A peptide-linked recombinant glucocerebrosidase for targeted neuronal delivery: Design, production, and assessment.

Although recombinant glucocerebrosidase (GCase) is the standard therapy for the inherited lysosomal storage disease Gaucher's disease (GD), enzyme replacement is not effective when the central nervous system is affected. We created a series of recombinant genes/proteins where GCase was linked to different membrane binding peptides including the Tat peptide, the rabies glycoprotein derived peptide (RDP), the binding domain from tetanus toxin (TTC), and a tetanus like peptide (Tet1). The majority of these proteins were well-expressed in a mammalian producer cell line (HEK 293F). Purified recombinant Tat-GCase and RDP-GCase showed similar GCase protein delivery to a neuronal cell line that genetically lacks the functional enzyme, and greater delivery than control GCase, Cerezyme (Genzyme). This initial result was unexpected based on observations of superior protein delivery to neurons with RDP as a vector. A recombinant protein where a fragment of the flexible hinge region from IgA (IgAh) was introduced between RDP and GCase showed substantially enhanced GCase neuronal delivery (2.5 times over Tat-GCase), suggesting that the original construct resulted in interference with the capacity of RDP to bind neuronal membranes. Extended treatment of these knockout neuronal cells with either Tat-GCase or RDP-IgAh-GCase resulted in an >90% reduction in the lipid substrate glucosylsphingosine, approaching normal levels. Further in vivo studies of RDP-IgAh-GCase as well as Tat-GCase are warranted to assess their potential as treatments for neuronopathic forms of GD. These peptide vectors are especially attractive as they have the potential to carry a protein across the blood-brain barrier, avoiding invasive direct brain delivery.

Tat-tetanus toxin fragment C: a novel protein delivery vector and its use with photochemical internalization.

Protein delivery vectors can be grouped into two classes, those with specific membrane receptors undergoing conventional endocytosis and cell penetrating peptides (CPP) that have the capacity to cross cell or endosomal membranes. For both forms of vectors, translocation across a membrane is usually an inefficient process. In the current study, a novel vector combining the widely used CPP, Tat and the non-toxic neuronal binding domain of tetanus toxin (fragment C or TTC) was assessed for its capacity to deliver GFP as a test cargo protein to human neural progenitor cells (NPCs). These two functional membrane interacting domains dramatically enhanced internalization of the conjugated cargo protein. Tat-TTC-GFP was found to be bound or internalized at least 83-fold more than Tat-GFP and 33-fold more than TTC-GFP in NPCs by direct fluorimetry, and showed enhanced internalization by quantitative microscopy of 18 - and 14-fold, respectively. This preferential internalization was observed to be specific to neuronal cell types. Photochemical internalization (PCI) was utilized to facilitate escape of the endosome-sequestered proteins. The combined use of the Tat-TTC delivery vector with PCI led to both enhancement of neural cell type specific delivery to an endosomal target, followed by the option of efficient release to the cytosol.

Genome-wide transcriptional dependence on conserved regions of Mot1.

TATA binding protein (TBP) plays a central role in transcription complex assembly and is regulated by a variety of transcription factors, including Mot1. Mot1 is an essential protein in Saccharomyces cerevisiae that exerts both negative and positive effects on transcription via interactions with TBP. It contains two conserved regions important for TBP interactions, another conserved region that hydrolyzes ATP to remove TBP from DNA, and a fourth conserved region with unknown function. Whether these regions contribute equally to transcriptional regulation genome-wide is unknown. Here, we employ a transient-replacement assay using deletion derivatives in the conserved regions of Mot1 to investigate their contributions to gene regulation throughout the S. cerevisiae genome. These four regions of Mot1 are essential for growth and are generally required for all Mot1-regulated genes. Loss of the ATPase region, but not other conserved regions, caused TBP to redistribute away from a subset of Mot1-inhibited genes, leading to decreased expression of those genes. A corresponding increase in TBP occupancy and expression occurred at another set of genes that are normally Mot1 independent. The data suggest that Mot1 uses ATP hydrolysis to redistribute accessible TBP away from intrinsically preferred sites to other sites of intrinsically low preference.