Adenovirus-mediated gene transfer to retinal cells was evaluated using the replication-defective recombinant adenovirus vector Ad2/CMVlacZ-1 (coding for /I-galactosidase) both in an in vitro murine culture model and in vivo in adult mice. In vitro, no difference in infectability of neuronal and glial cells was observed, and 5096 of neurons expressed the exogenous gene at low viral concentration (10 pfulcell). In vivo, intraocular injection of 3 x IO6 pfu Ad2/CMVlacZ-1 resulted in expression of the transferred /3galactosidase gene in retinal pigment epithelium and ganglion cells. These results demonstrate that Ad2lCMVkzcZ-1 is an effective vector for gene transfer into retinal cells. &y wor& Defective recombinant adenovirus; /I-Galactosidase; Retina; Gene transfer; Retinal cell culture Introductioll

Silencing individual C (constant region) genes in a؊/؊ background reduces mature B cell levels, and L chain-deficient ( ؊/؊ ؊/؊ ) mice attain a complete block in B cell development at the stage when L chain rearrangement, resulting in surface IgM expression, should be completed. L chain deficiency prevents B cell receptor association, and L chain function cannot be substituted (e.g., by surrogate L chain). Nevertheless, precursor cell levels, controlled by developmental progression and checkpoint apoptosis, are maintained, and B cell development in the bone marrow is fully retained up to the immature stage. L chain deficiency allows H chain retention in the cytoplasm, but prevents H chain release from the cell, and as a result secondary lymphoid organs are B cell depleted while T cell levels remain normal. (1). L chain genes are encoded by two different loci, and in the mouse there are an extensive number of V gene segments upstream of five J and one C gene (2). The mouse L chain locus contains within an ϳ200-kb region 2 sets of V, J, and C genes that can independently rearrange: V2-Vx-J2-C2-J4-C4 and V1-J3-C3-J1-C1 (3). C1 appears to be predominantly expressed, followed by C2 and then C3, which are less frequently expressed (4), while C4 is not found to be expressed due to a lack of functional splice sites (5). Although the locus is Ͼ10 times larger than the locus, with Ͼ100 V genes, this extensive complexity is not regarded as the reason why most mouse Abs carry a L chain. It may be that the mouse locus is simply more efficient in DNA rearrangement, which is supported by the finding that in the majority of cells with rearranged V the locus is still in germline configuration, while in most cells expressing L chain the locus is either nonproductively rearranged or deleted (6).Several mouse strains with silenced L chain locus have been described. They were generated by homologous integration of a selectable marker gene in C or targeted removal of C or J (7-11). Silencing expression of L chain shed light on isotype exclusion and L chain activation, and it was concluded that and expression are separate and independent events. Although homozygous Ϫ/Ϫ mice compensate for the deficiency with increased production, their splenic B cells and ϩ cells in the bone marrow are reduced compared with those in normal mice (7, 9), which may suggest that L chain rearrangement and expression are relatively inefficient. However, despite the lack of L chain, these mice are healthy and can mount an efficient immune response (11).During B cell development gene segments encoding Ig H chains rearrange first by D (diversity) to J H recombination at the pro-B cell stage. This is followed by V H to D-J H recombination at the pre-B-I stage; if a H chain can pair with a surrogate L chain, consisting of VpreB and 5 protein, this forms a surface expressed pre-B cell receptor (pre-BCR) 3 at the pre-B-II differentiation stage. Ordered rearrangement of Ig genes appears to direct B cell development and provides important checkpoints to establish ...

Duchenne and the less severe Becker form of muscular dystrophy (DMD,BMD) result from genetic deficiency in the level and/or activity of the protein dystrophin. The recent availability of cDNA based minigenes encoding recombinant dystrophin polypeptides has raised the possibility of somatic gene transfer as a therapeutic approach to treat dystrophin deficiency. In this respect, the mdx mouse provides a useful model of DMD exhibiting features characteristic of both the early myopathic and later fibrotic phases of the human disease. Using a mutated human cDNA, compatible in size with virus-based somatic gene transfer vectors, the pathophysiological consequences of restoring dystrophin expression have been examined in transgenic mdx mice. Transgene expression was correlated with a marked reduction of the skeletal myofibre necrosis and regeneration which is a major feature of the dystrophin-deficient phenotype in young mdx mice. The cDNA construct which is based on a very mild BMD phenotype thus encodes a highly functional dystrophin molecule whose reduced size renders it an attractive candidate for development as a therapeutic gene transfer reagent.

Adenovirus-mediated gene transfer has been used to promote efficient expression of various reporter and therapeutic transgenes such as minidystrophin in skeletal muscle tissue. However, down-regulation of the adenovirus internalisation receptors, a& and avp5, in adult myofibres and in mature cultured myotubes makes them less susceptible to infection than neonatal muscle or cultured myoblasts. It has been reported elsewhere that adenoviral transduction of cells that are normally refractory to infection can be enhanced by complexing virus particles with cationic lipids or cationic polymers. In this study we describe increased levels of adenovirusmediated transduction of cultured C2C12 myotubes, when the vector is complexed with either of the cationic lipids Lipofectamine or 1,3-dioleoyloxy-2-(6-carboxyspermy1)propylamide (DOSPER) or the cationic polymer polyethylenimine. The presence of polycations allowed a smaller dose of adenovirus vector to be used to attain the same level of infection seen with adenovirus alone, which has important relevance to future in vivo studies. Electron microscopic analysis of adenovirus/polycation complexes showed large aggregates as opposed to single adenovirus particles in the absence of polycations. Finally, by complexing adenovirus particles with polycations, partial protection against the neutralising effect of adenovirus antiserum was observed.

Ig are multifunctional molecules with distinct properties assigned to individual domains. To assess the importance of IgM domain assembly in B cell development we generated two transgenic mouse lines with truncated muH chains by homologous integration of the neomycin resistance gene (neo(r)) into exons C(mu)1 and C(mu)2. Upon DNA rearrangement shortened muH chain transcripts, V(H)-D-J(H)-C(mu)3-C(mu)4, are produced independent of the transcriptional orientation and termination signals provided by neo(r). The truncated muH chain of approximately 52 kDa associates non-covalently with the L chain to form a monovalent HL heterodimer. Surface IgM is assembled into a defective BCR complex which has lost important signalling capacity. In immunizations with T-dependent and T-independent antigens, specific IgM antibodies cannot be detected, whilst IgG responses remain normal. B cell development in the bone marrow is characterized by an increase in early B cells, but a decrease of B220(+) cells from the stage when muH chain rearrangement is completed. The peritoneal lymphocyte population has elevated levels of CD5(+) B cells and their expansion may be the result of a negative feedback mechanism. The results show that antigenic stimulation is compromised by truncated monovalent IgM and that this deficit in stimulation leads to reduced levels of conventional B-2 lymphocytes, but dramatically increased levels of B-1 cells.