Gene therapy of the central nervous system: general considerations on viral vectors for gene transfer into the brain.

The last decade has nourished strong doubts on the beneficial prospects of gene therapy for curing fatal diseases. However, this climate of reservation is currently being transcended by the publication of several successful clinical protocols, restoring confidence in the appropriateness of therapeutic gene transfer. A strong sign of this present enthusiasm for gene therapy by clinicians and industrials is the market approval of the therapeutic viral vector Glybera, the first commercial product in Europe of this class of drug. This new field of medicine is particularly attractive when considering therapies for a number of neurological disorders, most of which are desperately waiting for a satisfactory treatment. The central nervous system is indeed a very compliant organ where gene transfer can be stable and successful if provided through an appropriate strategy. The purpose of this review is to present the characteristics of the most efficient virus-derived vectors used by researchers and clinicians to genetically modify particular cell types or whole regions of the brain. In addition, we discuss major issues regarding side effects, such as genotoxicity and immune response associated to the use of these vectors.

The HIV-1 DNA flap stimulates HIV vector-mediated cell transduction in the brain.

During HIV-1 reverse transcription, central initiation of the plus-strand DNA at the central polypurine tract (cPPT) and central termination at the central termination sequence (CTS) lead to the formation of a three-stranded DNA structure: the HIV-1 central DNA flap. We recently reported that the DNA flap acts as a cis-active determinant of HIV-1 genome nuclear import. Commonly employed HIV-1-derived vectors (HR vectors) lack the central DNA flap. Here we report that the insertion of this DNA flap sequence into HR vectors (TRIP vectors) improves gene transduction in neural cells, ex vivo and in vivo, in rat brain. When neural cells are exposed to increasing concentrations of TRIP vector particles, transgene expression correlates with the dose of vector. This effect contrasts with the plateau observed when using an HR vector. We further demonstrate that the increase of in vivo transduction efficiency obtained with TRIP vectors is due to the stimulation of their genome nuclear import.

Control of erythropoietin secretion by doxycycline or mifepristone in mice bearing polymer-encapsulated engineered cells.

Cell encapsulation offers a safe and manufacturable method for the systemic delivery of therapeutic proteins from genetically engineered cells. However, control of dose delivery remains a major issue with regard to clinical application. We generated populations of immortalized murine NIH 3T3 fibroblasts that secrete mouse erythropoietin (Epo) in response to stimulation by doxycycline or mifepristone. Engineered cells were introduced into AN69 hollow fibers, which were implanted in the peritoneal cavity or recipient mice. Animals receiving doxycycline or mifepristone showed stable polyhemia and increased serum Epo concentrations over a 6-month observation period, whereas animals not receiving the inducer drug had normal hematocrits. Epo secretion could be switched on and off, depending on the presence of doxycycline in the drinking water. In contrast, polyhemia was hardly reversible after subcutaneous injections of mifepristone. These data show that a permanent and regulated systemic delivery of a therapeutic protein can be obtained by the in vivo implantation of engineered allogeneic cells immunoprotected in membrane polymers.

Primary adult human astrocytes as an ex vivo vehicle for beta-glucuronidase delivery in the brain.

Astrocytes are a good candidate cell type for brain transplantation: They are endogenous to the CNS, they have efficient secretory machinery, and they play a major role in neuronal support. We assessed the potential of genetically modified primary adult human astrocytes as vehicles for the delivery of secreted molecules in the mammalian CNS. We report that such cells can be efficiently transduced by a recombinant adenoviral vector carrying the human beta-glucuronidase cDNA (Ad/CMV*beta-glu) and that the transduced astrocytes produce large amounts of the enzyme. Released beta-glucuronidase could be captured, in vitro, by primary neurons and astrocytes and by a neuroblastoma cell line and beta-glucuronidase-deficient fibroblasts. Following grafting into the mouse striatum, adult human astrocytes survived and expressed the transgene for at least 8 weeks. Moreover, the dosage of beta-glucuronidase activity within the grafted brains revealed high enzymatic levels at a long distance from the graft. These experiments document the grafting of engineered primary adult human astrocytes, allowing the release of a secreted therapeutic factor throughout the brain.

Efficient transduction of neural cells in vitro and in vivo by a baculovirus-derived vector.

Gene delivery to the central nervous system is central to the development of gene therapy for neurological diseases. We developed a baculovirus-derived vector, the Bac-CMV-GFP vector, containing a reporter gene encoding for the green fluorescent protein (GFP) under the control of the cytomegalovirus (CMV) promoter. Two neuroblastomal cell lines and three human primary neural cultures could be efficiently transduced. In all cases, addition of butyrate, an inhibitor of histone deacetylase, increased the level of expression in terms of the number of GFP-expressing cells and the intensity of fluorescence. The level of expression in a human telencephalic culture was over 50% of transduced cells with a multiplicity of infection of 25. GFP expression was demonstrated to be genuine expression and not pseudotransduction of the reporter protein. Most interestingly, Bac-CMV-GFP could transduce neural cells in vivo when directly injected into the brain of rodents and was not inactivated by the complement system. Thus, baculovirus is a promising tool for gene transfer into the central nervous system both for studies of the function of foreign genes and the development of gene therapy strategies.