Modification of dendritic cells (DCs) is a promising avenue for gene therapy purposes, given the versatility and the multiplicity of functions of these cells. In this study, we show that preincubation of monocyte-derived DCs with low amounts of non-infectious virion-like particles derived from the simian immunodeficiency virus (SIV MAC VLPs) increases up to 10-fold the efficiency of transduction by HIV-1 lentiviral vectors at low multiplicity of infections yielding up to 90% of transduced cells, in the absence of alterations of DCs behavior. This effect is restricted to DCs and specified by the viral accessory protein Vpx. Thus, preincubation with empty VLPs of SIV MAC can be used in transduction protocols to increase the efficacy of HIV-1-mediated modification of DCs. Gene Therapy (2006) 13, 991-994.

Background: Vpx is a non-structural protein coded by members of the SIV SM /HIV-2 lineage that is believed to have originated by duplication of the common vpr gene present in primate lentiviruses. Vpx is incorporated into virion particles and is thus present during the early steps of viral infection, where it is thought to drive nuclear import of viral nucleoprotein complexes. We have previously shown that Vpx is required for SIV MAC -derived lentiviral vectors (LVs) infection of human monocyte-derived dendritic cells (DCs). However, since the requirement for Vpx is specific for DCs and not for other non-dividing cell types, this suggests that Vpx may play a role other than nuclear import.

Basal keratinocytes of the epidermis adhere to their underlying basement membrane through a specific interaction with laminin-5, which is composed by the association of ␣3, ␤3, and ␥2 chains. Laminin-5 has the ability to induce either stable cell adhesion or migration depending on specific processing of different parts of the molecule. One event results in the cleavage of the carboxyl-terminal globular domains 4 and 5 (LG4/5) of the ␣3 chain. In this study, we recombinantly expressed the human ␣3LG4/5 fragment in mammalian cells, and we show that this fragment induces adhesion of normal human keratinocytes and fibrosarcoma-derived HT1080 cells in a heparan-and chondroitin sulfate-dependent manner. Immunoprecipitation experiments with Na 2 35 SO 4 -labeled keratinocyte and HT1080 cell lysates as well as immunoblotting experiments revealed that the major proteoglycan receptor for the ␣3LG4/5 fragment is syndecan-1. Syndecan-4 from keratinocytes also bound to ␣3LG4/5. Furthermore we could show for the first time that unprocessed laminin-5 specifically binds syndecan-1, while processed laminin-5 does not. These results demonstrate that the LG4/5 modules within unprocessed laminin-5 permit its cell binding activity through heparan and chondroitin sulfate chains of syndecan-1 and reinforce previous data suggesting specific properties for the precursor molecule.Laminins (LNs) 1 are extracellular matrix glycoproteins composed of ␣, ␤, and ␥ chains assembled into a cross-shaped heterotrimer (␣␤␥) by forming a triple-stranded coiled-coil structure through their ␣-helical domains. At present, 15 heterotrimers termed LN-1 to LN-15 have been described with different subunit composition selected from five individual ␣ chains (␣1-␣5), three ␤ chains (␤1-␤3), and three ␥ chains (␥1-␥3) (1, 2). All LN ␣ chains comprise a large globular domain in their carboxylterminal region (G domain), which consists of five homologous globular subdomains of about 200 amino acids each (LG1-LG5). LN-5, with chain composition ␣3␤3␥2, is a component of basement membranes underlying specialized epithelia with secretory or protective function (3). In skin, LN-5 is synthesized by keratinocytes initially as a high molecular mass precursor protein of 460 kDa of which the ␣3 and ␥2 chains undergo specific processing to smaller forms after being secreted and deposited into the extracellular matrix (ECM) (4, 5). Processing of the ␣3 chain consists of cleavage of the carboxyl-terminal globular domains 4 and 5 (LG4/5) (6, 7). An additional cleavage within the aminoterminal domain IIIa of the ␣3 chain occurs subsequently and may also be important for LN-5 function (8, 9). Processing of the ␥2 chain occurs at the amino terminus with loss of a 50-kDa domain (9 -11). Processed LN-5 is the major component of anchoring filaments in skin (12) where it mediates cell adhesion via interaction of the ␣3 carboxyl-terminal LG1-3 triplet domain with both ␣ 3 ␤ 1 and ␣ 6 ␤ 4 integrins (13-16). Several studies have suggested that processed LN-5 functions both in the nuclea...

Virus families have evolved different strategies for genome uncoating, which are also followed by recombinant vectors. Vectors derived from adeno-associated viruses (AAV) are considered as leading delivery tools for in vivo gene transfer, and in particular gene therapy. Using a combination of atomic force microscopy (AFM), biochemical experiments, and physical modeling, we investigated here the physical properties and stability of AAV vector particles. We first compared the morphological properties of AAV vectors derived from two different serotypes (AAV8 and AAV9). Furthermore, we triggered ssDNA uncoating by incubating vector particles to increasing controlled temperatures. Our analyses, performed at the single-particle level, indicate that genome release can occur in vitro via two alternative pathways: either the capsid remains intact and ejects linearly the ssDNA molecule, or the capsid is ruptured, leaving ssDNA in a compact entangled conformation. The analysis of the length distributions of ejected genomes further revealed a two-step ejection behavior. We propose a kinetic model aimed at quantitatively describing the evolution of capsids and genomes along the different pathways, as a function of time and temperature. This model allows quantifying the relative stability of AAV8 and AAV9 particles.