rAAV-compatible MiniPromoters for restricted expression in the brain and eye

Abstract Background Small promoters that recapitulate endogenous gene expression patterns are important for basic, preclinical, and now clinical research. Recently, there has been a promising revival of gene therapy for diseases with unmet therapeutic needs. To date, most gene therapies have used viral-based ubiquitous promoters–however, promoters that restrict expression to target cells will minimize off-target side effects, broaden the palette of deliverable therapeutics, and thereby improve safety and efficacy. Here, we take steps towards filling the need for such promoters by developing a high-throughput pipeline that goes from genome-based bioinformatic design to rapid testing in vivo. Methods For much of this work, therapeutically interesting Pleiades MiniPromoters (MiniPs; ~4 kb human DNA regulatory elements), previously tested in knock-in mice, were “cut down” to ~2.5 kb and tested in recombinant adeno-associated virus (rAAV), the virus of choice for gene therapy of the central nervous system. To evaluate our methods, we generated 29 experimental rAAV2/9 viruses carrying 19 different MiniPs, which were injected intravenously into neonatal mice to allow broad unbiased distribution, and characterized in neural tissues by X-gal immunohistochemistry for icre, or immunofluorescent detection of GFP. Results The data showed that 16 of the 19 (84 %) MiniPs recapitulated the expression pattern of their design source. This included expression of: Ple67 in brain raphe nuclei; Ple155 in Purkinje cells of the cerebellum, and retinal bipolar ON cells; Ple261 in endothelial cells of brain blood vessels; and Ple264 in retinal Müller glia. Conclusions Overall, the methodology and MiniPs presented here represent important advances for basic and preclinical research, and may enable a paradigm shift in gene therapy

The H1P1 family of cytoskeletal-associated proteins

Huntingtin-interacting protein 1 (HJPT) is a membrane associated protein which interacts with huntingtin, the protein altered in Huntington Disease (HD). It was determined that the H1P1 gene consists of 32 exons spanning approximately 215 kb of genomic DNA and gives rise to two alternate splice forms termed HIPl-1 and HIP1- 2. Additionally, a novel homolog of HIP1, termed HIP12, was identified. HIP1 and HIP12 share significant amino acid identity and both proteins contain an epsin N-terminal homology (ENTH), coiled coil and talin-like domains. While overexpression of HTP1 is toxic in cell culture, HIP12 did not confer toxicity in the same assay systems. Interestingly, HIP12 did not interact with huntingtin but was able to interact with HJPT suggesting a potential interaction in vivo which may influence the function of each respective protein. Tissue distribution studies indicated that HIP1 was expressed predominantly within the brain whereas HIP12 expression was more ubiquitous. Within cells, HIP1 and HJPT2 displayed a similar punctate distribution at the plasma membrane and underlying cortical actin cytoskeleton. HIP1 was shown to interact with the endocytic proteins clathrin heavy chain and AP2. However, HIP12 displayed weak binding to clathrin heavy chain and no interaction with AP2. HIP1 and HIP12 were also shown to interact with the clathrin light chain. In addition, it was also revealed that HJPT and HIP12 were capable of inducing the assembly of the clathrin coat in vitro. Finally, it was determined that HIP12 interacts with F-actin suggesting that HIP12 may interact with the cortical actin cytoskeleton underlying clathrin-coated pits and vesicles. HIP1 may be indirectly associated with the actin cytoskeleton by forming heterodimers with HIP12. Thus, HTP1 and HTP12 may act as a molecular "link" between the endocytic machinery and the cortical actin cytoskeleton. Taken together, these results demonstrate that HIP1 and HIP12 are components of the endocytic machinery that participate in the recruitment, assembly and stabilization of the clathrin lattice structure. The observation that huntingtin associates with HIP1 suggests that it may function in endocytosis and transport. Since mutant huntingtin displays an altered interaction with HIP1, it raises the possibility that HD may be the result of defects in endocytosis and transport. The determination of the cellular functions of the HJP1 family and huntingtin in endocytosis and transport may provide valuable insights into the pathogenic mechanism(s) underlying HD.Medicine, Faculty ofMedical Genetics, Department ofGraduat

Differential effects of cysteamine on heat shock protein induction and cytoplasmic granulation in astrocytes and glioma cells

