skin and the chronic trauma of friction, RDEB patients often develop squamous cell carcinomas (a form of skin cancer). There is currently no cure for the disease, and palliative care includes complex bandaging, surgical removal of damaged tissue, and nutritional support.
Bauer presented the preliminary results of his latest research at the 50th annual meeting of the American Society for Hematology in San Francisco on Sunday, December 7, 2008. He and his UC Davis research team presented a poster detailing the development of a mouse model that allows pre-clinical testing of their new gene-therapy protocol, which they hope will pave the way for human clinical trials within five years.
Bauer, an assistant professor of hematology and oncology and director of a good manufacturing practice (GMP) laboratory now under construction in the new UC Davis Institute for Regenerative Cures in Sacramento, United States, explained: "The case in Germany was a natural gene-therapy experiment. We are working on a similar approach to genetically engineer a patient's own stem cells in a way that mimics this natural immunity. The German case offers further proof that genetic engineering provides a pathway to success, and gene therapy offers real hope as a cure for AIDS."
Last month, German doctors reported that they had cured a 42-year-old of acquired immune deficiency syndrome, or AIDS. The patient, an American living in Berlin, also had leukemia, which is best treated by a bone marrow transplant. Thinking they might be able to cure the man of both diseases, the physicians gave him a bone marrow transplant from a person with natural immunity to HIV. The patient has now lived for 20 months since the transplant without any detectable traces of HIV.
To establish similar immunity in HIV patients, the UC Davis team manipulated human skin cells to give these cells many of the same properties as stem cells. These transformed cells, called induced pluripotent stem (IPS) cells, are capable of differentiating into, among other cell types, hematopoietic stem cells, which are normally found in bone marrow and are responsible for producing the various types of immune cells.
"If we can replace normal immune cells with HIV-resistant ones, we can cure AIDS," Bauer said.
Bauer and stem cell program research associate Joseph Anderson have developed several anti-HIV genes that they plan to insert into IPS cells using standard gene-therapy techniques and viral vectors (viruses that efficiently insert the genes they carry into host cells). These engineered IPS cells could then be differentiated into bone marrow stem cells and introduced into the patient using a procedure similar to a bone marrow transplant.
"The hope is that one day we will use a patient's own skin cells to develop the engineered IPS cells to avoid possible rejection," said Bauer, who worked on clinical HIV gene therapy trials at Childrens Hospital Los Angeles before coming to UC Davis. "As in the German case, the end result would be an immune system that produces HIV-resistant immune cells."
In theory, the experimental treatment would result in an immune system that remains functional, even in the face of an HIV infection, but would halt or slow the progression toward AIDS.
"The anti-HIV genes take advantage of how HIV works," added Anderson, who is now writing a paper about the investigation. "The virus targets cells that are descendants of hemopoeitic stem cells."
During the first stages of infection, HIV targets macrophage cells, gaining entrance into the cell by binding to a receptor called CCR5 on the cell's surface. Later in the infection it targets CD4+ T cells, binding to the CXCR4 receptor on the surface of these cells and bringing on full-blown AIDS.
What researchers discovered is that there is a natural mutation in less than 1 percent of Caucasians that results in a lack of CCR5 receptors on any of their cells.
"We also found that these people are naturally resistant to HIV," said Bauer. "So, more than 10 years ago, we began our work creating a gene that would knock down expression of CCR5 and other key receptors and interfere with other routes of HIV infection."
For IPS-based anti-HIV gene therapy to become reality, UC Davis researchers must first conduct safety and efficacy trials. Researchers have created a mouse model that replicates a normally functioning human immune system.
"We can now move forward and test the safety of the viral vectors, as well as the ability of anti-HIV genes to inhibit HIV infection," noted Anderson. "The humanized mouse model is an important step toward bringing this possible cure to patients."
Bauer and Anderson are hoping to demonstrate in their mouse model that HIV-infection cannot occur following their gene therapy treatment, providing the needed confidence in safety before embarking on clinical trials. This work and studies on the clinical use of IPS cells, Bauer predicts, will lead to a cure for AIDS.
"A real cure will come when we can replace all the hematopoietic stem cells with HIV resistant stem cells. What is so exciting is that we're clearly on the path of doing just that," said Bauer.
Meanwhile, Prof. Derek Persons, assistant member in the St. Jude Department of Hematology said: "While this is a very useful treatment for the disease, our studies indicated that it might be possible to cure the disorder if we could use gene transfer to permanently increase fetal hemoglobin levels."
