Preclinical Evaluation of a Novel Lentiviral Vector Driving Lineage-Specific BCL11A Knockdown for Sickle Cell Gene Therapy.

In this work we provide preclinical data to support initiation of a first-in-human trial for sickle cell disease (SCD) using an approach that relies on reversal of the developmental fetal-to-adult hemoglobin switch. Erythroid-specific knockdown of BCL11A via a lentiviral-encoded microRNA-adapted short hairpin RNA (shRNA) leads to reactivation of the gamma-globin gene while simultaneously reducing expression of the pathogenic adult sickle β-globin. We generated a refined lentiviral vector (LVV) BCH-BB694 that was developed to overcome poor vector titers observed in the manufacturing scale-up of the original research-grade LVV.

Prostaglandin E Increases Lentiviral Vector Transduction Efficiency of Adult Human Hematopoietic Stem and Progenitor Cells.

Gene therapy currently in development for hemoglobinopathies utilizes ex vivo lentiviral transduction of CD34 hematopoietic stem and progenitor cells (HSPCs). A small-molecule screen identified prostaglandin E (PGE) as a positive mediator of lentiviral transduction of CD34 cells. Supplementation with PGE increased lentiviral vector (LVV) transduction of CD34 cells approximately 2-fold compared to control transduction methods with no effect on cell viability.

Preclinical evaluation of efficacy and safety of an improved lentiviral vector for the treatment of β-thalassemia and sickle cell disease.

A previously published clinical trial demonstrated the benefit of autologous CD34(+) cells transduced with a selfinactivating lentiviral vector (HPV569) containing an engineered β-globin gene (β(A-T87Q)-globin) in a subject with β thalassemia major. This vector has been modified to increase transduction efficacy without compromising safety. In vitro analyses indicated that the changes resulted in both increased vector titers (3 to 4 fold) and increased transduction efficacy (2 to 3 fold).

Synaptic glutamate release by ventromedial hypothalamic neurons is part of the neurocircuitry that prevents hypoglycemia.

The importance of neuropeptides in the hypothalamus has been experimentally established. Due to difficulties in assessing function in vivo, the roles of the fast-acting neurotransmitters glutamate and GABA are largely unknown. Synaptic vesicular transporters (VGLUTs for glutamate and VGAT for GABA) are required for vesicular uptake and, consequently, synaptic release of neurotransmitters.

Leptin directly activates SF1 neurons in the VMH, and this action by leptin is required for normal body-weight homeostasis.

Leptin, an adipocyte-derived hormone, acts directly on the brain to control food intake and energy expenditure. An important question is the identity of first-order neurons initiating leptin's anti-obesity effects. A widely held view is that most, if not all, of leptin's effects are mediated by neurons located in the arcuate nucleus of the hypothalamus.

Divergence of melanocortin pathways in the control of food intake and energy expenditure.

Activation of melanocortin-4-receptors (MC4Rs) reduces body fat stores by decreasing food intake and increasing energy expenditure. MC4Rs are expressed in multiple CNS sites, any number of which could mediate these effects. To identify the functionally relevant sites of MC4R expression, we generated a loxP-modified, null Mc4r allele (loxTB Mc4r) that can be reactivated by Cre-recombinase.