María Clemente

Characterization of carbosilane dendrimers as effective carriers of siRNA to HIV-infected lymphocytes.

One of the primary limitations of RNA interference as a technique for gene regulation is effective delivery of siRNA into the target cells. Dendrimers are nanoparticles that are increasingly being used as oligonucleotide and drug delivery vehicles. We have developed amino-terminated carbosilane dendrimers (CBS) as a means to protect and transport siRNA. Initially, stability studies showed that CBS bind siRNA via electrostatic interactions. Dendrimer-bound siRNA was found to be resistant to degradation by RNase. Cytotoxicity assays of CBS/siRNA dendriplexes with peripheral blood mononuclear cells (PBMC) and the lymphocytic cell line SupT1 revealed a maximum safe dendrimer concentration of 25 microg/ml. Next, utilizing flow cytometry and confocal microscopy, lymphocytes were seen to be successfully transfected by fluorochrome-labeled siRNA either naked or complexed with CBS. Dendriplexes with +/- charge ratio of 2 were determined to have the highest transfection efficiency while maintaining a low level of toxicity in these systems including hard-to-transfect HIV-infected PBMC. Finally, CBS/siRNA dendriplexes were shown to silence GAPDH expression and reduce HIV replication in SupT1 and PBMC. These results point to the possibility of utilizing dendrimers such as CBS to deliver and transfect siRNA into lymphocytes thus allowing the use of RNA interference as a potential alternative therapy for HIV infection.

Carbosilane Dendrimers to Transfect Human Astrocytes with Small Interfering RNA Targeting Human Immunodeficiency Virus

BackgroundHIV infection of the CNS is the principle cause of HIV-associated dementia in adults and encephalopathy in children. Gene therapy techniques such as small interfering RNA (siRNA) possess great potential in drug development, but first they must overcome the key obstacle of reaching the interior of the affected cells. A successful delivery vector for anti-HIV drugs that is capable of crossing the blood-brain barrier (BBB) could provide a way of addressing this issue. Non-viral vectors such as dendrimers offer a means for effectively delivering and transfecting siRNA to the target cells.ObjectiveTo evaluate the application of gene therapy for reducing HIV replication in human astrocytes.MethodsWe used the 2G-NN16 amino-terminated carbosilane dendrimer as a method for delivering siRNA to HIV-infected human astrocytes. We tested the cytotoxicity in human astrocytoma cells caused by 2G-NN16 and dendriplexes formed with siRNA (siRNA/2G-NN16) by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium-bromide (MTT) and lactate dehydrogenase assays. The ability to transfect human astrocytes with siRNA/2G-NN16 dendriplexes was tested by flow cytometry and immunofluorescence microscopy. To assess the potential capability of siRNA/2G-NN16 dendriplexes for crossing the BBB, we used an in vitro transcytosis assay with bovine brain microvascular endothelial cells. HIV-1 inhibition assays using 2G-NN16 and siRNA/2G-NN16 dendriplexes were determined by quantification of the viral load from culture supernatants of the astrocytes.ResultsA gradual time-controlled degradation of the 2G-NN16 dendrimer and liberation of its siRNA cargo between 12 and 24 hours was observed via gel electrophoresis. There was no cytotoxicity in HIV-infected or non-infected human astrocytoma cells when treated with up to 24 μg/mL of 2G-NN16 dendrimer or siRNA/2G-NN16 dendriplexes, and siRNA/2G-NN16 dendriplexes were seen to successfully transfect human astrocytes even after crossing an in vitro BBB model. More interestingly, transfected siRNA was observed to exert a biologic effect, as dendriplexes were shown to down-regulate the housekeeping gene GAPDH and to reduce replication of HIV-1 strains X4-HIV NL4-3 and R5-HIV BaL in human astrocytes.ConclusionsThe 2G-NN16 dendrimer successfully delivers and transfects siRNA to HIV-infected human astrocytes and achieves gene silencing without causing cytotoxicity.Both authors have contributed equally to this paper.

