New Gene Therapy Technique On Induced Pluripotent Stem Cells Holds Promise In Treating Immune System Disease

Researchers have developed an effective technique that uses gene therapy on stem cells to correct chronic granulomatous disease (CGD) in cell culture, which could eventually serve as a treatment for this rare, inherited immune disorder, according to a study published in Blood, the Journal of the American Society of Hematology.

CGD prevents neutrophils, a type of white blood cell of the immune system, from making hydrogen peroxide, an essential defense against life-threatening bacterial and fungal infections. Most cases of CGD are a result of a mutation on the X chromosome, a type of CGD that is called "X-linked" (X-CGD).

While antibiotics can treat infections caused by X-CGD, they do not cure the disease itself. Patients with X-CGD can be cured with a hematopoietic stem cell (HSC) transplant from healthy bone marrow; however, finding a compatible donor is difficult. Even with a suitable donor, patients are at risk of developing graft-versus-host disease (GVHD), a serious and often deadly post-transplant complication that occurs when newly transplanted donor cells recognize a recipient's own cells as foreign and attack the patient's body.

Another treatment option under development for X-CGD is gene therapy, a technique for correcting defective genes responsible for disease development that involves manipulation of genetic material within an individual's blood-forming stem cells using genetically engineered viruses. However, this gene therapy has so far proved to be inefficient at correcting X-CGD. In addition, these engineered viruses insert new genetic material at random locations in the blood-forming stem cell genome, putting patients at significantly higher risk for developing genetic mutations that may eventually lead to serious blood disorders, including blood cancer.

In order to develop a more effective and safer gene therapy for X-CGD, researchers from the National Institute of Allergy and Infectious Disease (NIAID) at the National Institutes of Health (NIH) and The Johns Hopkins University School of Medicine embarked on a study using a more precise method for performing gene therapy that did not use viruses for the gene correction. Researchers removed adult stem cells from the bone marrow of a patient with X-CGD and genetically reprogrammed them to become induced pluripotent stem cells (iPS cells). Like embryonic stem cells, these patient-specific iPS cells can be grown and manipulated indefinitely in culture while retaining their capacity to differentiate into any cell type of the body, including HSCs.

"HSCs that are derived from gene corrected iPS cells are tissue-compatible with the patient and may create a way for the patient's own cells to be used in a transplant to cure the disease, removing the risk of GVHD or the need to find a compatible donor," said Harry L. Malech, MD, senior study author, Chief of the Laboratory of Host Defenses and Head of the Genetic Immunotherapy Section of NIAID at the NIH. "However, turning iPS cells into a large number of HSCs that are efficently transplantable remains technically difficult; therefore, our study aimed at demonstrating that it is possible to differentiate gene corrected iPS cells into a large number of corrected neutrophils. These corrected neutrophils, grown in culture, are tissue-compatible with the patient and may be used to manage the life-threatening infections that are caused by the disease."

Typically, iPS cells from a patient with an inherited disorder do not express disease traits, despite the fact that the iPS cell genome contains the expected mutation. The researchers were able to prove, in culture, that iPS cells from a patient with X-CGD could be differentiated into mature neutrophils that failed to produce hydrogen peroxide, thus expressing the disease trait. This is the first study in which the disease phenotype has been reproduced in neutrophils differentiated from X-CGD patient-specific iPS cells.

After discovering that the disease could be reproduced in cell culture, the researchers then sought to correct the disease and produce healthy neutrophils in culture. They used synthetic proteins called zinc finger nucleases (ZFNs) to target a corrective gene at a specifically defined location in the genome of the X-CGD iPS cells. The iPS cells were then carefully screened to identify those containing a single copy of the corrective gene properly inserted only at the safe site. The researchers observed that some of the gene-corrected iPS cells could differentiate into neutrophils that produced normal levels of hydrogen peroxide, effectively "correcting" the disease.

"This is the first study that uses ZFNs in specific targeting gene transfer to correct X-CGD," said Dr. Malech. "Demonstrating that this approach to gene therapy works with a single-gene disease such as X-CGD means that the results from our study offer not only a potential treatment for this disease, but more importantly, a technique by which other single-gene diseases can be corrected using specifically targeted gene therapy on iPS cells."

