A fundamental challenge in gene therapy is to develop approaches for delivering
nucleic acid-based gene interfering agents, such as small interfering RNAs and
ribozymes, to the appropriate cells in a way that is tissue/cell specific, efficient, and safe. Using human cytomegalovirus (HCMV)
infection of differentiated macrophages as the model, we showed that Salmonella can efficiently deliver
RNase P-based
ribozyme sequence in specific human cells, leading to substantial
ribozyme expression and effective inhibition of
viral infection. We constructed a functional
RNase P ribozyme (M1GS
RNA) that targets the overlapping
mRNA region of two HCMV
capsid proteins, the capsid scaffolding
protein (CSP) and
assemblin, which are essential for viral capsid formation. Substantial expression of
ribozymes was observed in human differentiated macrophages that were treated with attenuated Salmonella strains carrying the
ribozyme sequence constructs. A reduction of 87-90% in viral CSP expression and a reduction of about 5,000-fold in viral growth were observed in cells that were treated with Salmonella carrying the sequence of the functional
ribozyme but not with those carrying the sequence of a control
ribozyme that contained mutations abolishing the catalytic activity. To our knowledge, this study showed for the first time that
ribozymes expressed following targeted gene transfer with Salmonella-based vectors are highly active and specific in blocking
viral infection. Moreover, these results demonstrate the feasibility to develop Salmonella-mediated gene transfer of
RNase P ribozymes as an effective approach for gene-targeting applications.