He and his colleagues developed a technique to insert the gene for gamma-globin into blood-forming cells using a harmless viral carrier. The researchers extracted the blood-forming cells, performed the viral gene insertion in a culture dish and then re-introduced the altered blood-forming cells into the body. The hope was that those cells would permanently generate red blood cells containing fetal hemoglobin, alleviating the disease.
The researchers used a strain of mouse with basically the same genetic defect and symptoms as humans with sickle cell disease. The scientists introduced the gene for gamma-globin into the mice's blood-forming cells and then introduced those altered cells into the mice.
The investigators found that months after they introduced the altered blood-forming cells, the mice continued to produce gamma-globin in their red blood cells.
"When we examined the treated mice, we could detect little, if any, disease using our methods," said Persons, the paper's senior author. "The mice showed no anemia, and their organ function was essentially normal."
The researchers also transplanted the altered blood-forming cells from the original treated mice into a second generation of sickle cell mice to show that the gamma-globin gene had incorporated itself permanently into the blood-forming cells. Five months after that transplantation, the second generation of mice also showed production of fetal hemoglobin and correction of their disease.
"We are very encouraged by our results," Persons said. "They demonstrate for the first time that it is possible to correct sickle cell disease with genetic therapy to produce fetal hemoglobin. We think that increased fetal hemoglobin expression in patients will be well tolerated and the immune system would not reject the hemoglobin, in comparison to other approaches."
While Persons believes that the mouse experiments will lead to treatments in humans, he cautioned that technical barriers still need to be overcome. "It is far easier to achieve high levels of gene insertion into mouse cells than into human cells," he said. "In our mouse experiments, we routinely saw one or two copies of the gamma-globin gene inserted into each cell. However, in humans this insertion rate is at least a hundred-fold less."
Persons' laboratory is currently working with other animal and human cells to develop methods to achieve a high enough gene insertion rate to make the gene therapy clinically useful.
Prof. Jakub Tolar, of the University of Minnesota and lead author of the study published in Blood said: "We have been looking into stem cells as viable treatment options for correction of conditions such as epidermolysis bullosa, because they can produce extracellular matrix proteins. In this condition, the skin, the largest organ in the body, can significantly benefit from a renewable source of healthy cells that can help improve the connection between the dermis and epidermis and strengthen the skin against everyday stresses."
In this study, researchers worked with a mouse model of RDEB-infused bone marrow cells to determine if they would increase production of the col7 protein and formation of anchoring fibrils, and improve survival in the mouse recipients. The research team used bone marrow cells enriched for hematopoietic (stem cells that can develop into most blood cell types) and progenitor cells to increase the concentration of cells with the capacity to produce col7. The team tested these cells against non-enriched stem cells to determine their benefit to the treated mice.
Results of the study found that when injected into mice with RDEB, these specially selected marrow-derived stem cells diminished the disease process. They traveled to the diseased skin areas, increased protein and anchoring fibrils, prevented blister formation and extended survival. In contrast to other marrow cells, the selected cells extended the median survival time versus untreated or non-enriched marrow-treated recipients (10.0 versus 5.6 versus 6.0 days, respectively). Three of the 20 mice treated with the enriched cells benefited enough from the treatment to survive longer than the treatment period (untreated RDEB mice usually die within two weeks). Importantly, each survivor demonstrated marked improvement of new blister formation (blisters develop consistently in the areas of trauma, including footpads due to walking or in the oral cavity due to eating) with some evidence of old blisters healing.
"Our data provide the first evidence that a selected population of marrow cells can connect the epidermis and dermis in a mouse model of the disease and offer a potentially valuable approach for treatment of human RDEB and other extracellular matrix disorders. These results provide proof of principle of bone marrow transfer to repair the basement membrane defect in RDEB, and they warrant a clinical trial to assess the safety and efficacy of treatment of human RDEB by means of hematopoietic cell transplantation," said Tolar.
Research suggests that the systemic infusion of wild-type bone marrow cells could provide benefit to other human disorders of the extracellular matrix. Efforts are underway to identify the requirements of bone marrow-derived stem cells capable of efficiently homing to wounded skin and producing an array of extracellular matrix proteins. As the principal advantage of systemic therapy is its potential to target not only the skin but also the mucosa of the mouth and gastrointestinal tract, the clinical testing of efficacy of human bone marrow for the treatment of human RDEB is underway to determine whether it is of more substantial benefit than local protein, gene, or cellular therapies currently being investigated by other researchers.
An estimated 50 in one million live births are diagnosed with EB. The disorder occurs in every racial and ethnic group throughout the world and affects both sexes.
