Delivery of the 135 kb human frataxin genomic DNA locus gives rise to different frataxin isoforms.

Friedreich's ataxia (FRDA) is the most common form of hereditary ataxia caused by recessive mutations in the FXN gene. Recent results have indicated the presence of different frataxin isoforms due to alternative gene expression mechanisms. Our previous studies demonstrated the advantages of using high-capacity herpes simplex virus type 1 (HSV-1) amplicon vectors containing the entire FXN genomic locus (iBAC-FXN) as a gene-delivery vehicle capable of ensuring physiologically-regulated and long-term persistence. Here we describe how expression from the 135 kb human FXN genomic locus produces the three frataxin isoforms both in cultured neuronal cells and also in vivo. Moreover, we also observed the correct expression of these frataxin isoforms in patient-derived cells after delivery of the iBAC-FXN. These results lend further support to the potential use of HSV-1 vectors containing entire genomic loci whose expression is mediated by complex transcriptional and posttranscriptional mechanisms for gene therapy applications.

TARDBP pathogenic mutations increase cytoplasmic translocation of TDP-43 and cause reduction of endoplasmic reticulum Ca²âº signaling in motor neurons.

The transactive response DNA binding protein (TDP-43) is a major component of the characteristic neuronal cytoplasmic inclusions seen in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Furthermore, pathogenic mutations in the gene encoding TDP-43, TARDBP, are found in sporadic and familial ALS cases. To study the molecular mechanisms of cellular toxicity due to TDP-43 mutations we generated a novel in vitro cellular model using a fluorescently tagged human genomic TARDBP locus carrying one of two ALS-associated mutations, A382T or M337V, which were used to generate site-specific bacterial artificial chromosome (BAC) human stable cell lines and BAC transgenic mice. In cell lines and primary motor neurons in culture, TDP-M337V mislocalized to the cytoplasm more frequently than wild-type TDP (wt-TDP) and TDP-A382T, an effect potentiated by oxidative stress. Expression of mutant TDP-M337V correlated with increased apoptosis detected by cleaved caspase-3 staining. Cells expressing mislocalized TDP-M337V spontaneously developed cytoplasmic aggregates, while for TDP-A382T aggregates were only revealed after endoplasmic reticulum (ER) stress induced by the calcium-modifying drug thapsigargin. Lowering Ca(2+) concentration in the ER of wt-TDP cells partially recapitulated the effect of pathogenic mutations by increasing TDP-43 cytoplasmic mislocalization, suggesting Ca(2+) dysregulation as a potential mediator of pathology through alterations in Bcl-2 protein levels. Ca(2+) signaling from the ER was impaired in immortalized cells and primary neurons carrying TDP-43 mutations, with a 50% reduction in the levels of luminal ER Ca(2+) stores content and delayed Ca(2+) release compared with cells carrying wt-TDP. The deficits in Ca(2+) release in human cells correlated with the upregulation of Bcl-2 and siRNA-mediated knockdown of Bcl-2 restored the amplitude of Ca(2+) oscillations in TDP-M337V cells. These results suggest that TDP-43 pathogenic mutations elicit cytoplasmic mislocalization of TDP-43 and Bcl-2 mediated ER Ca(2+) signaling dysregulation.

RNAi-mediated knockdown of HMG CoA reductase enhances gene expression from physiologically regulated low-density lipoprotein receptor therapeutic vectors in vivo.

The development of novel strategies to enhance gene expression from therapeutic vectors may prove advantageous for complementation gene therapy. This applies to therapeutic expression of the low-density lipoprotein receptor (LDLR) gene to treat familial hypercholesterolaemia (FH), where appropriate gene regulation could enhance therapeutic effect. We have previously reported that LDLR genomic DNA expression vectors can be regulated in vivo by pravastatin. In the current study, we investigated whether targeted knockdown of the mevalonate pathway in conjunction with LDLR delivery would lead to enhanced LDLR transgene expression and improved phenotype recovery. We demonstrated here that knockdown of HMG CoA reductase (HMGCR) by up to 70% using small interfering RNAs (siRNAs) led to a significant increase in binding and internalisation of LDL particles in vitro in mouse and human cells. In vivo co-injection of LDLR promoter luciferase expression plasmids with siRNAs or microRNA (miRNA) expression vectors targeting mouse Hmgcr led to at least a 10-fold increase in luciferase expression. Injection of Ldlr(-/-) mice with pLDLR-LDLR expression plasmids led to a significant reduction in plasmid LDL cholesterol, which was further enhanced by co-injection with miRNA expression vectors targeted to mouse Hmgcr. Our data suggest that targeted knockdown of HMGCR may enhance gene therapy outcomes for FH.