Highly Efficient Transfection of Rat Cortical Neurons Using Carbosilane Dendrimers Unveils a Neuroprotective Role for HIF-1α in Early Chemical Hypoxia-Mediated Neurotoxicity

PurposeTo study the effect of a non-viral vector (carbosilane dendrimer) to efficiently deliver small interfering RNA to postmitotic neurons to study the function of hypoxia-inducible factor-1α (HIF1-α) during chemical hypoxia-mediated neurotoxicity.MethodsChemical hypoxia was induced in primary rat cortical neurons by exposure to CoCl2. HIF1-α levels were determined by Western Blot and toxicity was evaluated by both MTT and LDH assays. Neurons were incubated with dendriplexes containing anti-HIF1-α siRNA and both uptake and HIF1-α knockdown efficiency were evaluated.ResultsWe report that a non-viral vector (carbosilane dendrimer) can deliver specific siRNA to neurons and selectively block HIF1-α synthesis with similar efficiency to that achieved by viral vectors. Using this method, we have found that this transcription factor plays a neuroprotective role during the early phase of chemical hypoxia-mediated neurotoxicity.ConclusionThis work represents a proof-of-concept for the use of carbosilane dendrimers to deliver specific siRNA to postmitotic neurons to block selected protein synthesis. This indicates that this type of vector is a good alternative to viral vectors to achieve very high transfection levels in neurons. This also suggests that carbosilane dendrimers might be very useful for gene therapy.

In vivo delivery of siRNA to the brain by carbosilane dendrimer.

Nanotechnology offers a new platform for therapeutic delivery of antiretrovirals to the central nervous system (CNS). Nanoformulated antiretroviral drugs offer multifunctionality, that is, the ability to package multiple diagnostic and therapeutic agents in the same nanocompose, along with the added provisions of site-directed delivery, delivery across the blood-brain-barrier (BBB), and controlled release of therapeutics. We studied the viability of dendrimers and dendriplexes in human primary astrocytes, as well as their uptake by these astrocytes. Functional validation was performed by using specific siRNA against HIV-1 Nef to interfere to HIV-1 infectivity. A high efficiency in Nef silencing, reducing HIV-1 infectivity was observed in astrocytes treated with dendriplexes compared with control or siRandom treated astrocytes. More interestingly, we studied the biodistribution of the second generation of carbosilane dendrimer loaded with FITC (2G-(SNMe3I)11-FITC) in vivo, in BALB/c mice. Dendriplexes were inoculated into BALB/c mice by the retro-orbital venous plexus, and their localization was determined after 1 and 24h post-injection. Dendriplexes were detected inside the brain by a sensitive imaging system of fluorescent imaging in vivo (IVIS Lumina), and by confocal microscopy analysis of sections of OCT-embedded tissues. The 2G-(SNMe3I)11-FITC dendrimer transported efficiently siRNA into the brain, crossing the BBB. Moreover, this dendrimer successfully delivered and transfected siRNA to HIV-infected human primary astrocytes and achieved gene silencing without causing cytotoxicity. These results highlight the potential of this nanoformulation in the treatment of neurological disorders.

Characterization of novel StAR (steroidogenic acute regulatory protein) mutations causing non-classic lipoid adrenal hyperplasia.

Context Steroidogenic acute regulatory protein (StAR) is crucial for transport of cholesterol to mitochondria where biosynthesis of steroids is initiated. Loss of StAR function causes lipoid congenital adrenal hyperplasia (LCAH). Objective StAR gene mutations causing partial loss of function manifest atypical and may be mistaken as familial glucocorticoid deficiency. Only a few mutations have been reported. Design To report clinical, biochemical, genetic, protein structure and functional data on two novel StAR mutations, and to compare them with published literature. Setting Collaboration between the University Childrens Hospital Bern, Switzerland, and the CIBERER, Hospital Vall dHebron, Autonomous University, Barcelona, Spain. Patients Two subjects of a non-consanguineous Caucasian family were studied. The 46,XX phenotypic normal female was diagnosed with adrenal insufficiency at the age of 10 months, had normal pubertal development and still has no signs of hypergonodatropic hypogonadism at 32 years of age. Her 46,XY brother was born with normal male external genitalia and was diagnosed with adrenal insufficiency at 14 months. Puberty was normal and no signs of hypergonadotropic hypogonadism are present at 29 years of age. Results StAR gene analysis revealed two novel compound heterozygote mutations T44HfsX3 and G221S. T44HfsX3 is a loss-of-function StAR mutation. G221S retains partial activity (∼30%) and is therefore responsible for a milder, non-classic phenotype. G221S is located in the cholesterol binding pocket and seems to alter binding/release of cholesterol. Conclusions StAR mutations located in the cholesterol binding pocket (V187M, R188C, R192C, G221D/S) seem to cause non-classic lipoid CAH. Accuracy of genotype-phenotype prediction by in vitro testing may vary with the assays employed.

