LDLR-Gene therapy for familial hypercholesterolaemia: Problems, progress, and perspectives

Coronary artery diseases (CAD) inflict a heavy economical and social burden on most populations and contribute significantly to their morbidity and mortality rates. Low-density lipoprotein receptor (LDLR) associated familial hypercholesterolemia (FH) is the most frequent Mendelian disorder and is a major risk factor for the development of CAD. To date there is no cure for FH. The primary goal of clinical management is to control hypercholesterolaemia in order to decrease the risk of atherosclerosis and to prevent CAD. Permanent phenotypic correction with single administration of a gene therapeutic vector is a goal still needing to be achieved. The first ex vivo clinical trial of gene therapy in FH was conducted nearly 18 years ago. Patients who had inherited LDLR gene mutations were subjected to an aggressive surgical intervention involving partial hepatectomy to obtain the patient's own hepatocytes for ex vivo gene transfer with a replication deficient LDLR-retroviral vector. After successful re-infusion of transduced cells through a catheter placed in the inferior mesenteric vein at the time of liver resection, only low-level expression of the transferred LDLR gene was observed in the five patients enrolled in the trial. In contrast, full reversal of hypercholesterolaemia was later demonstrated in in vivo preclinical studies using LDLR-adenovirus mediated gene transfer. However, the high efficiency of cell division independent gene transfer by adenovirus vectors is limited by their short-term persistence due to episomal maintenance and the cytotoxicity of these highly immunogenic viruses. Novel long-term persisting vectors derived from adeno-associated viruses and lentiviruses, are now available and investigations are underway to determine their safety and efficiency in preparation for clinical application for a variety of diseases. Several novel non-viral based therapies have also been developed recently to lower LDL-C serum levels in FH patients. This article reviews the progress made in the 18 years since the first clinical trial for gene therapy of FH, with emphasis on the development, design, performance and limitations of viral based gene transfer vectors used in studies to ameliorate the effects of LDLR deficiency

Perinatal Gene Transfer to the Liver

The liver acts as a host to many functions hence raising the possibility that any one may be compromised by a single gene defect. Inherited or de novo mutations in these genes may result in relatively mild diseases or be so devastating that death within the first weeks or months of life is inevitable. Some diseases can be managed using conventional medicines whereas others are, as yet, untreatable. In this review we consider the application of early intervention gene therapy in neonatal and fetal preclinical studies. We appraise the tools of this technology, including lentivirus, adenovirus and adeno-associated virus (AAV)-based vectors. We highlight the application of these for a range of diseases including hemophilia, urea cycle disorders such as ornithine transcarbamylase deficiency, organic acidemias, lysosomal storage diseases including mucopolysaccharidoses, glycogen storage diseases and bile metabolism. We conclude by assessing the advantages and disadvantages associated with fetal and neonatal liver gene transfer

Delivering efficient liver-directed AAV-mediated gene therapy.

Adeno-associated virus vectors (AAV) have become the leading technology for liver-directed gene therapy. After the pioneering trials using AAV2 and AAV8 to treat haemophilia B, D’Avola et al. recently reported the first-in-human clinical trial of adeno-associated virus vector serotype 5 (AAV5) in acute intermittent porphyria (AIP). Treatment was reported as safe, but the main surrogate biomarkers of AIP, porphobilinogen (PBG) and delta-aminolevulinate (ALA) were unchanged. This lack of efficacy contrasts with results from the haemophilia B trial using AAV8 capsid by Nathwani et al., which showed a significant and long-lasting improvement of the clinical phenotype. Haemophilia B is an amenable target for successful gene therapy as raising expression of plasma factor IX (FIX) level above 1% can modify the phenotype from severe to moderate. Development of a variety of capsids for clinical application is useful to overcome pre-existing neutralising antibodies. The differences in cell-specific transduction by different AAV serotypes are primarily owing to specificities in cellular uptake or post cell-entry processing. Indeed AAV5 presents several theoretical advantages as an alternative capsid to AAV8 for liver-directed gene therapy: suitable liver tropism, less off-target biodistribution, low seroprevalence in humans and minimal cross-reactivity with other serotypes

