Overview. Gene therapy for Alzheimer’s disease is a long way from being practicable, but some prototypical approaches are being explored. In April 2001, Ceregene, a subsidiary of Cell Genesys, began a Phase I trial for its nerve growth factor (nerve growth factor) gene delivery involving eight patients with early Alzheimer’s disease. Another promising approach pioneered by researchers at the Salk Institute and at the University of California-San Diego (UCSD) introduces the neprilysin gene into the brain cells of Alzheimer’s disease patients. Neprilysin has been shown to degrade Aβ-42 in vitro, and mice in which the gene has been knocked out have an increase in Aβ-42 levels in their brains. The researchers used a modified lentivirus (an HIV-like virus lacking the genes required for infection) to introduce the neprilysin gene into the cortex and hippocampus of mouse models of Alzheimer’s disease. The treatment showed a 50% reduction in levels of Aβ-42, and signs of neuronal degeneration were reduced (). These agents are still in discovery phase and have not been licensed, but they demonstrate that neprilysin may effectively reduce Aβ-42 levels in vivo and that in vivo gene therapy approaches may efficiently deliver genes to the cortex and the hippocampus. It is unclear, however, whether regions of the brain as large as those affected in Alzheimer’s disease patients can be effectively targeted by this method. This section profiles CERE-110, an ex vivo gene therapy that delivers the nerve growth factor gene to Alzheimer’s disease patients, because it is the one promising gene therapy approach that has completed Phase I clinical trials.
Mechanism of Action. Gene therapy delivers genes to a patient in an effort to stem the course of a disease by either increasing the levels of a gene, augmenting a natural process, or replacing a patient’s faulty gene. In ex vivo gene therapy, such as CERE-110, a gene is introduced (transfected) into cells taken from patient biopsies in the laboratory. The cells that have been most effectively modified are then selected and injected into a relevant brain region of the patient. The modified cells implant themselves near the injected region, then produce and secrete the gene product into the brain region. This strategy has the advantage of being cell-type specific and of insuring that the injected cells are immunocompatible with the patient’s. However, this approach is encumbered with the drawback of injecting dividing cells directly into the brain, an invasive procedure that could potentially generate brain tumors, although no tumors have been seen in animal studies so far.
It is noteworthy, however, that, although the prototypical gene therapy studies on severe-combined immunodeficiencies conducted in the late 1990s failed to detect any cancer-promoting activity in animal models, treated patients developed leukemia at a rate of 15%. More recent studies in patients using newer vectors and transplantation technology have not detected any further cases of cancer so far ().
As an alternative approach, in vivo gene therapy delivers a gene that has been packaged into viral particles by injecting the particles into a relevant site in the patient’s brain. The virus will then infect cells at the site of injection and deliver the gene of interest to the patient’s cells. This method presents the disadvantage of using viral particles, which may be toxic to certain cell types and may elicit immune responses. Lentiviruses, the viruses that look most promising, are particularly efficient at infecting neurons and have not elicited these side effects so far. An additional problem with this delivery is that many cell types will be infected by viral particles, including neurons, glial cells, and vascular cells. The adeno-associated viral vector (AAV) has also been shown to infect cells in the CNS (albeit predominantly nonneuronal cells such as astrocytes) (). Theoretically, the cell type transfected matters less than the efficient and stable expression of the gene in a cell and the synthesis of biologically active gene products. In addition, the proteins that interact with the transfected gene product (e.g., the trkA receptors in the case of transfected nerve growth factor) must be present in the vicinity of nerve growth factor-expressing cells. There are also problems surrounding the use of injection to deliver the therapy. A localized injection may not target a large enough region of the brain.
Both ex vivo and in vivo techniques require invasive brain injections, which may result in local bleeding and neuronal damage, as was the case in the death of one patient in the Phase I CERE-110 ex vivo trial. Finally, the implantation of genes into inappropriate regions at inappropriate developmental times may have unforeseen deleterious effects that would be impossible to reverse. The possibility of regulating a gene using promoters that respond to extraneously added agents such as tetracycline is currently being explored, but the technique remains in discovery phase.
CERE-110 (NeuroRescue Alzheimer’s disease). CERE-110 is an ex vivo gene therapy being developed by Ceregene for the treatment of patients with mild to moderate Alzheimer’s disease. It involves the transfection of patients’ fibroblasts with the nerve growth factor (nerve growth factor) gene, which itself was licensed from Genentech in March 2003. A Phase I trial was completed in November 2003. A further Phase I study was initiated in September 2004 for the use of CERE-110 as an in vivo approach using a different viral delivery method (the AAV vector), a technique that is less labor intensive than the ex vivo approach. The new Phase I trial will evaluate the safety of the technique in six patients with mild to moderate Alzheimer’s disease (MMSE scores, 16-24; i.e., patients with worse MMSE scores than those in the ex vivo trial) over the course of 24 months.
