ABSTRACT
Inefficient gene transfer has limited the success of gene therapy in the hematopoietic system. Here we develop a novel chimeric adenovirus (Ad) vector containing Ad serotype 11 fiber-modified capsids and E1/E3 deleted viral genomes (Ad5/11) or genomes devoid of all viral genes (DeltaAd5/11). The capsid-modified vectors transduced human hematopoietic cells more efficiently than the unmodified Ad5-based vector. The absence of viral genes from the DeltaAd5/11 vector allowed for transduction without the associated toxicity seen with the first-generation E1/E3 deleted vector. Chimeric vectors were used for transient expression of the ecotropic retrovirus receptor (ecoR) in Mo7e cells (a CD34-positive, c-Kit-positive, growth-factor-dependent human cell line) as a model for human hematopoietic progenitor cells. Expression of ecoR conferred susceptibility to subsequent retroviral transduction. The DeltaAd5/11 vector used to express ecoR allowed for expansion of retrovirally transduced cells, whereas transduction with the first-generation Ad5/11 vector resulted in cytotoxicity and, over time, loss of cells expressing the retrovirus-vector-derived transgene.
Subject(s)
Adenoviridae/genetics , Gene Transfer Techniques , Genetic Vectors , Genome, Viral , Hematopoietic Stem Cells , Animals , Antigens, CD34/biosynthesis , Apoptosis , Capsid/genetics , Capsid/metabolism , Capsid/physiology , Cell Line , Cell Survival , Cells, Cultured , Gene Deletion , Genetic Therapy/methods , Genetic Vectors/toxicity , Humans , Rats , Transduction, Genetic , TransgenesSubject(s)
Acyltransferases/metabolism , Alcohol Oxidoreductases/metabolism , Visual Pathways/enzymology , Animals , Carrier Proteins/metabolism , Cattle , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Esters , Hydrolysis , Recombinant Proteins/metabolism , Retinaldehyde/metabolism , Retinoids/isolation & purificationABSTRACT
Photoisomerization of 11-cis-retinal to all-trans-retinal triggers phototransduction in the retinal photoreceptor cells and causes ultimately the sensation of vision. 11-cis-Retinal is enzymatically regenerated through a complex set of reactions in adjacent retinal pigment epithelial cells (RPE). In this study using all-trans-9-desmethylretinol (lacking the C(19) methyl group) and all-trans-13-desmethylretinol (lacking the C(20) methyl group), we explored the effects of C(19) and C(20) methyl group removals on isomerization of these retinols in RPE microsomes. The C(19) methyl group may be involved in the substrate activation, whereas the C(20) methyl group causes steric hindrance with a proton in position C(10) of 11-cis-retinol; thus, removal of this group could accelerate isomerization. We found that all-trans-9-desmethylretinol and all-trans-13-desmethylretinol are isomerized to their corresponding 11-cis-alcohols, although with lower efficiencies than isomerization of all-trans-retinol to 11-cis-retinol. These findings make the mechanism of isomerization through the C(19) methyl group unlikely, because in the case of 9-desmethylretinol, the isomerization would have to progress by proton abstraction from electron-rich olefinic C(9). The differences between all-trans-retinol, all-trans-9-desmethylretinol, and all-trans-13-desmethylretinol appear to be a consequence of the enzymatic properties, and binding affinities of the isomerization system, rather than differences in the chemical or thermodynamic properties of these compounds. This observation is also supported by quantum chemical calculations. It appears that both methyl groups are not essential for the isomerization reaction and are not likely involved in formation of a transition stage during the isomerization process.
Subject(s)
Pigment Epithelium of Eye/chemistry , Retinaldehyde/chemistry , Alcohol Oxidoreductases/chemistry , Alcohol Oxidoreductases/metabolism , Animals , Apoproteins/chemistry , Apoproteins/genetics , Apoproteins/metabolism , Carrier Proteins/chemistry , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cattle , Diterpenes , Isomerism , Pigment Epithelium of Eye/cytology , Pigment Epithelium of Eye/metabolism , Protein Conformation , Quantum Theory , Retinaldehyde/metabolism , Retinoids/chemistry , Retinoids/metabolism , Serum Albumin, Bovine/physiologyABSTRACT
In photoreceptor cells of the retina, photoisomerization of 11-cis-retinal to all-trans-retinal triggers phototransduction. Regeneration of 11-cis-retinal proceeds via a complex set of reactions in photoreceptors and in adjacent retinal pigment epithelial cells where all-trans-retinol is isomerized to 11-cis-retinol. Our results show that isomerization in vitro only occurs in the presence of apo-cellular retinaldehyde-binding protein. This retinoid-binding protein may drive the reaction by mass action, overcoming the thermodynamically unfavorable isomerization. Furthermore, this 11-cis-retinol/11-cis-retinal-specific binding protein potently stimulates hydrolysis of endogenous 11-cis-retinyl esters but has no effect on hydrolysis of all-trans-retinyl esters. Apo-cellular retinaldehyde-binding protein probably exerts its effect by trapping the 11-cis-retinol product. When retinoid-depleted retinal pigment epithelial microsomes were preincubated with different amounts of all-trans-retinol to form all-trans-retinyl esters and then [3H]all-trans-retinol was added, as predicted, the specific radioactivity of [3H]all-trans-retinyl esters increased during subsequent reaction. However, the specific radioactivity of newly formed 11-cis-retinol stayed constant during the course of the reaction, and it was largely unaffected by expansion of the all-trans-retinyl ester pool during the preincubation. The absence of dilution establishes that most of the ester pool does not participate in isomerization, which in turn suggests that a retinoid intermediate other than all-trans-retinyl ester is on the isomerization reaction pathway.
