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regulated through modulation of cellular receptor interactions by 1 or more of the following approaches: alteration of virus docking proteins, utilization of alternate serotypes, pseudotyping, and introduction of cellular receptors into the viral envelope. Once a vector is optimally targeted to the brain region of interest, therapeutic transgene expression can be restricted to selected cell populations via the utilization of cell type-specific promoters and/or transcriptional elements (2-6). Strict spatial control of transgene expression is important to ensure the correct cells will manufacture the gene product. This control, in turn, reduces the risk that ectopic transgene expression will occur and lead to untoward effects on adjacent neurological pathways.

Because many neurodegenerative disorders are protracted in duration, gene-base modalities will be required to impart therapeutic benefit for several decades of an individual's lifetime. To this end, a vector genome should be stably maintained within the transduced cell of the neural pathway for extended periods of time. Vector genome maintenance is, therefore, an important factor in selection of an appropriate gene therapy vehicle for such disorders. Vector genomes can exist episom-ally and/or as integrated forms within nuclei of transduced cells. The postmitotic property of CNS neuronal populations does not exclude the utilization of episomal vectors because genomes can be maintained without the fear of progressive loss due to mitosis. Integrating vectors circumvent this issue but their use augments safety concerns, including potential to transactivate nearby proto-oncogenes and to disrupt essential host genes via insertional mutagenesis. This fear has apparently become a reality as evidenced in the case of the children in Europe treated for X-linked severe combined immune deficiency disease (X-SCID) with a murine retroviral vector expressing the T cell growth factor, gammaC. Two of 10 enrolled infants developed similar leukemia-like illnesses, and the trial was halted to determine the genesis of the adverse event (7).

Another similar issue regarding vector selection relates to the desired levels and duration of gene product expression for treatment of neurodegenerative disorders. Depending the vector and transcriptional elements chosen, pharmacological or physiological levels of transgene expression can be achieved for short- or long-term periods of duration. As with other selection criteria, the decision of which level/duration of expression is preferred rests heavily on the underlying molecular mechanisms to be targeted and at which time during the disease course the intervention is to be implemented. Early interventions may require maintenance of long-term physiological levels of transgene expression (i.e., neuroprotective strategies) because the neuronal system is likely to be intact at this time. A vector/promoter combination that safely and stably maintains gene expression at nearly physiological levels in the CNS would serve as a potential candidate for such early treatment approaches. Treatment modalities that are implemented after presentation of clinical neurodegenerative disease symptoms may require long-term pharmacological levels of transgene product to restore neurological function to a brain region decimated by disease.

Safety is of utmost concern regarding the application of novel gene therapeutic strategies to the treatment of neurode-generative disorders. Many presently available vectors trigger immunogenic and/or inflammatory responses when introduced into the CNS. These responses are known to arise from the humoral and/or cell-mediated arms of the immune system, and the magnitude differs depending which vector type is employed. For example, repeat administration of early generation viral vectors has been shown to lead to lower transgene expression and serious inflammation, likely the result of a primed immune system (8). Therefore, a vector that is stably maintained and that can express its encoded transgene for extended periods of time would likely prove to be a more favorable choice as a gene therapeutic vehicle for neurodegenerative disorders. Another aspect that is often overlooked regarding gene therapy safety is the role of transgene products in the elaboration of immune responses and toxicity. Transgene products that are of foreign origin, ectopically expressed, or pharmacologically expressed possess the potential to induce cytotoxicity and/or immune responses. Research addressing these issues is imperative to elucidate the role of transgene products in the elaboration of these potentially harmful responses, and how such responses can be successfully circumvented. Utilization of regulation-competent transcriptional or posttranscriptional elements in delivery vectors to provide ''fine-tuning'' of therapeutic transgene expression levels is a way to minimize harmful clinical outcomes.

C. Vector Delivery

Once a suitable vector has been chosen for treating a particular neurological disease, a safe means for delivery must be established to provide optimal therapeutic benefit. With recent refinements of stereotactic surgical procedures, highly precise and reproducible delivery to specific regions of the brain are now performed [reviewed in (9)]. By placing the patient's head within a rigid frame and using 3-dimensional cartesian reference points, delivery of vectors or cells can be made to a defined space within the coordinate system. Gene therapeutic vectors can be introduced into the brain via direct or indirect means. Direct gene transfer involves either local or global delivery of a selected vector to the brain. Direct local delivery using stereotactic methods is highly suitable for treatment of certain neurological diseases due to the fairly circumscribed region that may be afflicted. Figure 1 illustrates the finely tuned and restricted delivery of an herpes simplex virus (HSV) amplicon vector expressing the p-galactosidase gene within the striatum of a mouse (10).

Other investigative groups have used more global approaches to CNS gene delivery that have broader applications for many neurological diseases where the volume of affected brain region is beyond the feasibility of conventional stereo-tactic methods. Convection-enhanced delivery (CED) has been developed to distribute homogeneous tissue concentrations of vectors over a large region of rodent or primate brain (11-14). This method has been used extensively in the development of gene therapeutic strategies for neurodegenerative disorders, but further research is required to determine if global distribution of vectors produces deleterious effects on neural pathway functioning. For example, global expression of vector-derived neurotrophic factors may induce uncontrolled neuritic sprouting, and subsequently, altered neuronal activity and physiology.

Indirect gene transfer, or ex vivo therapy, uses transplantation of genetically altered (vector-transduced) or unaltered cells capable of restoring functionality to a diseased region. Although ex vivo therapy includes the use of neural stem cells for repopulation of the denervated dopaminergic system and restoration of pathway function, this type of therapy is be discussed in this review. Vectors used to genetically modify transplanted cells typically express either secreted trophic factors for neuroprotection/neuroaugmentation of surrounding host tissues or cell-intrinsic survival factors for protection of

Figure 1 Stereotactic delivery of an HSV amplicon vector expressing p-galactosidase into the mouse striatum. Mice were injected with 1 X 105 transduction units of HSVlac using a microprocessor-controlled pump. Animals were sacrificed and perfused 4 days posttransduction and X-gal histochemistry was performed on 40-^m sections. Sections representative of the injection site (A), a site anterior of the injection (B), and a site posterior of the injection (C). All sections were counterstained with thionin and acquired at a magnification of 2.5 X. The photomicrographs indicate that focal delivery of a viral vector can be achieved within the brain. (From Ref. 10, © 1998 Elsevier Science B.V.) See the color insert for a color version of this figure.

Figure 1 Stereotactic delivery of an HSV amplicon vector expressing p-galactosidase into the mouse striatum. Mice were injected with 1 X 105 transduction units of HSVlac using a microprocessor-controlled pump. Animals were sacrificed and perfused 4 days posttransduction and X-gal histochemistry was performed on 40-^m sections. Sections representative of the injection site (A), a site anterior of the injection (B), and a site posterior of the injection (C). All sections were counterstained with thionin and acquired at a magnification of 2.5 X. The photomicrographs indicate that focal delivery of a viral vector can be achieved within the brain. (From Ref. 10, © 1998 Elsevier Science B.V.) See the color insert for a color version of this figure.

grafted cells from the stress of transplantation. Vectors delivered via ex vivo therapy must be able to integrate into the host cell genome because transplanted cell populations are expanded prior to grafting. Utilization of gene transfer technologies may allow for the expansion of the graft cell population or increased graft survival via expression of a growth or survival factor, thereby minimizing the amount of starting fetal tissue (15,16).

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