Spanlastics a Novel Nanovesicular Carrier: Its Potential Application and Emerging Trends in Therapeutic Delivery.

Nanotechnology-based drug delivery system has played a very crucial role in overpowering the tasks allied with the conventional dosage form. Spanlastics, an elastic nanovesicle with an ability to carry wide range of drug molecules, make it a potential drug delivery carrier. Spanlastics have extended rising curiosity for diverse sort of route of administration. They can squeeze themselves through the skin pore due to elastic and deformable nature which makes them favorable for transdermal delivery. Spanlastics consist of non-ionic surfactant or blend of surfactants. Many researchers proved that spanlastics have been significantly augment therapeutic efficacy, enhanced drug bioavailability, and reduced drug toxicity. This review summarizes various vesicular systems, composition and structure of spanlastics, advantages of spanlastics over other drug delivery systems, and mechanism of drug penetration through skin. It also gives a brief on different types of drug encapsulated in spanlastics vesicles for the treatment of various diseases.

Evaluation of Orthodontic Mini-implant-anchored en masse Retraction of Maxillary Anterior Teeth with Reduced Bone Support: A Prospective Finite Element Analysis Study.

AIM: The purpose of this study was to evaluate force systems to bring about the en masse retraction of maxillary anterior teeth having reduced bone levels using finite element analysis. MATERIALS AND METHODS: This is a prospective study. Three-dimensional finite element models of maxillary dentition having normal alveolar bone level and 2, 4, and 6 mm bone loss with first premolar extraction were constructed from a spiral CT scan of a skull. Archwire and brackets were modeled on the facial surfaces of teeth. Retraction force of 175 gm was applied from an orthodontic mini-implant placed bilaterally between the second premolar and first molar and 12 mm above plane of the archwire to anterior retraction hook (ARH) fixed at two heights of 6 and 10 mm above the archwire. RESULTS: Maximum displacement and periodontal ligament (PDL) stress were calculated for different combinations of bone levels and ARH. As the bone loss increased, the tipping tendency, amount of intrusion, and maximum von Mises stress in PDL also increased, showing a direct correlation. CONCLUSION: To minimize tipping and PDL stress, the height of ARH should be increased in alveolar bone loss conditions to allow retraction force to pass through or even above the center of resistance of anterior teeth. Even then, pure bodily retraction may not be achieved, but tipping tendency can be reduced. Nevertheless, it may not be suitable to increase ARH beyond a limit owing to chances of irritation to the vestibular mucosa. Alternative methods should be contemplated to reduce the tipping behavior. CLINICAL SIGNIFICANCE: The alternative is to apply a lighter retraction force to reduce lingual tipping. A higher counter-moment in the archwire or bracket can also be incorporated.

Atypical radiological findings of primary central nervous system lymphoma.

PURPOSE: Primary central nervous system lymphoma (PCNSL) presenting with atypical radiological findings often leads to delayed diagnosis. We aim to characterize the radiological features and apparent diffusion coefficient (ADC) values of PCNSL with atypical neuroimaging presentation in our local population. METHODS: We retrospectively reviewed all patients with histological diagnosis of CNS lymphoma at our tertiary center from 2005 to 2016. We screened all initial pre-treatment MRIs and excluded cases with typical imaging findings of contrast-enhancing lesions without intra-lesional susceptibility and central non-enhancement. Additional exclusion criteria included (i) relapsed PCNSL, (ii) secondary CNS lymphoma, and (iii) positive HIV status. Two independent raters scored MRI and CT scans at presentation. We computed ADC values in the tumors by 2 methods: single region of interest (ROI1) and multiple ROI (ROI2). RESULTS: Sixteen (25.4%) of 63 patients with CNS lymphoma met inclusion criteria. There were 8 men; median age was 61 (range 22-81) years. Histological diagnoses were diffuse large B cell lymphoma (n = 14) and intravascular lymphoma (n = 2). Fifteen (93%) patients had enhancing lesions (5 solitary; 10 multifocal); most enhancing lesions had T1 hypointense (67%) and T2 mixed (53%) signals, and 6 (40%) had central non-enhancing regions. Nine (56%) patients had lesions with susceptibility. Using the ROI methods, median values for minimum ADC and mean ADC ranged 0.65-0.71 × 10-3 mm2/s and 0.79-0.84 × 10-3 mm2/s respectively. CONCLUSION: PCNSL with atypical radiological features represented one-fourth of our histologically diagnosed lymphoma cases; low ADC values in atypical lesions should prompt clinicians to consider early biopsy for definitive diagnosis.

Correction to: atypical radiological findings of primary central nervous system lymphoma.

The above article was published with incorrect list of authors. We have added Seyed Ehasan Saffari and his affiliation as the addition of the new author to the author list was requested at revision stage.

Engineering Protein-Secreting Plasma Cells by Homology-Directed Repair in Primary Human B Cells.

The ability to engineer primary human B cells to differentiate into long-lived plasma cells and secrete a de novo protein may allow the creation of novel plasma cell therapies for protein deficiency diseases and other clinical applications. We initially developed methods for efficient genome editing of primary B cells isolated from peripheral blood. By delivering CRISPR/CRISPR-associated protein 9 (Cas9) ribonucleoprotein (RNP) complexes under conditions of rapid B cell expansion, we achieved site-specific gene disruption at multiple loci in primary human B cells (with editing rates of up to 94%). We used this method to alter ex vivo plasma cell differentiation by disrupting developmental regulatory genes. Next, we co-delivered RNPs with either a single-stranded DNA oligonucleotide or adeno-associated viruses containing homologous repair templates. Using either delivery method, we achieved targeted sequence integration at high efficiency (up to 40%) via homology-directed repair. This method enabled us to engineer plasma cells to secrete factor IX (FIX) or B cell activating factor (BAFF) at high levels. Finally, we show that introduction of BAFF into plasma cells promotes their engraftment into immunodeficient mice. Our results highlight the utility of genome editing in studying human B cell biology and demonstrate a novel strategy for modifying human plasma cells to secrete therapeutic proteins.