Infectious delivery and long-term persistence of transgene expression in the brain by a 135-kb iBAC-FXN genomic DNA expression vector.

Novel gene-based therapies for disease will depend in many cases on long-term persistent transgene expression. To develop gene therapy strategies for Friedreich's ataxia (FRDA), we have examined the persistence of transgene expression in the brain in vivo provided by the entire 135 kb FXN genomic DNA locus delivered as an infectious bacterial artificial chromosome (iBAC) herpes simplex virus type 1 (HSV-1)-based vector injected in the adult mouse cerebellum. We constructed genomic DNA-reporter fusion vectors carrying a complete 135 kb FXN genomic locus with an insertion of the Escherichia coli lacZ gene at the ATG start codon (iBAC-FXN-lacZ). SHSY5Y human neuroblastoma cells transduced by iBAC-FXN-lacZ showed high efficiency of vector delivery and LacZ expression. Direct intracranial injection of iBAC-FXN-lacZ into the adult mouse cerebellum resulted in a large number of easily detectable transduced cells, with LacZ expression driven by the FXN genomic locus, which persisted for at least 75 days. Green fluorescent protein expression driven from the same vector but by the strong HSV-1 IE4/5 promoter was transient. Our data demonstrate for the first time sustained transgene expression in vivo by infectious delivery of a genomic DNA locus >100 kb in size. Such an approach may be suitable for gene rescue strategies in neurological disease, such as FRDA.

Lentiviral gene transfer to reduce atherosclerosis progression by long-term CC-chemokine inhibition.

CC-chemokines are important mediators in the pathogenesis of atherosclerosis. Atherosclerosis progression is reduced by high-level, short-term inhibition of CC-chemokine activity, for example by adenoviral gene transfer. However, atherosclerosis is a chronic condition where short-term effects, while demonstrating proof-of-principle, are unlikely to provide maximum therapeutic benefit. Accordingly, we generated a recombinant lentivirus, lenti35K, encoding the broad-spectrum CC chemokine inhibitor, 35K, derived from the vaccinia virus. To investigate the effects of prolonged broad-spectrum chemokine inhibition on atherosclerosis, lenti35K, or lentiGFP or PBS were delivered to 6-week-old ApoE knockout (ApoE-KO) mice by hydrodynamic injection. Sustained lentiviral transduction and transgene expression were demonstrated by 35K mRNA and viral DNA in liver tissue, and recombinant 35K protein circulating in the plasma, 3 months after gene transfer. Plasma from lenti35K animals had reduced chemokine activity compared with plasma from lentiGFP or PBS-treated animals. Histologic analysis of aortic sinus sections revealed that atherosclerotic plaque area in lenti35K mice was significantly reduced compared with both lentiGFP and PBS controls. Furthermore, plaque macrophage content was substantially reduced in lenti35K mice. Lentiviral 35K gene transfer is a promising experimental strategy to reduce atherosclerosis progression, and demonstrates the potential of long-term CC-chemokine inhibition as a potential therapeutic target in atherosclerosis.

LRRK2 is a component of granular alpha-synuclein pathology in the brainstem of Parkinson's disease.

Classical Parkinson's disease (PD) is characterized by the appearance of Lewy bodies (LBs) in affected brain regions, showing mostly compact alpha-synuclein deposition, in contrast with punctate or granular deposition, hypothesized to represent early stages of aggregation. Leucine-rich repeat kinase 2 (LRRK2) is the commonest mutated gene in inherited and idiopathic PD. LRRK2 mutation carriers display a diverse neuropathology, including alpha-synuclein and tau inclusions, suggesting an upstream role for LRRK2 in protein aggregation. We studied LRRK2 expression throughout the normal human brain with three different antibodies. We also examined the pattern of LRRK2 expression in relation to alpha-synuclein aggregation and LB formation in the brainstem of sporadic LB disease. Physiological LRRK2 expression was not restricted to regions preferentially affected in PD and LRRK2 often localized to the nuclear envelope in addition to the known cytoplasmic expression. In PD, we were able to consistently detect LRRK2 in the halo of a minority (approximately 10%) of nigral LBs using three different antibodies. Only one antibody detected LRRK2 in the core of approximately 80% of classic LBs. In the lower brainstem, most notably in the dorsal motor nucleus of the vagus, we found previously unrecognized LRRK2 labelling of complex globular lesions, filled with LB-like matter showing a punctate or granular staining for alpha-synuclein. This was often accompanied by strong LRRK2 expression within dystrophic neurites. Our findings confirm widespread physiological LRRK2 expression in the human brain and suggest an association of LRRK2 with possible early-stage alpha-synuclein pathology in the brainstem of PD.

