Designing lentiviral gene vectors

Lentiviral gene vectors are an important tool in gene therapy and basic biomedical research. They are transducing viral particles, normally replication defective, which are generated using the packaging machinery of lentiviruses. These vectors are used to deliver the encapsidated payload genes to the nuclei of the target cells, offering stable transgene expression in many settings in vitro and in vivo. Successful generation of high-titre lentiviral vectors capable of efficiently expressing transgenes over long period of time is governed by a number of vector design rules, some of which are common to all gene vectors while others are specific to lentiviral vectors. Construction of lentiviral vectors with the cargo genes driven by tissue-specific promoters is a particular challenge. This review focuses both on the guiding principles and the technical know-how of the lentiviral gene vector design.Published versio

Chronically shortened rod outer segments accompany photoreceptor cell death in Choroideremia

X-linked choroideremia (CHM) is a disease characterized by gradual retinal degeneration caused by loss of the Rab Escort Protein, REP1. Despite partial compensation by REP2 the disease is characterized by prenylation defects in multiple members of the Rab protein family that are master regulators of membrane traffic. Remarkably, the eye is the only organ affected in CHM patients, possibly because of the huge membrane traffic burden of the post mitotic photoreceptors, which synthesise outer segments, and the adjacent retinal pigment epithelium that degrades the spent portions each day. In this study, we aimed to identify defects in membrane traffic that might lead to photoreceptor cell death in CHM. In a heterozygous null female mouse model of CHM (Chmnull/WT), degeneration of the photoreceptor layer was clearly evident from increased numbers of TUNEL positive cells compared to age matched controls, small numbers of cells exhibiting signs of mitochondrial stress and greatly increased microglial infiltration. However, most rod photoreceptors exhibited remarkably normal morphology with well-formed outer segments and no discernible accumulation of transport vesicles in the inner segment. The major evidence of membrane trafficking defects was a shortening of rod outer segments that was evident at 2 months of age but remained constant over the period during which the cells die. A decrease in rhodopsin density found in the outer segment may underlie the outer segment shortening but does not lead to rhodopsin accumulation in the inner segment. Our data argue against defects in rhodopsin transport or outer segment renewal as triggers of cell death in CHM

Sequential and compartmentalized action of Rabs, SNAREs, and MAL in the apical delivery of fusiform vesicles in urothelial umbrella cells

Uroplakins (UPs) are major differentiation products of urothelial umbrella cells and play important roles in forming the permeability barrier and in the expansion/stabilization of the apical membrane. Further, UPIa serves as a uropathogenic Escherichia coli receptor. Although it is understood that UPs are delivered to the apical membrane via fusiform vesicles (FVs), the mechanisms that regulate this exocytic pathway remain poorly understood. Immunomicroscopy of normal and mutant mouse urothelia show that the UP-delivering FVs contained Rab8/11 and Rab27b/Slac2-a, which mediate apical transport along actin filaments. Subsequently a Rab27b/Slp2-a complex mediated FV–membrane anchorage before SNARE-mediated and MAL-facilitated apical fusion. We also show that keratin 20 (K20), which forms a chicken-wire network ∼200 nm below the apical membrane and has hole sizes allowing FV passage, defines a subapical compartment containing FVs primed and strategically located for fusion. Finally, we show that Rab8/11 and Rab27b function in the same pathway, Rab27b knockout leads to uroplakin and Slp2-a destabilization, and Rab27b works upstream from MAL. These data support a unifying model in which UP cargoes are targeted for apical insertion via sequential interactions with Rabs and their effectors, SNAREs and MAL, and in which K20 plays a key role in regulating vesicular trafficking

Rab27a and Rab27b control different steps of the exosome secretion pathway

Exosomes are secreted membrane vesicles that share structural and biochemical characteristics with intraluminal vesicles of multivesicular endosomes (MVEs). Exosomes could be involved in intercellular communication and in the pathogenesis of infectious and degenerative diseases. The molecular mechanisms of exosome biogenesis and secretion are, however, poorly understood. Using an RNA interference (RNAi) screen, we identified five Rab GTPases that promote exosome secretion in HeLa cells. Among these, Rab27a and Rab27b were found to function in MVE docking at the plasma membrane. The size of MVEs was strongly increased by Rab27a silencing, whereas MVEs were redistributed towards the perinuclear region upon Rab27b silencing. Thus, the two Rab27 isoforms have different roles in the exosomal pathway. In addition, silencing two known Rab27 effectors, Slp4 (also known as SYTL4, synaptotagmin-like 4) and Slac2b (also known as EXPH5, exophilin 5), inhibited exosome secretion and phenocopied silencing of Rab27a and Rab27b, respectively. Our results therefore strengthen the link between MVEs and exosomes, and introduce ways of manipulating exosome secretion in vivo

