Artificial miRNAs mitigate shRNA-mediated toxicity in the brain: implications for the therapeutic development of RNAi.

Huntington's disease (HD) is a fatal, dominant neurodegenerative disease caused by a polyglutamine repeat expansion in exon 1 of the HD gene, which encodes the huntingtin protein. We and others have shown that RNAi is a candidate therapy for HD because expression of inhibitory RNAs targeting mutant human HD transgenes improved neuropathology and behavioral deficits in HD mouse models. Here, we developed shRNAs targeting conserved sequences in human HD and mouse HD homolog (HDh) mRNAs to initiate preclinical testing in a knockin mouse model of HD. We screened 35 shRNAs in vitro and subsequently narrowed our focus to three candidates for in vivo testing. Unexpectedly, two active shRNAs induced significant neurotoxicity in mouse striatum, although HDh mRNA expression was reduced to similar levels by all three. Additionally, a control shRNA containing mismatches also induced toxicity, although it did not reduce HDh mRNA expression. Interestingly, the toxic shRNAs generated higher antisense RNA levels, compared with the nontoxic shRNA. These results demonstrate that the robust levels of antisense RNAs emerging from shRNA expression systems can be problematic in the mouse brain. Importantly, when sequences that were toxic in the context of shRNAs were placed into artificial microRNA (miRNA) expression systems, molecular and neuropathological readouts of neurotoxicity were significantly attenuated without compromising mouse HDh silencing efficacy. Thus, miRNA-based approaches may provide more appropriate biological tools for expressing inhibitory RNAs in the brain, the implications of which are crucial to the development of RNAi for both basic biological and therapeutic applications.

Optimization of feline immunodeficiency virus vectors for RNA interference.

RNA interference (RNAi) occurs naturally in plant and animal cells as a means for modulating gene expression. This process has been experimentally manipulated to achieve targeted gene silencing in cells, tissues, and animals, using a variety of vector systems. Here, we tested the hypothesis that vectors based on feline immunodeficiency virus (FIV) could be used for coexpression of reporter constructs and RNAi expression cassettes. We found, unexpectedly, in our initial constructs that placement of RNAi expression cassettes downstream from a polymerase II (pol II)-expressed reporter gene inhibited reporter expression but not vector titer. Through a series of intermediate vector constructs, we found that placement of the RNAi expression cassette relative to the Rev response element and the pol II expression cassette was critical for efficient RNAi and reporter gene expression. These results suggested that steric factors, including RNA structure and recruitment of competing transcriptional machinery, may affect gene expression from FIV vectors. In a second series of studies, we show that target sequence silencing can be achieved in cells transduced by FIV vectors coexpressing reporter genes and 3' untranslated region resident microRNAs. The optimized FIV-based RNAi expression vectors will find broad use given the extensive tropism of pseudotyped FIV vectors for many cell types in vitro and in vivo.

RNA interference improves motor and neuropathological abnormalities in a Huntington's disease mouse model.

Huntington's disease (HD) is a fatal, dominant neurogenetic disorder. HD results from polyglutamine repeat expansion (CAG codon, Q) in exon 1 of HD, conferring a toxic gain of function on the protein huntingtin (htt). Currently, no preventative treatment exists for HD. RNA interference (RNAi) has emerged as a potential therapeutic tool for treating dominant diseases by directly reducing disease gene expression. Here, we show that RNAi directed against mutant human htt reduced htt mRNA and protein expression in cell culture and in HD mouse brain. Importantly, htt gene silencing improved behavioral and neuropathological abnormalities associated with HD. Our data provide support for the further development of RNAi for HD therapy.

Targeted viral delivery of Cre recombinase induces conditional gene deletion in cardiovascular circuits of the mouse brain.

The Cre/loxP system has shown promise for investigating genes involved in nervous system function and pathology, although its application for studying central neural regulation of cardiovascular function and disease has not been explored. Here, we report for the first time that recombination of loxP-flanked genes can be achieved in discrete cardiovascular regulatory nuclei of adult mouse brain using targeted delivery of adenovirus (Ad) or feline immunodeficiency virus (FIV) bearing Cre recombinase (Ad-Cre, FIV-Cre). Single stereotaxic microinjections of Ad-Cre or FIV-Cre into specific nuclei along the subfornical organ-hypothalamic-hypophysial and brain stem-parabrachial axes resulted in robust and highly localized gene deletion as early as 7 days and for as long as 3 wk in a reporter mouse model in which Cre recombinase activates beta-galactosidase expression. An even greater selectivity in Cre-mediated gene deletion could be achieved in unique subpopulations of cells, such as vasopressin-synthesizing magnocellular neurons, by delivering Ad-Cre via retrograde transport. Moreover, Ad-Cre and FIV-Cre induced gene recombination in differential cell populations within these cardiovascular nuclei. FIV-Cre infection resulted in LacZ activation selectively in neurons, whereas both neuronal and glial cell types underwent gene recombination upon infection with Ad-Cre. These results establish the feasibility of using a combination of viral and Cre/loxP technologies to target specific cardiovascular nuclei in the brain for conditional gene modification and suggest the potential of this approach for determining the functional role of genes within these sites.