Gene Therapy in HIV‐Infected Cells to Decrease Viral Impact by Using an Alternative Delivery Method

The ability of dendrimer 2G‐[Si{O(CH2)2N(Me)2+(CH2)2NMe3+(I−)2}]8 (NN16) to transfect a wide range of cell types, as well as the possible biomedical application in direct or indirect inhibition of HIV replication, was investigated. Cells implicated in HIV infection such as primary peripheral blood mononuclear cells (PBMC) and immortalized suspension cells (lymphocytes), primary macrophages and dendritic cells, and immortalized adherent cells (astrocytes and trophoblasts) were analyzed. Dendrimer toxicity was evaluated by mitochondrial activity, cell membrane rupture, release of lactate dehydrogenase, erythrocyte hemolysis, and the effect on global gene expression profiles using whole‐genome human microarrays. Cellular uptake of genetic material was determined using flow cytometry and confocal microscopy. Transfection efficiency and gene knockdown was investigated using dendrimer‐delivered antisense oligonucleotides and small interfering RNA (siRNA). Very little cytotoxicity was detected in a variety of cells relevant to HIV infection and erythrocytes after NN16 dendrimer treatment. Imaging of cellular uptake showed high transfection efficiency of genetic material in all cells tested. Interestingly, NN16 further enhanced the reduction of HIV protein 24 antigen release by antisense oligonucleotides due to improved transfection efficiency. Finally, the dendrimer complexed with siRNA exhibited therapeutic potential by specifically inhibiting cyclooxygenase‐2 gene expression in HIV‐infected nervous system cells. NN16 dendrimers demonstrated the ability to transfect genetic material into a vast array of cells relevant to HIV pathology, combining high efficacy with low toxicity. These results suggest that NN16 dendrimers have the potential to be used as a versatile non‐viral vector for gene therapy against HIV infection.

Synergistic Activation of Latent HIV-1 Expression by Novel Histone Deacetylase Inhibitors and Bryostatin-1.

Viral reactivation from latently infected cells has become a promising therapeutic approach to eradicate HIV. Due to the complexity of the viral latency, combinations of efficient and available drugs targeting different pathways of latency are needed. In this work, we evaluated the effect of various combinations of bryostatin-1 (BRY) and novel histone deacetylase inhibitors (HDACIs) on HIV-reactivation and on cellular phenotype. The lymphocyte (J89GFP) or monocyte/macrophage (THP89GFP) latently infected cell lines were treated with BRY, panobinostat (PNB) and romidepsin (RMD) either alone or in combination. Thus, the effect on the viral reactivation was evaluated. We calculated the combination index for each drug combination; the BRY/HDACIs showed a synergistic HIV-reactivation profile in the majority of the combinations tested, whereas non-synergistic effects were observed when PNB was mixed with RMD. Indeed, the 75% effective concentrations of BRY, PNB and RMD were reduced in these combinations. Moreover, primary CD4 T cells treated with such drug combinations presented similar activation and proliferation profiles in comparison with single drug treated cells. Summing up, combinations between BRY, PNB and/or RMD presented a synergistic profile by inducing virus expression in HIV-latently infected cells, rendering these combinations an attractive novel and safe option for future clinical trials.

Retinol-Binding Protein 4 Levels in Obese Children and Adolescents with Glucose Intolerance

Background: Retinol-binding protein 4 (RBP4) is known to be involved in obesity-associated insulin resistance. Aims: To study the relationships between the degree of adiposity, insulin resistance indices, plasma lipids, inflammatory parameters, glucose intolerance (GI) status and plasma RBP4 levels in obese children and adolescents. Patients and Methods: Prospective study comprising 199 obese patients (95 boys) aged 8–16 years (11.8 ± 1.9). Fifty-three subjects (23 boys) of similar mean age, 11.3 ± 2.1 years, served as controls. BMI, waist and hip circumferences, plasma lipids, and inflammatory parameters were measured and patients underwent an oral glucose tolerance test. Plasma RBP4 levels were determined by nephelometry. Results: Plasma RBP4 levels (pg/ml) in obese patients with GI (n = 15) were higher (45.0 ± 14.1) compared with those of obese patients without GI (35.9 ± 11.7, p = 0.02; n = 184) and controls (31.5 ± 12.3, p = 0.04) in a generalized linear model adjusted for age, sex, BMI and pubertal status. A negative correlation was found between the skeletal muscle insulin resistance index and RBP4; positive correlations were found between the RBP4 and BMI Z-score (r = 0.213, p < 0.001), waist circumferences (r = 0.135, p < 0.05), plasma triglycerides (r = 0.187, p = 0.005) and apolipoprotein B (0.187, p = 0.007). Conclusions: Our results suggest a direct relationship between circulating insulin and RBP4 levels, which indicates that this protein might contribute to the development of muscle insulin resistance.