A subpopulation of astrocytes in the limbic and periventricular brain regions of many vertebrates, including humans, progressively accumulates cytoplasmic inclusions with advancing age which are morphologically and histochemically distinct from the aging pigment, lipofuscin. The sulfhydryl compound, cysteamine (CSH), promotes the accumulation of identical autofluorescent, peroxidase-positive granules in primary astrocyte cultures. In the present study, we demonstrated that within 6 hours of CSH-treatment, long before cytoplasmic granulation occurs, cultured astroglia exhibit increased levels of HSP 27, HSP 90 and heme oxygenase (HO-1) protein and mRNA, relative to control (untreated) cultures. Moreover, CSH treatment elicited relatively minor increases in HO-1 mRNA and had no effect on HSP 27 and HSP 90 mRNA and protein in the glioma cells. Untreated glioma cells exhibited yellow autofluorescent cytoplasmic granules consistent with lipofuscin which was not affected by CSH exposure. Moreover, CSH treatment failed to induce the accumulation of red autofluorescent, peroxidase-positive inclusions in these cells. Taken together, these findings support the notion that the biogenesis of peroxidase-positive astrocytic granules in CSH-treated cultures and in the aging periventricular brain is dependent on an antecedent stress (heat shock) response. In further support of this contention, we demonstrated that CSH treatment up-modulates the expression of the mitochondrial antioxidant MnSOD in cultured astroglia. Augmented levels of MnSOD mRNA and protein were only noted after 5 days CSH exposure. The relatively late induction of MnSOD suggests that it is not part of generalized cellular stress response. It may serve as a late cytoprotective mechanism in astrocytes against secondary superoxide injury resulting from mitochondrial damage rather than due to the direct effects of CSH. (Abstract shortened by UMI.

HIP1 and HIP12 display differential binding to F-actin, AP2, and clathrin : identification of a novel interaction with clathrin light chain

Huntingtin-interacting protein 1 (HIP1) and HIP12 are orthologues of Sla2p, a yeast protein with essential functions in endocytosis and regulation of the actin cytoskeleton. We now report that HIP1 and HIP12 are major components of the clathrin coat that interact but differ in their ability to bind clathrin and the clathrin adaptor AP2. HIP1 contains a clathrin-box and AP2 consensus-binding sites that display high affinity binding to the terminal domain of the clathrin heavy chain and the ear domain of the AP2 alpha subunit, respectively. These consensus sites are poorly conserved in HIP12 and correspondingly, HIP12 does not bind to AP2 nor does it demonstrate high affinity clathrin binding. Moreover, HIP12 co-sediments with F-actin in contrast to HIP1, which exhibits no interaction with actin in vitro. Despite these differences, both proteins efficiently stimulate clathrin assembly through their central helical domain. Interestingly, in both HIP1 and HIP12, this domain binds directly to the clathrin light chain. Our data suggest that HIP1 and HIP12 play related yet distinct functional roles in clathrin-mediated endocytosis

Identification of a set of genes showing regionally enriched expression in the mouse brain

Background: The Pleiades Promoter Project aims to improve gene therapy by designing human mini-promoters (< 4 kb) that drive gene expression in specific brain regions or cell-types of therapeutic interest. Our goal was to first identify genes displaying regionally enriched expression in the mouse brain so that promoters designed from orthologous human genes can then be tested to drive reporter expression in a similar pattern in the mouse brain. Results: We have utilized LongSAGE to identify regionally enriched transcripts in the adult mouse brain. As supplemental strategies, we also performed a meta-analysis of published literature and inspected the Allen Brain Atlas in situ hybridization data. From a set of approximately 30,000 mouse genes, 237 were identified as showing specific or enriched expression in 30 target regions of the mouse brain. GO term over-representation among these genes revealed co-involvement in various aspects of central nervous system development and physiology. Conclusion: Using a multi-faceted expression validation approach, we have identified mouse genes whose human orthologs are good candidates for design of mini-promoters. These mouse genes represent molecular markers in several discrete brain regions/cell-types, which could potentially provide a mechanistic explanation of unique functions performed by each region. This set of markers may also serve as a resource for further studies of gene regulatory elements influencing brain expression.Medical Genetics, Department ofMedicine, Faculty ofMolecular Medicine and Therapeutics, Centre forNon UBCReviewedFacult

Identification of a set of genes showing regionally enriched expression in the mouse brain-1

Ers of tissues is plotted on the X-axis. At each node in the dendrogram, the number of genes shared between libraries in the tissue cluster is indicated. A threshold of 50% of maximum EDwas chosen for coloring of branch lines in the dendrogram.<p><b>Copyright information:</b></p><p>Taken from "Identification of a set of genes showing regionally enriched expression in the mouse brain"</p><p>http://www.biomedcentral.com/1471-2202/9/66</p><p>BMC Neuroscience 2008;9():66-66.</p><p>Published online 14 Jul 2008</p><p>PMCID:PMC2483290.</p><p></p

Identification of a set of genes showing regionally enriched expression in the mouse brain-2

Ntain expression information for a gene in a specific region of the brain.<p><b>Copyright information:</b></p><p>Taken from "Identification of a set of genes showing regionally enriched expression in the mouse brain"</p><p>http://www.biomedcentral.com/1471-2202/9/66</p><p>BMC Neuroscience 2008;9():66-66.</p><p>Published online 14 Jul 2008</p><p>PMCID:PMC2483290.</p><p></p