Remodeling of the Basal Labyrinth of Retinal Pigment Epithelial Cells With Osmotic Challenge, Age, and Disease

PURPOSE: The basal surface of the retinal pigment epithelium (RPE) is folded into a complex basal labyrinth thought to facilitate solute and water transport. We aimed to analyze and define the structural organization of the basal labyrinth of the RPE to enable quantitative analysis of structural changes in age and disease and to better understand the relationship between basal labyrinth structure and efficiency of transepithelial transport. METHODS: Conventional transmission and serial block-face scanning electron microscopy and electron tomography were used to examine the structure of the basal labyrinth in mouse eyes of different ages and genotypes and with and without osmotic shock before fixation. RESULTS: We identified structurally distinct zones (stacked and ribbon-like) within the RPE basal labyrinth that are largely organelle free and cisternal elements that make contact with the endoplasmic reticulum (ER) and mitochondria. These zones are lost in a hierarchic fashion with age and prematurely in a model of the progressive retinal degenerative disease, choroideremia. Junctional complexes crosslink closely opposed infoldings. Spacing between the basal infoldings was affected by subtle osmotic changes while osmotic shock induced dramatic remodeling of the infoldings. CONCLUSION: The basal labyrinth has complex but ordered structural elements that break down with age and in choroideremia. The geometry of these elements and site of contact with ER and mitochondria likely facilitate the ion transport that drives water transport across the basal RPE surface. Changes in structure in response to local osmotic variation may allow transport to be modulated in order to maintain RPE volume

Conditional ablation of the choroideremia gene causes age-related changes in mouse retinal pigment epithelium.

The retinal pigment epithelium (RPE) is a pigmented monolayer of cells lying between the photoreceptors and a layer of fenestrated capillaries, the choriocapillaris. Choroideremia (CHM) is an X-linked progressive degeneration of these three layers caused by the loss of function of Rab Escort protein-1 (REP1). REP1 is involved in the prenylation of Rab proteins, key regulators of membrane trafficking. To study the pathological consequences of chronic disruption of membrane traffic in the RPE we used a cell type-specific knock-out mouse model of the disease, where the Chm/Rep1 gene is deleted only in pigmented cells (Chm(Flox), Tyr-Cre+). Transmission electron microscopy (TEM) was used to quantitate the melanosome distribution in the RPE and immunofluorescent staining of rhodopsin was used to quantitate phagocytosed rod outer segments in retinal sections. The ultrastructure of the RPE and Bruch's membrane at different ages was characterised by TEM to analyse age-related changes occurring as a result of defects in membrane traffic pathways. Chm/Rep1 gene knockout in RPE cells resulted in reduced numbers of melanosomes in the apical processes and delayed phagosome degradation. In addition, the RPE accumulated pathological changes at 5-6 months of age similar to those observed in 2-year old controls. These included the intracellular accumulation of lipofuscin-containing deposits, disorganised basal infoldings and the extracellular accumulation of basal laminar and basal linear deposits. The phenotype of the Chm(Flox), Tyr-Cre+ mice suggests that loss of the Chm/Rep1 gene causes premature accumulation of features of aging in the RPE. Furthermore, the striking similarities between the present observations and some of the phenotypes reported in age-related macular degeneration (AMD) suggest that membrane traffic defects may contribute to the pathogenesis of AMD

Дослідження протизапальної активності полісахаридного комплексу з пагонів Багна звичайного