Lentivirus vectors pseudotyped with filoviral envelope glycoproteins transduce airway epithelia from the apical surface independently of folate receptor alpha.

The practical application of gene therapy as a treatment for cystic fibrosis is limited by poor gene transfer efficiency with vectors applied to the apical surface of airway epithelia. Recently, folate receptor alpha (FR alpha), a glycosylphosphatidylinositol-linked surface protein, was reported to be a cellular receptor for the filoviruses. We found that polarized human airway epithelia expressed abundant FR alpha on their apical surface. In an attempt to target these apical receptors, we pseudotyped feline immunodeficiency virus (FIV)-based vectors by using envelope glycoproteins (GPs) from the filoviruses Marburg virus and Ebola virus. Importantly, primary cultures of well-differentiated human airway epithelia were transduced when filovirus GP-pseudotyped FIV was applied to the apical surface. Furthermore, by deleting a heavily O-glycosylated extracellular domain of the Ebola GP, we improved the titer of concentrated vector severalfold. To investigate the folate receptor dependence of gene transfer with the filovirus pseudotypes, we compared gene transfer efficiency in immortalized airway epithelium cell lines and primary cultures. By utilizing phosphatidylinositol-specific phospholipase C (PI-PLC) treatment and FR alpha-blocking antibodies, we demonstrated FR alpha-dependent and -independent entry by filovirus glycoprotein-pseudotyped FIV-based vectors in airway epithelia. Of particular interest, entry independent of FR alpha was observed in primary cultures of human airway epithelia. Understanding viral vector binding and entry pathways is fundamental for developing cystic fibrosis gene therapy applications.

In vivo gene transfer using a nonprimate lentiviral vector pseudotyped with Ross River Virus glycoproteins.

Vectors derived from lentiviruses provide a promising gene delivery system. We examined the in vivo gene transfer efficiency and tissue or cell tropism of a feline immunodeficiency virus (FIV)-based lentiviral vector pseudotyped with the glycoproteins from Ross River Virus (RRV). RRV glycoproteins were efficiently incorporated into FIV virions, generating preparations of FIV vector, which after concentration attain titers up to 1.5 x 10(8) TU/ml. After systemic administration, RRV-pseudotyped FIV vectors (RRV/FIV) predominantly transduced the liver of recipient mice. Transduction efficiency in the liver with the RRV/FIV was ca. 20-fold higher than that achieved with the vesicular stomatitis virus G protein (VSV-G) pseudotype. Moreover, in comparison to VSV-G, the RRV glycoproteins caused less cytotoxicity, as determined from the levels of glutamic pyruvic transaminase and glutamic oxalacetic transaminase in serum. Although hepatocytes were the main liver cell type transduced, nonhepatocytes (mainly Kupffer cells) were also transduced. The percentages of the transduced nonhepatocytes were comparable between RRV and VSV-G pseudotypes and did not correlate with the production of antibody against the transgene product. After injection into brain, RRV/FIV preferentially transduced neuroglial cells (astrocytes and oligodendrocytes). In contrast to the VSV-G protein that targets predominantly neurons, <10% of the brain cells transduced with the RRV pseudotyped vector were neurons. Finally, the gene transfer efficiencies of RRV/FIV after direct application to skeletal muscle or airway were also examined and, although transgene-expressing cells were detected, their proportions were low. Our data support the utility of RRV glycoprotein-pseudotyped FIV lentiviral vectors for hepatocyte- and neuroglia-related disease applications.

Selective gene transfer to key cardiovascular regions of the brain: comparison of two viral vector systems.

The systemic renin-angiotensin system (RAS) plays a critical role in cardiovascular (CV) homeostasis. All components of the RAS are also known to be produced cell-specifically within specific brain regions, although the role of the brain RAS relative to the systemic RAS has remained a puzzle due to the difficulty of dissecting these two systems. Selectively targeting these regions with genes that modify the RAS could help unravel this puzzle. We compared the ability of adenovirus (Ad) and lentivirus (feline immunodeficiency virus, FIV) vectors to mediate gene delivery in vivo to the supraoptic nucleus (SON) and subfornical organ (SFO), two important CV control regions known to express the various RAS genes. SON or SFO of adult C57BL/6 mice (n=37) were stereotaxically injected with replication-deficient recombinant Ad or FIV harboring a beta-galactosidase (beta-gal) reporter gene. At 1, 3, or 8 weeks post-injection, brain sections were processed for beta-Gal activity, double immunofluorescence to verify cell-type specificity of viral transduction, or immunohistochemical detection of inflammatory mediators. Our results demonstrate that: (1) murine SFO and SON can be selectively targeted for gene transfer in vivo;(2) FIV mediated neuron-specific gene delivery, whereas Ad transduced both neuronal and glial cell types in SFO and SON; (3) Ad injected into the SON transduced neurons within the SFO through retrograde transport, whereas FIV did not; (4) beta-gal activity remained stable for 3 weeks but then declined by 8 weeks with Ad, while minimal decline occurred with FIV; (5) FIV did not cause inflammatory responses, whereas infiltrate was detectable in Ad-injected SFO and SON. These vectors are potentially important tools for dissecting the cell- and site-specific components of the brain RAS and other important CV regulatory systems within this circuitry, and may have therapeutic applications for centrally mediated CV diseases.