Source:
American Society of Hematology

Periodontal Stem Cell Transplantation Shows Promise

Periodontal ligament stem cells (PDLSCs) have been found to be the most efficacious of three kinds of clinically tested dental tissue-derived stem cells, reports a study published in the current issue of Cell Transplantation (20:2), freely available on-line here.

According to researchers in Seoul, South Korea, transplantation of PDLSCs into beagle dogs modeled with advanced periodontal (gum) disease that affected their premolars and molars, which are morphologically similar to the corresponding areas in human dentition, was most effective. PDLSCs showed the best regenerating capacity of the periodontal ligament (which attaches the tooth to the alveolar bone in which the teeth sit), alveolar bone, cementum (material that comprises the surface of a tooth's root), peripheral nerve and blood vessels when compared to similar transplants using dental pulp stem cells (taken from the center of teeth) or periapical follicular stem cells (taken from the developing root).

"Periodontitis, characterized by bone resorption, periodontal pocketing and gingival inflammation, is the most common cause of tooth loss in adults and affects 10 to 15 percent of adults worldwide," said corresponding author Dr. Pill-Hoon Choung of the Seoul National University School of Dentistry. "Our study sought to evaluate the effectiveness of autologous stem cell transplantation (i.e. transplant of a patient's own cells) using three kinds of autologous dental stem cells similar to mensenchymal stem cells."

Past efforts at improving periodontal regeneration included xenogenic (from a different species) bone particle graft using growth factors, but the clinical results were generally unsatisfactory, said the researchers.

In their stem cell transplant study, Dr. Choung's group found PDLSCs to be most efficacious of the three cell types since they offered the best results with respect to the quality and quantity of regenerated tissues.

"PDLSCs made more calcium nodules and showed higher alkaline phosphatase (ALP) activity than did the other two stem cell varieties," added Dr. Choung.

The researchers concluded that further studies should investigate which factors influence the stabilization and differentiation in the diseased periodontal microenvironment and which factors make the three kinds of dental stem cells react differently in vivo.

"This study highlights the diverse sources of stem cells available in the tissues of the body for repair and how the optimal cell type for possible treatments needs to be determined - in this case for the treatment of dental-related disorders such as gum disease" said Dr. Paul Sanberg, coeditor-in-chief of Cell Transplantation and executive director of the University of South Florida Center of Excellence for Aging and Brain Repair.

Citation:
Park, J-Y.; Jeon, S. H.; Choung, P-H. Efficacy of periodontal stem cell transplantation in the treatment of advanced periodontitis. Cell Transplantation. 20(2):271-285; 2011.

Source:
David Eve
Cell Transplantation Center of Excellence for Aging and Brain Repair

 

Stem Cells May Show Promise For People With Rapidly Progressing MS

A long term study reports about the effectiveness of replacing bone marrow, purposely destroyed by chemotherapy, with autologous (self) stem cell rescue for people with aggressive forms of multiple sclerosis (MS). The study is published in the March 22, 2011, print issue of Neurology®, the medical journal of the American Academy of Neurology.

For the treatment, chemotherapy drugs are used to kill all of the patient's blood cells, including the immune cells that are believed to be attacking the body's own central nervous system. Bone marrow stem cells removed from the patient are purified and transplanted back into the body, which saves life by replacing the blood cells and also is proposed to 'reboot' the immune system.

The study followed 35 people for an average of 11 years after transplant. The study involved people with rapidly progressive MS who had tried a number of other treatments for MS with little or no effect. All were severely disabled by the disease, with an average score of six on a scale of disease activity that ranges from zero being a normal neurological examination to 10 meaning death due to MS. A score of six means able to walk with a cane or crutch; a seven is mainly in a wheelchair. All had worsened by at least one point on the scale in the year prior to the transplant.

After the transplants, the probability of participants having no worsening of their disease for 15 years was 25 percent. The probability was higher 44 percent for those who had active brain lesions, which are a sign of disease activity, at the time of the transplant.

For 16 people, symptoms improved by an average of one point on the scale after the transplant, and the improvements lasted for an average of two years. The participants also had a reduction in the number and size of lesions in their brains. Two people (six percent) died from complications related to the transplant at two months and 2-1/2 years post-transplant.

Study author Vasilios Kimiskidis, MD, of Aristotle University of Thessaloniki Medical School in Thessaloniki, Greece noted that more research is needed on this treatment, including studies that compare people receiving the treatment to a control group that does not receive the treatment.