Stable correction of a genetic deficiency in human cells by an episome carrying a 115 kb genomic transgene.

Persistent expression of a transgene at therapeutic levels is required for successful gene therapy, but many small vectors with heterologous promoters are prone to vector loss and transcriptional silencing. The delivery of genomic DNA would enable genes to be transferred as complete loci, including regulatory sequences, introns, and native promoter elements. These elements may be critical to ensure prolonged, regulated, and tissue-specific transgene expression. Many studies point to considerable advantages to be gained by using complete genomic loci in gene expression. Large-insert vectors incorporating elements of the bacterial artificial chromosome (BAC) cloning system, and the episomal maintenance mechanisms of Epstein-Barr virus (EBV), can shuttle between bacteria and mammalian cells, allowing large genomic loci to be manipulated conveniently. We now demonstrate the potential utility of such vectors by stably correcting a human genetic deficiency in vitro. When the complete hypoxanthine phosphoribosyltransferase (HPRT) locus of 115 kilobases (kb) was introduced into deficient human cells, the transgene was both maintained as an episome and expressed stably for six months in rapidly dividing cell cultures. The results demonstrate for the first time that gene expression from an episomal genomic transgene can correct a cell culture disease phenotype for a prolonged period.

An infectious transfer and expression system for genomic DNA loci in human and mouse cells.

The recent completion of the human genome sequence allows genomics research to focus on understanding gene complexity, expression, and regulation. However, the routine-use genomic DNA expression systems required to investigate these phenomena are not well developed. Bacterial artificial chromosomes (BACs) and P1-based artificial chromosomes (PACs) have proved excellent tools for the human genome sequencing projects. We describe a system to rapidly and efficiently deliver and express BAC and PAC library clones in human and mouse cells by converting them into infectious amplicon vectors. We show packaging and intact delivery of genomic inserts of >100 kilobases with efficiencies of up to 100%. To demonstrate that genomic loci transferred in this way are functional, the complete human hypoxanthine phosphoribosyltransferase (HPRT) locus contained within a 115-kilobase BAC insert was shown to be expressed when delivered by infection into both a human HPRT-deficient fibroblast cell line and a mouse primary hepatocyte culture derived from Hprt-/- mice. Efficient gene delivery to primary cells is especially important, as these cells cannot be expanded using antibiotic selection. This work is the first demonstration of infectious delivery and expression of genomic DNA sequences of >100 kilobases, a technique that may prove useful for analyzing gene expression from the human genome.

Parkinson's disease in a dish - Using stem cells as a molecular tool.

Parkinson's disease (PD) is the second most common neurodegenerative disease, with a strong genetic component to both the familial and sporadic forms. The cardinal motor symptoms of the disease result from the loss of dopamine (DA) neurons in the midbrain. There is currently no cure for PD and improved methods for modelling the disease are required in order to develop more effective therapeutic interventions. Patient-derived induced pluripotent stem cells (iPSCs) carry the genetic background of the donor, enabling accurate modelling of genetic diseases in vitro. Various human iPSCs from patients suffering different genetic forms of PD have been differentiated into DA neurons and demonstrated signs of the pathophysiology of PD in vitro. The examination of key cellular pathways such as calcium regulation and autophagy indicate that disease-associated genetic variants may have important implications for cellular function. This review examines and critiques how DA neurons from patient iPSCs have been used to model PD in vitro, and what iPSCs might hold for the future of PD research. This article is part of the Special Issue entitled 'The Synaptic Basis of Neurodegenerative Disorders'.

Assessing the functional characteristics of synonymous and nonsynonymous mutation candidates by use of large DNA constructs.

As we identify more and more genetic changes, either through mutation studies or population screens, we need powerful tools to study their potential molecular effects. With these tools, we can begin to understand the contributions of genetic variations to the wide range of human phenotypes. We used our catalogue of molecular changes in patients with carbamyl phosphate synthetase I (CPSI) deficiency to develop such a system for use in eukaryotic cells. We developed the tools and methods for rapidly modifying bacterial artificial chromosomes (BACs) for eukaryotic episomal replication, marker expression, and selection and then applied this protocol to a BAC containing the entire CPSI gene. Although this CPSI BAC construct was suitable for studying nonsynonymous mutations, potential splicing defects, and promoter variations, our focus was on studying potential splicing and RNA-processing defects to validate this system. In this article, we describe the construction of this system and subsequently examine the mechanism of four putative splicing mutations in patients deficient in CPSI. Using this model, we also demonstrate the reversible role of nonsense-mediated decay in all four mutations, using small interfering RNA knockdown of hUPF2. Furthermore, we were able to locate cryptic splicing sites for the two intronic mutations. This BAC-based system permits expression studies in the absence of patient RNA or tissues with relevant gene expression and provides experimental flexibility not available in genomic DNA or plasmid constructs. Our splicing and RNA degradation data demonstrate the advantages of using whole-gene constructs to study the effects of sequence variation on gene expression and function.