CERE-110 is an ex vivo gene therapy approach based on the AAV vector. Patients’ fibroblasts that have been transfected with the nerve growth factor would be injected into the nucleus basalis of Meynert, an area of the brain known to undergo significant degeneration in Alzheimer’s disease. Researchers hope the injected fibroblasts will implant in the nucleus or its vicinity and synthesize and release nerve growth factor. The released nerve growth factor would delay or reverse the degeneration of cholinergic neurons and prevent their death. The decline of acetylcholine (acetylcholine) levels in the hippocampus and the cortex, into which the cholinergic neurons project, would be reversed, and the cognitive and functional decline seen in Alzheimer’s disease patients may be stemmed. The technique would have the advantage of keeping nerve growth factor expression spatially restricted, which would prevent the occurrence of side effects such as pain and appetite suppression seen with the more systemic injection of nerve growth factor into the cerebral ventricles ().
Preclinical data in mice, rats, and monkeys suggest that nerve growth factor gene delivery may improve memory (). In addition, data presented at the 2nd Annual SMi Cell and Gene Therapy Conference on September 24-25, 2003, in London, suggest that nerve growth factor gene delivery restores a threefold level of activity in the cholinergic neurons of treated animals compared with controls injected with the vector alone. Scientists also demonstrated the durability of this technique; levels of nerve growth factor remained constant over the course of a year in these animals and the Phase I trial results indicate a similar stability of the nerve growth factor gene expression over the course of the trial (1.5 to 2 years). No toxic side effects were noted in this study. These experiments demonstrated that neurons did not redirect their original axon projections toward the implanted cells but still projected into the brain cortex, a requirement that is critical because redirection of neurons from their original projections to inappropriate projections would have rendered the technique useless.
In an ongoing Phase I trial, fibroblasts originally extracted from the patient’s skin were genetically modified to express nerve growth factor. These modified cells were then surgically implanted into the nucleus basalis. In addition to determining the safety of such a procedure, the researchers sought to determine whether these implanted cells could prevent the death of cholinergic neurons, enhance the function of remaining brain cells, and perhaps delay the cognitive decline seen in Alzheimer’s disease patients. Eight patients with mild to moderate Alzheimer’s disease (MMSE scores, 20-28) underwent surgical implantation of the fibroblasts. The patients were assessed for improvements in cognitive function using the MMSE and ADAS-cog tests and metabolic function was monitored using brain imaging (PET analysis).
Preliminary trial results for this Phase I trial were presented at the Society for Neuroscience Conference in San Diego in October 2004 (). A brain autopsy of a patient who had died of unrelated causes demonstrated that the fibroblasts had implanted into the nucleus basalis region and that there was a robust sprouting of the axons of cholinergic neurons in the vicinity of the injection site, suggesting that the elevated levels of nerve growth factor in the region had interrupted the degeneration of cholinergic neurons and induced a measure of neuronal regrowth. These results are encouraging because they indicate that the nerve growth factor produced by the fibroblasts is biologically active and may maintain the connections of cholinergic neurons that had not yet degenerated. It is unclear, however, whether connections that had already degenerated could be re-established. The procedure appeared to be well tolerated, although two patients suffered bleeding caused by movement during the injections; one of these patients died five weeks after the injection. Such bleeding did not occur once patients were anesthetized during the injection.
The results from the trial, which was insufficiently powered to detect cognitive improvement with six patients, nevertheless suggested a modest degree of clinical efficacy. The mean annualized rate of overall decline for patients prior to the surgery was 6.0±2.8 points on the MMSE while the decline after the surgery was reduced to 3.0 ±1.2 points per year. Importantly, the reduction in decline persisted over the 1.5- to 2-year study period, a period comparable to that seen in AChEI trials. Results were similar for the ADAS-cog, with an annualized decline of 4 points post-surgery, compared with 6 to 12 points in historical controls. The results failed to reach significance however, in part because of the small number of patients in the trial and the interpatient variability. Two of the six patients showed cognitive improvement 6 to 18 months after surgery, one showed no decline, and two declined mildly. PET analysis also showed increases in glucose metabolism by cells in the vicinity of the injection site, indicating that metabolic decline had been slowed in these cells (although the identify of the responding cells cannot be determined by PET). The lag time required to detect an effect after injection is expected, because nerve growth factor must be captured by neurons and transported to the neuron cell body, where genetic alterations will upregulate the levels of neuronal “building blocks” (cytoskeletal elements), and finally the building blocks must be transported to the axon’s terminals (a process requiring several weeks) before an effect is detected.
Previous experiments using nerve growth factor have shown that its expression in inappropriate regions of the central nervous system can produce significant side effects. When nerve growth factor was injected into the CSF of patients, side effects included pain and weight loss. These symptoms are believed to have been a result of the inappropriate sprouting of the axons of pain-sensing neurons and the suppression of appetite center function, respectively (). Additionally, neurotrophic factors such as nerve growth factor have been shown to have effects on cell morphology and differentiation and to possibly interfere with neurotransmission. It is also not clear how the spatial and temporal expression of nerve growth factor should be regulated. Finally, it is not possible to interrupt gene therapy if needed.