"Keeping that in mind, our feeling is that stem cell transplants may benefit people with rapidly progressive MS," he said. "This is not a therapy for the general population of people with MS but should be reserved for aggressive cases that are still in the inflammatory phase of the disease."

Source: American Academy of Neurology (AAN)

 

Patient's Own Cells May Hold Therapeutic Promise After Reprogramming, Gene Correction

Scientists from the Morgridge Institute for Research, the University of Wisconsin-Madison, the University of California and the WiCell Research Institute moved gene therapy one step closer to clinical reality by determining that the process of correcting a genetic defect does not substantially increase the number of potentially cancer-causing mutations in induced pluripotent stem cells.

Their work, scheduled for publication the week of April 4 in the online edition of the journal Proceedings of the National Academy of Sciences and funded by a Wynn-Gund Translational Award from the Foundation Fighting Blindness, suggests that human induced pluripotent stem cells altered to correct a genetic defect may be cultured into subsequent generations of cells that remain free of the initial disease. However, although the gene correction itself does not increase the instability or the number of observed mutations in the cells, the study reinforced other recent findings that induced pluripotent stem cells themselves carry a significant number of genetic mutations.

"This study showed that the process of gene correction is compatible with therapeutic use," says Sara Howden, primary author of the study, who serves as a postdoctoral research associate in James Thomson's lab at the Morgridge Institute for Research. "It also was the first to demonstrate that correction of a defective gene in patient-derived cells via homologous recombination is possible."

Like human embryonic stem cells, induced pluripotent stem cells can become any of the 220 mature cell types in the human body. Induced pluripotent stem cells are created when skin or other mature cells are reprogrammed to a pluripotent state through exposure to select combinations of genes or proteins.

Since they can be derived from a patient's own cells, induced pluripotent stem cells may offer some clinical advantages over human embryonic stem cells by avoiding problems with rejection. However, scientists are still working to understand subtle differences between human embryonic and induced pluripotent stem cells, including a higher rate of genetic mutations among the induced pluripotent cells and evidence that the cells may retain some "memory" of their previous lineage.

Gene therapy using induced pluripotent stem cells holds promise for treating many inherited and acquired diseases such as Huntington's disease, degenerative retinal disease or diabetes. The patient in this study suffers from a degenerative eye disease known as gyrate atrophy, which is characterized by progressive loss of visual acuity and night vision leading to eventual blindness. While diseases such as genetic retinal disorders and diabetes offer attractive targets for induced pluripotent stem cell-based transplant therapies, concerns have been raised over the commonly occurring mutations in the cells and their potential to become cancerous.

Howden says that because gene targeting to correct specific genetic defects typically requires an extended culture period beyond initial induced pluripotent stem cell generation, researchers have been interested to learn whether the process would increase the number of mutations in the cells. The team set out to determine if it was possible to correct defects without introducing a level of mutations that would be incompatible with clinical applications.

In the study, the researchers used a technique called episomal reprogramming to generate the induced pluripotent stem cells. In contrast to techniques that use retroviruses, episomal reprogramming doesn't involve inserting DNA into the genome. This technique allowed them to produce cells that were free of potentially harmful transgene sequences.

The scientists then corrected the actual retinal disease-causing gene defect using a technique called homologous recombination. The stem cells were extensively "characterized" or studied before and after the process to assess whether they developed significant additional mutations or variations. The results showed that the culture conditions required to correct a genetic defect did not substantially increase the number of mutations.

"By showing that the process of correcting a genetic defect in patient-derived induced pluripotent cells is compatible with therapeutic use, we eliminated one barrier to gene therapy based on these cells," Howden says. "There is still much work to be done."

David Gamm, an author of the study and an assistant professor with the Department of Ophthalmology and the Waisman Center Stem Cell Research Program, says the ability to correct gene defects in a patient's own induced pluripotent stem cells should increase the appeal of stem cell technology to researchers striving to improve vision in patients with inherited blinding disorders.

"Although further development certainly is needed before such techniques may reach the clinical trial stage, our findings offer reason for continued hope," Gamm says. "Dr. Howden and our collaborative group have overcome an important hurdle which, when considered in the context of other recent developments, may lead to personalized stem cell therapies that benefit people with genetic visual disorders."

Source: University of Wisconsin-Madison