Influencia de la edad de inicio del brote de crecimiento puberal en la talla adulta

Fundamento y objetivo: En ambos sexos la duracion del crecimiento posnatal difiere entre los sujetos sanos debido a diferencias en la edad a la que inician el crecimiento puberal. Sin embargo, poco se conoce sobre la influencia de este hecho en la estatura adulta. Nuestro objetivo ha sido comparar la talla adulta entre cada uno de los 5 grupos madurativos en los que se inicia el crecimiento puberal. Sujetos y metodo: Dos pediatras realizaron un seguimiento longitudinal de 230 personas (115 mujeres y 115 varones) sanas y sin medicaciones cronicas desde el nacimiento hasta la talla adulta. Se midio la estatura 1-3 veces/ano y se obtuvieron las correspondientes curvas de crecimiento. A partir de ellas se evaluaron la velocidad de crecimiento (cm/ano), la edad al inicio del crecimiento puberal (anos), el crecimiento puberal (cm) y la talla adulta (cm). Segun la edad de inicio del crecimiento puberal, los participantes se agruparon en 5 grupos: 8-9 anos (n = 10), 9-10 anos (n = 29), 10-11 anos (n = 45), 11-12 anos (n = 23) y 12-13 anos (n = 8) en las mujeres, y 10-11 anos (n = 10), 11-12 anos (n = 26), 12-13 anos (n = 45), 13-14 anos (n = 27) y 14-15 anos (n = 7) en los varones. Resultados: En ambos sexos se observaron diferencias estadisticamente significativas para la media de las estaturas al inicio del crecimiento puberal (p < 0,01) y para la media del crecimiento puberal total (p < 0,0001) cuando se comparan entre si los 5 grupos madurativos. Sin embargo, estas diferencias no se observaron entre la media de las tallas adultas cuando se comparan los 5 grupos madurativos entre si, ni cuando se comparo cada grupo con el conjunto de la muestra, ni cuando se comparo cada grupo con las medias obtenidas en estudios recientes de crecimiento de la poblacion espanola. En ambos sexos se observo una correlacion positiva y estadisticamente significativa (p # 0,03) entre la talla al inicio del crecimiento puberal y la talla adulta. Sin embargo, esta correlacion no se observo entre las edades de inicio del crecimiento puberal y las correspondientes estaturas adultas. Conclusiones: En ambos sexos, los factores geneticos, pero no la edad al inicio del crecimiento puberal, influyen en la talla adulta. Aunque la duracion del crecimiento posnatal es menor en las personas con maduracion precoz que en aquellas con maduracion mas tardia, las primeras tienen un desarrollo puberal mas prolongado, ganan mas centimetros de altura y finalizan su crecimiento con una estatura adulta similar.

Prostaglandin E2 reduces the release and infectivity of new cell-free virions and cell-to-cell HIV-1 transfer.

Background The course of human immunodeficiency virus type-1 (HIV-1) infection is influenced by a complex interplay between viral and host factors. HIV infection stimulates several proinflammatory genes, such as cyclooxigense-2 (COX-2), which leads to an increase in prostaglandin (PG) levels in the plasma of HIV-1-infected patients. These genes play an indeterminate role in HIV replication and pathogenesis. The effect of prostaglandin E2 (PGE2) on HIV infection is quite controversial and even contradictory, so we sought to determine the role of PGE2 and the signal transduction pathways involved in HIV infection to elucidate possible new targets for antiretrovirals. Results Our results suggest that PGE2 post-infection treatment acts in the late stages of the viral cycle to reduce HIV replication. Interestingly, viral protein synthesis was not affected, but a loss of progeny virus production was observed. No modulation of CD4 CXCR4 and CCR5 receptor expression, cell proliferation, or activation after PGE2 treatment was detected. Moreover, PGE2 induced an increase in intracellular cAMP (cyclic AMP) levels through the EP2/EP4 receptors. PGE2 effects were mimicked by dbcAMP and by a specific Epac (exchange protein directly activated by cyclic AMP) agonist, 8-Cpt-cAMP. Treatment with PGE2 increased Rap1 activity, decreased RhoA activity and subsequently reduced the polymerization of actin by approximately 30% compared with untreated cells. In connection with this finding, polarized viral assembly platforms enriched in Gag were disrupted, altering HIV cell-to-cell transfer and the infectivity of new virions. Conclusions Our results demonstrate that PGE2, through Epac and Rap activation, alters the transport of newly synthesized HIV-1 components to the assembly site, reducing the release and infectivity of new cell-free virions and cell-to-cell HIV-1 transfer.