High-efficiency transduction of spinal cord motor neurons by intrauterine delivery of integration-deficient lentiviral vectors

Integration-deficient lentiviral vectors (IDLVs) are promising gene delivery tools that retain the high transduction efficiency of standard lentiviral vectors, yet fail to integrate as proviruses and are instead converted into episomal circles. These episomes are metabolically stable and support long-term expression of transgenes in non-dividing cells, exhibiting a decreased risk of insertional mutagenesis. We have embarked on an extensive study to compare the transduction efficiency of IDLVs pseudotyped with different envelopes (vesicular stomatitis, Rabies, Mokola and Ross River viral envelopes) and self-complementary adeno-associated viral vectors, serotype-9 (scAAV-9) in spinal cord tissues after intraspinal injection of mouse embryos (E16). Our results indicate that IDLVs can transduce motor neurons (MNs) at extremely high efficiency regardless of the envelope pseudotype while scAAV9 mediates gene delivery to ~40% of spinal cord motor neurons, with other non-neuronal cells also transduced. Long-term expression studies revealed stable gene expression at 7months post-injection. Taken together, the results of this study indicate that IDLVs may be efficient tools for in utero cord transduction in therapeutic strategies such as for treatment of inherited early childhood neurodegenerative diseases

Proof-of-concept: neonatal intravenous injection of adeno-associated virus vectors results in successful transduction of myenteric and submucosal neurons in the mouse small and large intestine

BACKGROUND: Despite the success of viral vector technology in the transduction of the central nervous system in both preclinical research and gene therapy, its potential in neurogastroenterological research remains largely unexploited. This study asked whether and to what extent myenteric and submucosal neurons in the ileum and distal colon of the mouse were transduced after neonatal systemic delivery of recombinant adeno-associated viral vectors (AAVs). METHODS: Mice were intravenously injected at postnatal day one with AAV pseudotypes AAV8 or AAV9 carrying a cassette encoding enhanced green fluorescent protein (eGFP) as a reporter under the control of a cytomegalovirus promoter. At postnatal day 35, transduction of the myenteric and submucosal plexuses of the ileum and distal colon was evaluated in whole-mount preparations, using immunohistochemistry to neurochemically identify transduced enteric neurons. KEY RESULTS: The pseudotypes AAV8 and AAV9 showed equal potential in transducing the enteric nervous system (ENS), with 25-30% of the neurons expressing eGFP. However, the percentage of eGFP-expressing colonic submucosal neurons was significantly lower. Neurochemical analysis showed that all enteric neuron subtypes, but not glia, expressed the reporter protein. Intrinsic sensory neurons were most efficiently transduced as nearly 80% of calcitonin gene-related peptide-positive neurons expressed the transgene. CONCLUSIONS & INFERENCES: The pseudotypes AAV8 and AAV9 can be employed for gene delivery to both the myenteric and the submucosal plexus, although the transduction efficiency in the latter is region-dependent. These findings open perspectives for novel preclinical applications aimed at manipulating and imaging the ENS in the short term, and in gene therapy in the longer term

Intra-amniotic delivery of CFTR-expressing adenovirus does not reverse cystic fibrosis phenotype in inbred CFTR-knockout mice

This article is available open access through the publisher’s website at the link below. Copyright © 2008 The American Society of Gene Therapy.Due to its early onset and severe prognosis, cystic fibrosis (CF) has been suggested as a candidate disease for in utero gene therapy. In 1997, a study was published claiming that to how transient prenatal expression of CF transmembrane conductance regulator (CFTR) from an in utero –injected adenovirus vector could achieve permanent reversal of the CF intestinal pathology in adult CF knockout mice, despite the loss of CFTR transgene expression by birth. This would imply that the underlying cause of CF is a prenatal defect for which lifelong cure can be achieved by transient prenatal expression of CFTR. Despite criticism at the time of publication, no independent verification of this contentious finding has been published so far. This is vital for the development of future therapeutic strategies as it may determine whether CF gene therapy should be performed prenatally or postnatally. We therefore reinvestigated this finding with an identical adenoviral vector and a knockout CF mouse line (CftrtmlCam) with a completely inbred genetic background to eliminate any effects due to genetic variation. After delivery of the CFTR-expressing adenovirus to the fetal mouse, both vector DNA and transgenic CFTR expression were detected in treated animals postpartum but statistically no significant difference in survival was observed between the Cftr–/– mice treated with the CFTR-adenovirus and those treated with the control vector.Sport Aiding Medical Research for Kids, the Cystic Fibrosis Trust, and the Katharine Dormandy Trust

