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Familial hypercholesterolemia (FH) is a condition caused by mutations in the low-density lipoprotein receptor (LDLR) gene. Expression of LDLR is highly regulated and excess receptor expression is cytotoxic. To incorporate essential gene regulation into a gene therapy vector for FH, we generated vectors in which the expression of therapeutic human LDLR gene, or luciferase reporter gene, is driven by 10 kb of human LDLR genomic DNA encompassing the promoter region including elements essential for physiologically regulated expression. Using luciferase expression and specific LDL binding and internalization assays, we have shown in vitro that the genomic promoter element confers long-term, physiologically regulated gene expression and complementation of receptor deficiency in culture for 240 cell-generations. This was demonstrated in the presence of sterols or statins, modifiers of LDLR promoter activity. In vivo, we demonstrate efficient liver-specific delivery and expression of luciferase following hydrodynamic tail-vein injection and confirm that expression from the LDLR promoter element is sensitive to statin administration. We also demonstrate long-term LDLR expression from the 10-kb promoter element up to 9 months following delivery. The vector system that we describe provides the efficient delivery, long-term expression, and physiological regulation required for a successful gene therapy intervention for FH. [ABSTRACT FROM AUTHOR]

The lysine-specific demethylase KDM1A is a key regulator of stem cell potential in acute myeloid leukemia (AML). ORY-1001 is a highly potent and selective KDM1A inhibitor that induces H3K4me2 accumulation on KDM1A target genes, blast differentiation, and reduction of leukemic stem cell capacity in AML. ORY-1001 exhibits potent synergy with standard-of-care drugs and selective epigenetic inhibitors, reduces growth of an AML xenograft model, and extends survival in a mouse PDX (patient-derived xenograft) model of T cell acute leukemia. Surrogate pharmacodynamic biomarkers developed based on expression changes in leukemia cell lines were translated to samples from patients treated with ORY-1001. ORY-1001 is a selective KDM1A inhibitor in clinical trials and is currently being evaluated in patients with leukemia and solid tumors., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Friedreich's ataxia (FRDA) is caused by large GAA expansions in intron 1 of the frataxin gene (FXN), which lead to reduced FXN expression through a mechanism not fully understood. Understanding such mechanism is essential for the identification of novel therapies for FRDA and this can be accelerated by the development of cell models which recapitulate the genomic context of the FXN locus and allow direct comparison of normal and expanded FXN loci with rapid detection of frataxin levels. Here we describe the development of the first GAA-expanded FXN genomic DNA reporter model of FRDA. We modified BAC vectors carrying the whole FXN genomic DNA locus by inserting the luciferase gene in exon 5a of the FXN gene (pBAC-FXN-Luc) and replacing the six GAA repeats present in the vector with an ∼310 GAA repeat expansion (pBAC-FXN-GAA-Luc). We generated human clonal cell lines carrying the two vectors using site-specific integration to allow direct comparison of normal and expanded FXN loci. We demonstrate that the presence of expanded GAA repeats recapitulates the epigenetic modifications and repression of gene expression seen in FRDA. We applied the GAA-expanded reporter model to the screening of a library of novel small molecules and identified one molecule which up-regulates FXN expression in FRDA patient primary cells and restores normal histone acetylation around the GAA repeats. These results suggest the potential use of genomic reporter cell models for the study of FRDA and the identification of novel therapies, combining physiologically relevant expression with the advantages of quantitative reporter gene expression.

Herpes simplex type 1 (HSV-1) amplicon vectors possess a number of features that make them excellent vectors for the delivery of transgenes into stem cells. HSV-1 amplicon vectors are capable of efficiently transducing both dividing and nondividing cells and since the virus is quite large, 152 kb, it is of sufficient size to allow for incorporation of entire genomic DNA loci with native promoters. HSV-1 amplicon vectors can also be used to incorporate and deliver to cells a variety of sequences that allow extrachromosomal retention. These elements offer advantages over integrating vectors as they avoid transgene silencing and insertional mutagenesis. The construction of amplicon vectors carrying extrachromosomal retention elements, their packaging into HSV-1 viral particles, and the use of HSV-1 amplicons for stem cell transduction will be described.

