Patricia F. Fitzpatrick Dimond Ph.D. Technical Editor of Clinical OMICs President of BioInsight Communications

With some of the safety kinks worked out, gene-based treatments are making more progress.

The prospect of curing human diseases by replacing a disease-related gene with a normal version remains the ultimate goal of gene therapy. But in its early days, attempts at gene therapy met with unpredictable and occasionally fatal outcomes. The field sustained a serious setback in 2000 following the death of 18-year old Jesse Gelsinger after receiving gene therapy to treat orinthine trascarbamlase deficiency (OTCD), a rare metabolic disorder that prevents the body from breaking down ammonia.

Gene therapy took another blow in September 2003, when the FDA placed a temporary halt on all gene therapy trials using retroviral vectors in blood stem cells. The agency was responding to the development of a leukemia-like disorder that developed in a three-year-old boy following successful gene therapy for to X-linked severe combined immunodeficiency disease (X-SCID). Subsequently, the disease developed in three children, one of whom died from it.

Now, bolstered by the development of enabling technologies and recent clinical successes, gene therapy is making a significant comeback. Effective gene delivery has been established in multiple formats including direct DNA delivery, genetically engineered autologous cells, and specifically targeted gene modification or insertion.


In 2009, international teams of researchers reported the successful treatment of two children suffering from adrenoleukodystrophy (ALD). ALD is a severe hereditary condition caused by mutations in ABCD1 gene, which encodes the adrenoleukodystrophy protein (ALDP), a protein involved in fatty acid degradation.

Over the course of the disease, afflicted individuals steadily lose the myelin sheath that surrounds nerve cells. Myelin loss results in loss of nerve function, leading to increasing physical and mental disability. X-linked ALD, the most common form of the disease, affects boys as early as age six, with death usually occurring before the patients reach adolescence.

While ALD progression can be halted by allogeneic hematopoietic cell transplantation (HCT), finding correctly matched donors and the inherent dangers in the procedure present problems.

Investigators in France reported successful treatment of two ALD patients for whom there were no matched donors. They first removed CD34+ cells, then transfected the cells ex vivo with a lentiviral vector encoding the wild-type correct form of the gene encoding ALDP, and finally re-infusing them into the patients after they had received myeloablative treatment.

Over a span of 24 to 30 months of follow-up, the authors said, they could detect polyclonal reconstitution, with 9 to 14% of granulocytes, monocytes, and T and B lymphocytes expressing the ALD protein.

Beginning 14 to 16 months after infusion of the genetically corrected cells, progressive cerebral demyelination in the two patients stopped, a clinical outcome comparable to that achieved by allogeneic HCT. Thus, the authors said, lentiviral-mediated gene therapy of hematopoietic stem cells can provide clinical benefits in ALD.

Leber Congenital Amaurosis

Another successful area for gene therapy has been Leber congenital amaurosis (LCA), a heritable form of progressive blindness. LCA, the result of a mutation in the RPE65 gene, may be treatable by introducing a normal copy of the mutated gene directly into the retinas of affected individuals.

Normally, the RPE65 protein converts dietary vitamin A into a retina-specific form of vitamin A needed for rhodopsin formation. Rhodopsin is a visual pigment that absorbs light after it enters the eye, and it requires the RPE65 protein to regenerate after light exposure. Therefore, mutations in the RPE65 gene seen in LCA disrupt the visual cycle and prevent normal vision.

Several groups have reported progress in treating the disease in individuals with the specific mutation. In 2008, these researchers administered subretinal injections of recombinant adeno-associated virus (AAV) vector expressing RPE65 complementary DNA (cDNA) under the control of a human RPE65 promoter.

Investigators concluded that the safety, extent, and stability of improvement in vision in all patients support the use of AAV-mediated gene therapy for treatment of inherited retinal diseases, with early intervention resulting in the best potential gain.


Apart from advancements in DNA delivery and in vectors for gene delivery into patients’ cells, zinc finger nuclease technology may prove truly transformative to gene therapy in general. Zinc finger nucleases (ZFNs) are synthetic proteins consisting of an engineered zinc finger DNA-binding domain fused to the cleavage domain of a restriction endonuclease. These engineered molecules allow cellular DNA to be cut at specific points, with gene modification then occurring via the cell’s own natural repair mechanisms. They may also allow the insertion of entire genes at desired cleavage sites to replace missing or mutated genes.

Sangamo and colleagues from the University of Pennsylvania announced positive preliminary data from their Phase I trial being conducted in HIV-infected immunologic nonresponders. The patients enrolled in this study were HIV-infected individuals on highly active antiretroviral therapy with undetectable levels of virus but low T-cell counts.

The investigators used zinc fingers custom-designed to bind to specific DNA sequences in the CCR5 gene in HIV-infected patients’ T cells. The zinc finger proteins act as molecular scissors, bringing a DNA enzyme to the CCR5 gene to cut its sequence. During the repair process, a new mutation arises in the CCR5 protein, rendering it nonfunctional. Since the HIV virus uses the normal, unmodified version of CCR5 to gain access to T cells, the engineered cells became completely resistant to infection.

The data showed that a single infusion of the engineered cells was well tolerated, and the CCR5-modified cells successfully engrafted in all of the patients. The treatment also resulted in a durable improvement in total CD4+ T-cell counts in five of six patients analyzed.

The ZFN-CCR5-modified cells also exhibited normal T-cell growth kinetics and trafficking and underwent selective expansion in the gut mucosa, a major reservoir of virus in the body, suggesting, as predicted, that the cells were resistant to HIV infection.

Sangamo’s Philip Gregory, CSO and vp, research, told GEN that the goal of developing the company’s zinc finger nuclease program “has been to give investigators the ability to perform precision engineering directly on the genome itself.”

In the HIV application, he pointed out, no new DNA is being introduced. “We are really introducing a mutation ourselves just with the nucleases. We were able to do this because DNA repair mechanisms themselves are error prone. When the cells repair the break generated by the ZFN, the process occurs without error checking and mutations are introduced specifically at the site of the break. We can use this to achieve one desired outcome, knockout of the gene that’s been cleaved by the nuclease—in this case, the CCR5 gene. When the cell repairs it, it will create mutations.”

In explaining the production process for the autologous cells carrying the mutated CCR5 gene, Gregory said that the cells are collected from patients and sent to a processing facility, where they are exposed to the ZFN. The cells are expanded, and then re-infused into the patient, where they engraft, expand in the patients, and get trafficked to the normal place in the body.

“These cells are noninfectable by HIV,” Gregory said. He further explained that “just by protecting T cells, we aimed to create a reservoir of these cells that couldn’t be infected. These cells protect against loss of cells in HIV-infected patients.”

Gregory emphasized that CCR5 itself as a target “is one of the few situations in which we know the biology of the protein from the situation that exists in the natural population of patients. Patients with the Delta 32 CCR5 mutation don’t have the receptor on their cells and are completely normal but are resistant to infection.

“It turns out,” he said, “that an important feature of that mutation is that it eliminates CCR5 completely, giving rise to cells with no receptor on their surfaces.”

Sangamo says it is testing its product across a full range of HIV patients including those for whom current drug regimens are failing. “We are making good progress and are letting the data tell us where to focus our further clinical trials.”

All this progress, investigators point out, represents the culmination of years of experience, encompassing multiple disciplines from molecular biology through clinical science. And hopefully as positive clinical results continue to emerge, effective gene therapy, in whatever format, will become a real therapeutic option for intractable human diseases.

Patricia F. Dimond, Ph.D. ([email protected]), is a principal at BioInsight Consulting.

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