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Abstract
RNAi is an innate cellular mechanism that results in sequence specific gene silencing. This mechanism has been exploited for use as both a tool in biological research and as a therapeutic drug against a variety of diseases. The use of RNAi based gene therapies against HIV is considered a viable option to prevent or control infection. However, for gene therapy to be effective in the treatment of HIV, several barriers must be overcome. First, the intransigence of primary T cells to traditional transfection/transduction methods leads to inefficient delivery of small si/shRNAs to the main target cells of HIV (Goffinet and Keppler, 2006). Second, once inside the cell, the majority of siRNA remains trapped within the endosome where it is unavailable for loading onto the cytoplasmically located RISC complex (Gilleron et al., 2013). Third, targeting even conserved sequences of the HIV genome still allows for the emergence of escape mutants (Das et al., 2004; Westerhout et al., 2005). The work presented in this thesis aims to overcome these limitations by investigating efficient si/shRNA delivery vehicles and novel host genes that may be potential targets for RNAi mediated knockdown in the prevention of HIV.
For development of an siRNA delivery vector that may be more suitable for temporary knockdown of target genes, we aimed to adapt a cell penetrating peptide (CPP) for use as an siRNA delivery vehicle. CPPs such as nona-arginine (9R) are ineffective for siRNA-induced gene silencing as siRNA uptake is poor and siRNA complexes remain trapped within endosomes (Gilleron et al., 2013; Sahay et al., 2013). Our studies demonstrate that attaching 9R to ligands, such as the rabies virus glycoprotein or a single chain antibody directed against human CD7, that induce endocytosis quantitatively increases siRNA uptake and allows functional delivery of complexed siRNA. The intracellular trafficking pathway of ligand-9R allows cytoplasmic localization of delivered siRNA at early time points. Further, the L isoform of 9R in the ligand-9R format, in contrast to the protease-resistant D isoform, enables continued siRNA escape from late Rab 7+ endosomes in a protease dependent manner, prolonging mRNA knockdown. The data imply ligand-mediated endocytosis and siRNA dissociation by partial CPP degradation in the late endosome critically contribute to cytoplasmic siRNA delivery. These insights into siRNA uptake, trafficking and release mechanisms will help guide development of effective CPP-based siRNA delivery systems.
One of the major obstacles in the treatment and prevention of HIV infection is the viral sequence diversity, both globally and that which develops within an individual (Hu and Temin, 1990; Preston et al., 1988). However, it appears that in most instances only a single or a few founder viruses are capable of crossing the mucosal interface and initiating infection within the human host (Derdeyn et al., 2004; Keele et al., 2008). Hindering viral replication within the mucosal tissue during the initial phase of viral infection and entry into the human host may effectively block systemic infection and spread of HIV-1 and thus the establishment of a viral reservoir. The mucosal interface, therefore, represents a site of viral infection at a time point prior to viral evolution where treatment approaches, like RNAi targeting conserved viral sequences, may be harnessed to effectively block viral transmission. We therefore developed a humanized antibody to human CD7 for the delivery of anti-viral siRNAs to HIV susceptible CD7- T cells within the vaginal mucosa.
For a chronic infection like HIV, a gene therapy approach that elicits a stable knockdown of the target gene is considered favorable (ter Brake et al., 2008). We therefore sought to develop a lentiviral vector that can deliver integrating shRNA to primary T cells with high efficiency for long-term reduction of host gene expression. We used a monoclonal antibody to the pan T cell-surface molecule CD7 to guide a chimeric Sindbis envelope-pseudotyped lentiviral vector to T cells. We achieved selective transduction of human primary T cells both in culture and also following systemic injection of humanized mice. Transfer of anti-CCR5 shRNA to T cells within humanized mice protected against HIV-1 infection.
An alternative to initiating knockdown of viral genes with RNAi is to target host proteins critically required for viral replication. This may prove more effective as it is accompanied with a low risk of inducing mutations and viral escape is therefore less likely (Santa-Marta et al., 2013). A number of host genes have been associated with a role in the HIV lifecycle, however, in vivo validation of the dependence of HIV on these factors is required for them to be considered viable options for gene therapy. We investigated four genes implicated as HIV host dependency factors as potential targets for HIV gene therapy. After preliminary studies of target knockdown with shRNA in cell lines, we selected two for validation in a humanized mouse model for HIV challenge. Unfortunately, our in vivo experiments were not wholly reflective of the promising in vitro data and future investigations are required to clarify our observations.