We have optimised an efficient cationic lipoplex method for delivery of siRNA into the newborn mouse brain. Specific inhibition of exogenous target gene expression is obtained with picomolar amounts of siRNA.

The subventricular zone (SVZ) neural stem cell niche contains mixed populations of stem cells, transit-amplifying cells, and migrating neuroblasts. Deciphering how endogenous signals, such as hormones, affect the balance between these cell types is essential for understanding the physiology of niche plasticity and homeostasis. We show that Thyroid Hormone (T(3)) and its receptor, TRα1, are directly involved in maintaining this balance. TRα1 is expressed in amplifying and migrating cells. In vivo gain- and loss-of-function experiments demonstrate first, that T(3)/TRα1 directly repress Sox2 expression, and second, that TRα1 overexpression in the niche favors the appearance of DCX+ migrating neuroblasts. Lack of TRα increases numbers of SOX2+ cells in the SVZ. Hypothyroidism increases proportions of cells in interphase. Thus, in the adult SVZ, T(3)/TRα1 together favor neural stem cell commitment and progression toward a migrating neuroblast phenotype; this transition correlates with T(3)/TRα1-dependent transcriptional repression of Sox2.

Thyroid hormones (TH) are essential for brain development. However, information on if and how this key endocrine factor affects adult neurogenesis is fragmentary. We thus investigated the effects of TH on proliferation and apoptosis of stem cells in the subventricular zone (SVZ), as well as on migration of transgene-tagged neuroblasts out of the stem cell niche. Hypothyroidism significantly reduced all three of these processes, inhibiting generation of new cells. To determine the mechanisms relaying TH action in the SVZ, we analyzed which receptor was implicated and whether the effects were played out directly at the level of the stem cell population. The α TH receptor (TRα), but not TRβ, was found to be expressed in nestin positive progenitor cells of the SVZ. Further, use of TRα mutant mice showed TRα to be required to maintain full proliferative activity. Finally, a direct TH transcriptional effect, not mediated through other cell populations, was revealed by targeted gene transfer to stem cells in vivo. Indeed, TH directly modulated transcription from the c-myc promoter reporter construct containing a functional TH response element containing TRE but not from a mutated TRE sequence. We conclude that liganded-TRα is critical for neurogenesis in the adult mammalian brain.Key words: subventricular zone • NSC • TH nderstanding the genetic regulation of neural stem cell (NSC) maintenance, division, and differentiation is a central problem in NSC biology. The identification of subventricular astrocytes as NSCs in the subventricular zone (SVZ) of the adult rodent brain contributed to the confirmation of the existence of a neurogenic niche in this region (1). Although relatively quiescent and slow dividing, the NSC population residing in this site gives rise to rapidly dividing neural progenitors, which in turn generate neural precursors that have the ability to generate both neurons and glia. However, most cells emerging from the SVZ undergo apoptosis (2), while many of the remaining cells migrate to form new interneurons in the U Page 1 of 17 (page number not for citation purposes) olfactory lobe (3). Some cells may also generate glial cells in the corpus callosum (4). To exploit the therapeutic potential of endogenous stem cells, knowledge of the intrinsic and extrinsic mechanisms affecting NSC population size and expansion is needed. However, few studies have addressed endocrine control of NSC function, particularly in vivo (5).The thyroid hormones (TH) are essential for brain development in all vertebrates (6). The main secreted form of TH is thyroxine (T 4 ), which is converted to the biologically more active form triiodothyronine (T 3 ), in peripheral tissues. In each species studied, TH levels are highest during the period of most rapid brain growth and development, which in humans occurs during the first 2 yr of postnatal life. Lack of TH in early development causes severe neurological damage and results in cretinism in man (7). At the cellular level, TH action on specific cells and at specific phas...

Recent progress on the fronts of design, formulation and delivery with synthetic vectors has increased the potential of non-viral approaches to attain therapeutic applications. However, a number of hurdles remain to be cleared. On the one hand, we have the problem of producing and formulating stable, diffusible complexes and on the other hand, we have to face the fact that in vivo delivery is probably too complex and multifaceted to be achieved successfully with a single carrier molecule in most instances. This review concentrates on this latter aspect and the design of artificial multi-component vectors aimed at providing solutions to membrane crossing, endosomal escape and navigation through the nuclear pore. In many instances the solutions proposed have been inspired by natural mechanisms exploited by bacteria and viruses. However, entirely novel chemical approaches such as monomolecular DNA condensation via detergent dimerization, or endosome disruption by osmotic swelling, are also being investigated and developed. The combination of these naturally inspired and chemically-originated approaches is bringing us continually closer to the concept of constructing an artificial virus capable of delivering viable nucleic acid-based pharmaceuticals to defined cells in vivo. This review considers the most successful current solutions to the main biological barriers to gene delivery including appropriate DNA compaction, cell targeting and entry, vacuole escape, nuclear import and in vivo delivery. In the latter section, emphasis is placed on one of the most versatile non-viral vectors currently available, linear polyethylenimine.