Differentiation of human fetal mesenchymal stem cells into cells with an oligodendrocyte phenotype

This article is available open access through the publisher’s website at the link below. Copyright @ 2009 Landes Bioscience.The potential of mesenchymal stem cells (MSC) to differentiate into neural lineages has raised the possibility of autologous cell transplantation as a therapy for neurodegenerative diseases. We have identified a population of circulating human fetal mesenchymal stem cells (hfMSC) that are highly proliferative and can readily differentiate into mesodermal lineages such as bone, cartilage, fat and muscle. Here, we demonstrate for the first time that primary hfMSC can differentiate into cells with an oligodendrocyte phenotype both in vitro and in vivo. By exposing hfMSC to neuronal conditioned medium or by introducing the pro-oligodendrocyte gene, Olig-2, hfMSC adopted an oligodendrocyte-like morphology, expressed oligodendrocyte markers and appeared to mature appropriately in culture. Importantly we also demonstrate the differentiation of a clonal population of hfMSC into both mesodermal (bone) and ectodermal (oligodendrocyte) lineages. In the developing murine brain transplanted hfMSC integrated into the parenchyma but oligodendrocyte differentiation of these naïve hfMSC was very low. However, the proportion of cells expressing oligodendrocyte markers increased significantly (from 0.2% to 4%) by pre-exposing the cells to differentiation medium in vitro prior to transplantation. Importantly, the process of in vivo differentiation occurred without cell fusion. These findings suggest that hfMSC may provide a potential source of oligodendrocytes for study and potential therapy

Haemophilia B Curative FIX Production from a Low Dose UCOE-based Lentiviral Vector Following Hepatic Pre-natal Delivery

The ubiquitous chromatin opening element from the human HNRPA2B1-CBX3 housekeeping gene locus (A2UCOE) is able to provide stable and cell-to-cell reproducible levels of transgene expression regardless of target cell genome integration site with efficacy demonstrated in adult, embryonic and induced pluripotent stem cells and their differentiated progeny in vitro and in vivo. Here we evaluate the ability of A2UCOE-based lentiviral vectors to confer stable expression following pre-natal delivery in mice. Our results show stable post-natal A2UCOE-eGFP and A2UCOE-luciferase lentiviral vector presence in both the liver and haematopoietic system with concomitant persistence of expression demonstrating efficient transduction of both fetal liver and haematopoietic stem cells. In addition, we find that an A2UCOE-FIX lentiviral vector produces comparable amounts of plasma FIX protein to that obtained from a SFFV-FIX construct. Furthermore, the A2UCOE-FIX vector shows that at a low (0.19) average vector copy number per liver cell, it can provide stable levels of plasma FIX production, which would convert severe haemophilia B (<1%) to a mild phenotype (≈20%). Our results provide proof-ofprinciple for low dose pre-natal A2UCOE-based LV delivery to the liver as a therapeutic option for haemophilia B and potentially other metabolic conditions

Continual Conscious Bioluminescent Imaging in Freely Moving Mice

In vivo bioluminescent imaging allows the detection of reporter gene expression in rodents in real time. Here we describe a novel technology whereby we can generate somatotransgenic rodents with the use of a viral vector carrying a luciferase transgene. We are able to achieve long term luciferase expression by a single injection of lentiviral or adeno-associated virus vectors to newborn mice. Further, we describe whole body bioluminescence imaging of conscious mice in a noninvasive manner, thus enforcing the 3R’s (replacement, reduction, and refinement) of biomedical animal research