Safety Aspects of Gene Transfer

Gene transfer inserts a copy of the particular gene into the nuclear DNA. It could potentially revolutionise medicine, as a working copy of a defective gene could be inserted to treat genetic metabolic disorders, such as lysoso-mal storage diseases that account for mental retardation and affects the CNS in young children. Nowadays inserting genes into brain cells may also offer ways to slow down, or even reverse the damage from neurological disorders and stimulate brain reconstruction upon cellular therapy. Gene transfer to post-mitotic neurons (and other neural cells) has been achieved by several classes of viral vectors (Hermens and Verhaagen 1998). Herpes simplex viral and adenoviral vectors for gene transfer into brain cells must be banned for clinical trials (except perhaps for the killing of a brain tumor), because of the toxicity for several neuronal cell types. However, the use of AAV and LV vectors may be feasible in patients as absence of toxicity and destructive immunological responses (though immune responses are induced against the virus components) as well as long-term expression of the transgene are hallmarks for these vectors (Kordower et al. 1999; Tenenbaum et al. 2003). However, due to the death of patients in clinical trials outside the field of neural deficiencies, gene transfer has come under a cloud (Gansbacher 2003). Thus, even more than in cell therapy research, safety aspects are an important focus in the field of experimental gene transfer in the brain.

Aberrant insertion of the transgene in the genome of the host (i), the occurrence of integration of virus sequences at uncharacterised integration sites (insertional mutagenesis) (ii), as well as the insertion of the transcrip-tional active sequences that could result in activation of otherwise silent genes (iii), may have devastating effects or may perhaps even lead to disorganisation or tumor formation in the CNS. Furthermore, this cannot be corrected afterwards. Even the application of co-transduction with killer genes could have traumatic effects due to the inflammatory and immunogenic responses that will occur when activation of this safety system is indicated. Insertional mutagenesis by random gene addition has, so far, not been observed. Moreover the spread of viral vectors in the CNS is very limited so that the chance of major transduction of cells outside the area of injection is minimal (this is also of importance to ascertain absence of genetic alterations in germ line cells). Biosafety of AAV vectors is especially accepted, and the use of (Parvovirus family) AAV subtype 2 vector has been cleared for phase I studies in humans, also because the wild type AAV2 is not known to be an etiological agent in any disease in human beings (Tenenbaum et al. 2003). Therefore, safety aspects then mainly have to deal with side effects of the over-expression of the gene of expected therapeutic value.

Gene transfer by viral vectors in the brain has some specificity as not all vector types transduce all cell types of the brain, and not all AAV vector types have the same efficiency in transducing cells. The main side effect of a therapeutic gene may be the constitutive over-expression of the synthesised protein, its effect for the transduced cell and, if released, for cells that do not need the treatment. In other words, an uncontrollable, long term local effect that changes the functional balance of nervous pathways and circuits not affected by the disease, which may lead to unwanted structural or molecular alterations and thus to unwanted activities in the CNS. The application of NGF, for instance, has been shown to induce neuropathic pain in human patients (Pezet and McMahon 2006). This quite likely results from the neu-rite growth-stimulatory potency of this protein, which could have led to an abnormal pattern of synaptic connectivity between nerve cells or its modulatory role on the action of BDNF also involved in pain. However, as the brain is a very heterogeneous structure each substructure of which is acting in many physiological and mental functions, one cannot say that gene therapy with, for example, the gene for NGF expression in one area of the brain will have the same side effect when applied in another area. Other side effects on the basis of (newly) established, but misdirected, neuronal contacts will affect different functions. Considering aspects mentioned above, any new clinical trial of gene transfer for molecular neurosurgery of the diseased or traumatised brain should not be limited to tests of the primary outcome for the disease parameters but always include safety issues and tests on personality.

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