Topicality. Most of the respiratory diseases are accompanied by cough. In addition, many etiological cough factors are associated with the respiratory inflammatory process.Aim. To investigate the anti-inflammatory properties of the new Ledum palustre shoots polysaccharides complex at the most effective dose (100 mg/kg).Materials and methods. Experimental investigations of anti-inflammatory properties of Ledum palustre shoots polysaccharides complex at usual dose of 100 mg/kg on the model of carrageenan and zymosan edema in rats.Results and discussion. According to the results of the study, the mechanism of anti-exudative action of the first obtained polysaccharides of Ledum palustre shoots was discovered. The phytocomplex at a dose of 100 mg/kg on the model of zymosan swelling reliably reduced swelling of the paw in rats by 66.71 % compared with the control group. On the carragine edema model, the polysaccharides complex from the basal sprouts moderately reduced swelling. The anti-inflammatory activity was 31.32 % in comparison with the controled group, which resulted in the phyto-complex taking the referent drug action, whose effectiveness was 44.21 %.Conclusions. The polysaccharides of Ledum palustre shoots in a dose of 100 mg/kg demonstrated a pronounced anti-edema and ability to suppress the synthesis of leukotrienes and moderately affect the prostaglandins synthesis.Актуальність. Більша частина захворювань дихальних шляхів супроводжується кашлем. До того ж чимало етіологічних факторів кашлю пов’язані з запальним процесом органів дихання.Метою даної роботи було дослідити протизапальні властивості нового полісахаридного комплексу з пагонів Багна звичайного у найефективнішій дозі (100 мг/кг).Матеріали та методи. Експериментально дослідили протизапальні властивості полісахаридного комплексу з пагонів Багна звичайного у дозі 100 мг/кг на моделі карагенінового та зимозанового набряку у щурів.Результати та їх обговорення. За результатами дослідження було розкрито механізм антиексудативної дії вперше отриманого полісахаридного комплексу з пагонів Багна звичайного. Фітокомплекс у дозі 100 мг/кг на моделі зимозанового набряку достовірно зменшував набряк лапи у щурів на 66,71 % у порівнянні з контрольною групою. На моделі карагенінового набряку полісахаридний комплекс з пагонів Багна звичайного помірно зменшував набряк. Протизапальна активність склала 31,32 % у порівнянні з групою контролю, саме цим фітокомплекс і поступився дії препарату-референту, ефективність якого склала 44,21 %.Висновки. Полісахаридний комплекс з пагонів Багна звичайного у дозі 100 мг/кг продемонстрував виражену протинабрякову дію та здатність пригнічувати синтез лейкотрієнів і помірно впливати на синтез простагландинів

Beneficial effects on vision in patients undergoing retinal gene therapy for choroideremia

Retinal gene therapy is increasingly recognized as a novel molecular intervention that has huge potential in treating common causes of blindness, the majority of which have a genetic aetiology1,2,3,4,5. Choroideremia is a chronic X-linked retinal degeneration that was first described in 18726. It leads to progressive blindness due to deficiency of Rab-escort protein 1 (REP1). We designed an adeno-associated viral vector to express REP1 and assessed it in a gene therapy clinical trial by subretinal injection in 14 patients with choroideremia. The primary endpoint was vision change in treated eyes 2 years after surgery compared to unoperated fellow eyes. Despite complications in two patients, visual acuity improved in the 14 treated eyes over controls (median 4.5 letter gain, versus 1.5 letter loss, P = 0.04), with 6 treated eyes gaining more than one line of vision (>5 letters). The results suggest that retinal gene therapy can sustain and improve visual acuity in a cohort of predominantly late-stage choroideremia patients in whom rapid visual acuity loss would ordinarily be predicted

An engineered cereblon optimized for high throughput screening and molecular glue discovery

The majority of clinical degraders utilize an immunomodulatory imide drug (IMiD)-based derivative that directs their target to the E3 ligase receptor cereblon (CRBN); however, identification of IMiD molecular glue substrates has remained underexplored. To tackle this, we design human CRBN constructs, which retain all features for ternary complex formation, while allowing generation of homogenous and cost-efficient expression in E. coli. Extensive profiling of the construct shows it to be the "best of both worlds" in terms of binding activity and ease of production. We next designed the "Enamine focused IMiD library" and demonstrated applicability of the construct to high-throughput screening, identifying binders with high potency, ligand efficiency, and specificity. Finally, we adapt our construct for proof of principle glue screening approaches enabling IMiD cellular interactome determination. Coupled with our IMiD binding landscape the methods described here should serve as valuable tools to assist discovery of next generation CRBN glues

Ultra-large library docking for discovering new chemotypes

Despite intense interest in expanding chemical space, libraries containing hundreds-of-millions to billions of diverse molecules have remained inaccessible. Here we investigate structure-based docking of 170 million make-on-demand compounds from 130 well-characterized reactions. The resulting library is diverse, representing over 10.7 million scaffolds that are otherwise unavailable. For each compound in the library, docking against AmpC β-lactamase (AmpC) and the D 4 dopamine receptor were simulated. From the top-ranking molecules, 44 and 549 compounds were synthesized and tested for interactions with AmpC and the D 4 dopamine receptor, respectively. We found a phenolate inhibitor of AmpC, which revealed a group of inhibitors without known precedent. This molecule was optimized to 77 nM, which places it among the most potent non-covalent AmpC inhibitors known. Crystal structures of this and other AmpC inhibitors confirmed the docking predictions. Against the D 4 dopamine receptor, hit rates fell almost monotonically with docking score, and a hit-rate versus score curve predicted that the library contained 453,000 ligands for the D 4 dopamine receptor. Of 81 new chemotypes discovered, 30 showed submicromolar activity, including a 180-pM subtype-selective agonist of the D 4 dopamine receptor