Worldwide, human adenoviruses (Ad) cause 8% of viral infections responsible for a spectrum of diseases such as respiratory, gastro-intestinal, and urogenital tract infections, and keratoconjunctivitis. Epidemics affect school attendance and work-place productivity. Life-threatening disseminated infections have been reported among high-risk populations such as pediatric and adult bone marrow transplant (BMT) patients. Based on our observation that vitreous/hyaluronan (HA) enhances adenovirus (Ad) vector transduction not only in a dose dependent manner but also in a CD44 dependent manner, we hypothesize that modulating the interaction between CD44 and its ligand HA will inhibit adenoviral infections by several serotypes. Since degradation of CD44 by metalloproteinases and by the v-secretase/PS1 complex plays an important role in regulating protein transcription, we also hypothesize that this enhancement is related to degradation of CD44 with resultant changes in the CD44-dependent signaling pathways. To examine these hypotheses, the following Specific Aims are proposed:
Aim 1) Determine the role of CD44 in the enhancement of transgene expression. This will be accomplished by a) elucidating the role of CD44 degradation by using PCR to delete the matrix metalloproteinase (MMP) and y-secretase cleavage sites and by over-expressing the degradation product of CD44 (the CD44 intracellular domain) to determine its role in the HA induced enhancement of Ad transduction;b) elucidating the role of CD44 phosphorylation using PCR mutagenesis to alter critical phosphorylation sites in the cytoplasmic domain of CD44 and studying their effects on Ad5 transduction. Inhibitors of downstream signaling molecules associated with CD44 will also be studied;and c) determining if Ad5 can bind to the HA receptor CD44 using saturating concentrations of RGD peptide and low incubation temperatures to block viral endocytosis. Binding will be determined in the presence and absence of HA using CHO cells that do not express the CAR or CD46 adenoviral receptors but do express CD44. Digested HA, anti-CD44 and soluble CD44 will be used to study binding of Ad5 to CHO cells.
Aim 2) Establish if infections by wild type Ad of different serotypes can be influenced by modulation of the CD44 signaling pathway using Ads of different serotypes to infect human conjunctiva explants and measuring the viral particles released in the presence or absence of HA with Q-PCR. The effect of CD44 related signaling molecule inhibitors will also be tested. Unraveling the role that CD44 plays in adenoviral infections may uncover potential targets that can be used to design new drugs to control these infections thus impacting not only the lives of at-risk patients but also the health and productivity of the general population.
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