LRRTM1 on chromosome 2p12 is a maternally suppressed gene that is associated paternally with handedness and schizophrenia.

Left-right asymmetrical brain function underlies much of human cognition, behavior and emotion. Abnormalities of cerebral asymmetry are associated with schizophrenia and other neuropsychiatric disorders. The molecular, developmental and evolutionary origins of human brain asymmetry are unknown. We found significant association of a haplotype upstream of the gene LRRTM1 (Leucine-rich repeat transmembrane neuronal 1) with a quantitative measure of human handedness in a set of dyslexic siblings, when the haplotype was inherited paternally (P=0.00002). While we were unable to find this effect in an epidemiological set of twin-based sibships, we did find that the same haplotype is overtransmitted paternally to individuals with schizophrenia/schizoaffective disorder in a study of 1002 affected families (P=0.0014). We then found direct confirmatory evidence that LRRTM1 is an imprinted gene in humans that shows a variable pattern of maternal downregulation. We also showed that LRRTM1 is expressed during the development of specific forebrain structures, and thus could influence neuronal differentiation and connectivity. This is the first potential genetic influence on human handedness to be identified, and the first putative genetic effect on variability in human brain asymmetry. LRRTM1 is a candidate gene for involvement in several common neurodevelopmental disorders, and may have played a role in human cognitive and behavioral evolution.

Sequences adjacent to oriP improve the persistence of Epstein-Barr virus-based episomes in B cells.

Epstein-Barr virus (EBV) oriP and the EBV nuclear antigen 1 (EBNA-1) protein allow persistence of EBV-based episomes. A nuclear matrix attachment region (MAR) spans oriP and the adjacent region of the EBV genome containing the EBV-expressed RNAs. Here, we show that episomes with the MAR are retained significantly more efficiently in EBV-positive B cells than episomes containing oriP alone.

Long-term stability of large insert genomic DNA episomal shuttle vectors in human cells.

We have constructed an episomal shuttle vector which can transfer large (>100 kb) human genomic DNA inserts back and forth between bacteria and human cells and which can be tracked in rapidly dividing human cells using a live cell assay. The vector (p5170) is based on the F factor-derived bacterial artificial chromosome cloning vector used in Escherichia coli, with the addition of the family of repeats element from the Epstein-Barr virus (EBV) latent origin of replication. This element provides nuclear retention in cells expressing the EBV protein EBNA-1. We have subcloned a series of genomic DNA inserts into p5170 and transfected the constructs into an EBNA-1(+) human cell line. Episomal mitotic stability was quantitatively analysed using flow cytometry. The episomes were also tracked by time course photography of expanding colonies. A 117 kb episome was retained at approximately 2 copies/cell and could be shuttled unrearranged from the human cells into bacterial cells after 15 months of continuous cell growth. Furthermore, the episome could still be rescued from human cells cultured in the absence of selection for 198 days. Such a trackable E.coli /human cell line shuttle vector system capable of carrying >100 kb of genomic DNA in human cells could prove a valuable tool in gene expression studies.

Infectious delivery of the 132 kb CDKN2A/CDKN2B genomic DNA region results in correctly spliced gene expression and growth suppression in glioma cells.

The expression of genes from genomic loci can be relatively complex, utilizing exonic, intronic and flanking sequences to regulate tissue and developmental specificity. Infectious bacterial artificial chromosomes (iBACs) have been shown to deliver and express large genomic loci (up to 135 kb) into primary cells for functional analyses. The delivery of large genomic DNA inserts allows the expression of complex loci and of multiple splice variants. Herein, we demonstrate for the first time that an iBAC will deliver and correctly express in human glioma cells the entire CDKN2A/CDKN2B genomic region, which encodes for at least three important cell-cycle regulatory proteins (p16(INK4a), p14(ARF) and p15(INK4b)). Two of these proteins are expressed from overlapping genes, utilizing alternative splicing and promoter usage. The delivered locus expresses each gene at physiological levels and cellular responses (apoptosis versus growth arrest) occur dependent on cellular p53 status, as expected. The work further demonstrates the potential of the iBAC system for the delivery of genomic loci whose expression is mediated by complex splicing and promoter usage both for gene therapy applications and functional genomics studies.