Leucine rich repeat kinase 2 (LRRK2) mutations are the most common genetic cause of Parkinson's disease (PD) although LRRK2 function remains unclear. We report a new role for LRRK2 in regulating autophagy and describe the recruitment of LRRK2 to the endosomal-autophagic pathway and specific membrane subdomains. Using a novel human genomic reporter cellular model, we found LRRK2 to locate to membrane microdomains such as the neck of caveolae, microvilli/filopodia and intraluminal vesicles of multivesicular bodies (MVBs). In human brain and in cultured human cells LRRK2 was present in cytoplasmic puncta corresponding to MVBs and autophagic vacuoles (AVs). Expression of the common R1441C mutation from a genomic DNA construct caused impaired autophagic balance evident by the accumulation of MVBs and large AVs containing incompletely degraded material and increased levels of p62. Furthermore, the R1441C mutation induced the formation of skein-like abnormal MVBs. Conversely, LRRK2 siRNA knockdown increased autophagic activity and prevented cell death caused by inhibition of autophagy in starvation conditions. The work necessitated developing a new, more efficient recombineering strategy, which we termed Sequential insertion of Target with ovErlapping Primers (STEP) to seamlessly fuse the green fluorescent protein-derivative YPet to the human LRRK2 protein in the LRRK2 genomic locus carried by a bacterial artificial chromosome. Taken together our data demonstrate the functional involvement of LRRK2 in the endosomal-autophagic pathway and the recruitment to specific membrane microdomains in a physiological human gene expression model suggesting a novel function for this important PD-related protein.

Recent developments in extrachromosomal vector technology have offered new ways of designing safer, physiologically regulated vectors for gene therapy. Extrachromosomal, or episomal, persistence in the nucleus of transduced cells offers a safer alternative to integrating vectors which have become the subject of safety concerns following serious adverse events in recent clinical trials. Extrachromosomal vectors do not cause physical disruption in the host genome, making these vectors safe and suitable tools for several gene therapy targets, including stem cells. Moreover, the high insert capacity of extrachromosomal vectors allows expression of a therapeutic transgene from the context of its genomic DNA sequence, providing an elegant way to express normal splice variants and achieve physiologically regulated levels of expression. Here, we describe past and recent advances in the development of several different extrachromosomal systems, discuss their retention mechanisms, and evaluate their use as expression vectors to deliver and express genomic DNA loci. We also discuss a variety of delivery systems, viral and nonviral, which have been used to deliver episomal vectors to target cells in vitro and in vivo. Finally, we explore the potential for the delivery and expression of extrachromosomal transgenes in stem cells. The long-term persistence of extrachromosomal vectors combined with the potential for stem cell proliferation and differentiation into a wide range of cell types offers an exciting prospect for therapeutic interventions.

Episomal gene expression vectors offer a safe and attractive alternative to integrating vectors. Here we describe the development of a high capacity episomal vector system exploiting human episomal retention sequences to provide efficient vector maintenance and regulated gene expression through the delivery of a genomic DNA locus. The iBAC-S/MAR vector is capable of the infectious delivery and retention of large genomic DNA transgenes by exploiting the high transgene capacity of herpes simplex virus type 1 (HSV-1) and the episomal retention properties of the scaffold/matrix attachment region (S/MAR). The iBAC-S/MAR vector was used to deliver and maintain a 135 kb genomic DNA insert carrying the human low density lipoprotein receptor (LDLR) genomic DNA locus at high efficiency in CHO ldlr(-/-) a7 cells. Long-term studies on CHO ldlr(-/-) a7 clonal cell lines carrying iBAC-S/MAR-LDLR demonstrated low copy episomal stability of the vector for >100 cell generations without selection. Expression studies demonstrated that iBAC-S/MAR-LDLR completely restored LDLR function in CHO ldlr(-/-) a7 cells to physiological levels and that this expression can be repressed by approximately 70% by high sterol levels, recapitulating the same feedback regulation seen at the endogenous LDLR locus. This vector overcomes the major problems of vector integration and unregulated transgene expression.