Rodent and primate studies in cholinergic denervation indicate that NGF is important in rescuing cells from axotomy-induced cell death as well as potentially augmenting cholinergic function. Therefore, to evaluate the effectiveness of NGF as a therapy for AD, patients to be treated would ideally be in the early stages of the disease, so that the rate of disease progression can be assessed. Outcomes should be assessed with careful preoperative and postoperative monitoring with standard psychological batteries, AD and other dementia rating scales, and magnetic resonance imaging (MRI) of the brain.
To implement ex vivo gene therapy for AD, a population of cells needs to be genetically modified to secrete NGF. For reasons relating to their immunology, autografts of patient's skin fibroblasts appear to be the most promising vector. Animal experiments have confirmed the practicability of this strategy. The transduction of these cells can be effected by the culturing of cells harvested from patient's own skin, genetically modifying them with the application of retroviral constructs, and expanding and selecting the cell population of interest (Fig. 2). The level of trophic factor production per cell can be determined. These cells are then concentrated into a small volume for injection in a dose-specific manner.
Next, the genetically modified cells must be implanted into the NBM. To successfully introduce the cells, the exact location of the NBM must be determined and readily confirmed using radiological scanning techniques. The nucleus is situated between the anterior commissure and the base of the brain. An appreciation of its location and morphology can be achieved by examining immunostained brain sections. This anatomic knowledge can then be correlated with MRI of the appropriate sections of the brain in individual patients (Fig. 3). This then permits the determination of the coordinates of each target in the basal nucleus. The major consideration in the design of the operative procedure is the need for precise targeting of the implants while minimizing the risks of implantation. Any implantation procedure carries with it, by its necessarily invasive nature, risks of hemorrhage and infection. Stereotaxic implantation of these cells affords the most precise targeting. This involves placement of a stereotactic head frame, imaging of the brain while in the frame, determination of the coordinates for each specific target, placement of a bur hole or bur holes, and introduction of an injection catheter under precise control. Accurate graft implantation can be complicated by the potential for brain shift after the subarachnoid space is entered and CSF egress occurs. This consideration is especially germane in AD patients, who usually have substantial brain atrophy and consequent enlargement of the sub-
arachnoid space. To minimize this source of error, during surgical implantation every attempt must be made to minimize CSF egress through the bur hole. Design of the surgical procedure must also take into account the shape and orientation of the basal nucleus of Meynert. The NBM exhibits an anterior/ mesial/ventral to posterior/lateral/dorsal orientation between the anterior commissure and the base of the brain. This orientation does not permit a single needle passage, depositing implanted cells along the axis of the needle, to populate the entire NBM with grafted cells. Multiple deposits therefore have to be made by passing the injection needle from the cortex to the target along multiple separate tracts. This increases the risks of hemorrhage as well as potential risk of damage to nerve fiber tracts. To decrease this risk, the planning of the operative procedure must involve planning a trajectory that does not require the needle to traverse the ventricles or any major groups of vessels. In addition, multiple injections are mandated by the fact that NGF secreted by the engineered fibroblasts exhibits limited diffusion into the surrounding brain. The entire operative procedure must also be undertaken
under deep enough anesthesia to prevent movement of the patient because trauma to the brain could occur if the head and brain move while the injection needle is inside the brain. Last, the injected cells are genetically altered and the potential for tumorigenic transformation exists. The ability to visualize the implant after injection would be essential to detect any growth of the graft. Provision for elimination of any tumor that may form (e.g., Gamma Knife radiosurgery) must be in place prior to clinical application.
These considerations must be accounted for in the design of any clinical trial of ex vivo gene therapy for AD. Such a clinical trial is currently in progress (48).
Was this article helpful?