Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UMLS:C0001486 (Adenovirus)
 3,125 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

This report identifies formulation and processing factors that influence stability of viral preparations such as selection of appropriate buffer systems, cryoprotectants, and cooling rates. Adenovirus type 5 containing the lacZ marker gene was suspended in combinations of trehalose, sorbitol, sucrose, mannitol, glycine, CaCl2, and gelatin. X-gal stains of 293 cells were used to determine the lac-forming units (lfu)/ml of each preparation before and after treatments. Phosphate-buffered solutions (except those containing sucrose or trehalose) demonstrated a drop of 3 pH units upon freezing regardless of cryoprotectant used. Tris-buffered solutions demonstrated a variation in pH which was dependent upon chosen cryoprotectant, with 1 M trehalose exhibiting no change and a 5% mannitol/10 mM CaCl2 combination showing a 3-unit drop in pH. 4-[2-Hydroxyethyl]-1-piperazine ethanesulfonic acid (HEPES)-buffered solutions showed little change in initial pH when frozen regardless of cryoprotectant chosen. In solution, adenovirus was not affected by incubation for 24 hr in buffers ranging from pH 4 to 8. However, when the solutions were frozen, the number of remaining infectious virions was dependent upon the final pH of the suspending medium. Cryoprotectant solutions that significantly maintained viral stability during a single freeze--thaw cycle were 0.5 M sucrose, 0.5 M trehalose, and 10% sorbitol/0.4% gelatin. Long-term stability studies were performed at 4 degrees C with lyophilized sorbital/gelatin and sucrose preparations. Both formulations provided adequate stability for the adenovirus, with 2.6 and 5.6 x 10(11) lfu/ml detected 150 days after drying, respectively.
 ...
PMID:Factors that influence stability of recombinant adenoviral preparations for human gene therapy. 974 58

Replication-deficient adenovirus vectors are efficient vehicles for delivering therapeutic genes into mammalian cells. However, the high doses required to produce effective gene transfer in vivo can also cause unwanted cellular toxicity. To improve replication-deficient adenovirus transgene expression while minimizing adverse reactions, we have tested polycationic compounds for their ability to enhance adenovirus adsorption. We demonstrate increased transgene expression after mixing adenovirus preparations with polycations, cationic lipids, and CaCl2 prior to transduction in vitro. An E1-deleted adenovirus vector was admixed with various polycations, and beta-galactosidase (beta-gal) activity was evaluated. The optimal polycation concentrations for augmenting adenovirus-mediated gene transfer were 5-10 microg/mL polybrene, 400 microg/mL protamine sulfate, 10 microg/mL N-(1-[2,3-dioleoyloxy]propyl)-N,N,N-trimethylammonium methylsulfate (DOTAP), 2.5 microg/mL Lipofectamine, and 62.5 mM CaCl2. Polycations enhanced beta-gal expression in three of six established cell lines. Similar results were obtained using primary tumor cell cultures, where beta-gal expression was increased 1.5- to 10.7-fold (mean = 3.6) by polybrene, 1.8- to 7.5-fold (mean = 3.4) by DOTAP, and 2.3- to 10.4-fold (mean = 4.8) by protamine sulfate. Adenovirus transduction efficiency in two primary leukemia isolates was improved by 3- and 4.5-fold. We were unable to demonstrate any benefit when adenovirus was admixed with protamine sulfate prior to intratumoral injection in a xenogeneic severe combined immunodeficient mouse melanoma tumor model. Further studies will determine whether polycations can improve intratumoral gene transfer.
 ...
PMID:Polycations and cationic lipids enhance adenovirus transduction and transgene expression in tumor cells. 1050 54