Targeted gene editing restores regulated CD40L function in X-linked hyper-IgM syndrome.

Loss of CD40 ligand (CD40L) expression or function results in X-linked hyper-immunoglobulin (Ig)M syndrome (X-HIGM), characterized by recurrent infections due to impaired immunoglobulin class-switching and somatic hypermutation. Previous attempts using retroviral gene transfer to correct murine CD40L expression restored immune function; however, treated mice developed lymphoproliferative disease, likely due to viral-promoter-dependent constitutive CD40L expression. These observations highlight the importance of preserving endogenous gene regulation in order to safely correct this disorder. Here, we report efficient, on-target, homology-directed repair (HDR) editing of the CD40LG locus in primary human T cells using a combination of a transcription activator-like effector nuclease-induced double-strand break and a donor template delivered by recombinant adeno-associated virus. HDR-mediated insertion of a coding sequence (green fluorescent protein or CD40L) upstream of the translation start site within exon 1 allowed transgene expression to be regulated by endogenous CD40LG promoter/enhancer elements. Additionally, inclusion of the CD40LG 3'-untranslated region in the transgene preserved posttranscriptional regulation. Expression kinetics of the transgene paralleled that of endogenous CD40L in unedited T cells, both at rest and in response to T-cell stimulation. The use of this method to edit X-HIGM patient T cells restored normal expression of CD40L and CD40-murine IgG Fc fusion protein (CD40-muIg) binding, and rescued IgG class switching of naive B cells in vitro. These results demonstrate the feasibility of engineered nuclease-directed gene repair to restore endogenously regulated CD40L, and the potential for its use in T-cell therapy for X-HIGM syndrome.

High Efficiency CRISPR/Cas9-mediated Gene Editing in Primary Human T-cells Using Mutant Adenoviral E4orf6/E1b55k "Helper" Proteins.

Many future therapeutic applications of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 and related RNA-guided nucleases are likely to require their use to promote gene targeting, thus necessitating development of methods that provide for delivery of three components-Cas9, guide RNAs and recombination templates-to primary cells rendered proficient for homology-directed repair. Here, we demonstrate an electroporation/transduction codelivery method that utilizes mRNA to express both Cas9 and mutant adenoviral E4orf6 and E1b55k helper proteins in association with adeno-associated virus (AAV) vectors expressing guide RNAs and recombination templates. By transiently enhancing target cell permissiveness to AAV transduction and gene editing efficiency, this novel approach promotes efficient gene disruption and/or gene targeting at multiple loci in primary human T-cells, illustrating its broad potential for application in translational gene editing.

Rapamycin relieves lentiviral vector transduction resistance in human and mouse hematopoietic stem cells.

Transplantation of genetically modified hematopoietic stem cells (HSCs) is a promising therapeutic strategy for genetic diseases, HIV, and cancer. However, a barrier for clinical HSC gene therapy is the limited efficiency of gene delivery via lentiviral vectors (LVs) into HSCs. We show here that rapamycin, an allosteric inhibitor of the mammalian target of rapamycin complexes, facilitates highly efficient lentiviral transduction of mouse and human HSCs and dramatically enhances marking frequency in long-term engrafting cells in mice. Mechanistically, rapamycin enhanced postbinding endocytic events, leading to increased levels of LV cytoplasmic entry, reverse transcription, and genomic integration. Despite increasing LV copy number, rapamycin did not significantly alter LV integration site profile or chromosomal distribution in mouse HSCs. Rapamycin also enhanced in situ transduction of mouse HSCs via direct intraosseous infusion. Collectively, rapamycin strongly augments LV transduction of HSCs in vitro and in vivo and may prove useful for therapeutic gene delivery.

Intraosseous delivery of lentiviral vectors targeting factor VIII expression in platelets corrects murine hemophilia A.

Intraosseous (IO) infusion of lentiviral vectors (LVs) for in situ gene transfer into bone marrow may avoid specific challenges posed by ex vivo gene delivery, including, in particular, the requirement of preconditioning. We utilized IO delivery of LVs encoding a GFP or factor VIII (FVIII) transgene directed by ubiquitous promoters (a MND or EF-1α-short element; M-GFP-LV, E-F8-LV) or a platelet-specific, glycoprotein-1bα promoter (G-GFP-LV, G-F8-LV). A single IO infusion of M-GFP-LV or G-GFP-LV achieved long-term and efficient GFP expression in Lineage(-)Sca1(+)c-Kit(+) hematopoietic stem cells and platelets, respectively. While E-F8-LV produced initially high-level FVIII expression, robust anti-FVIII immune responses eliminated functional FVIII in circulation. In contrast, IO delivery of G-F8-LV achieved long-term platelet-specific expression of FVIII, resulting in partial correction of hemophilia A. Furthermore, similar clinical benefit with G-F8-LV was achieved in animals with pre-existing anti-FVIII inhibitors. These findings further support platelets as an ideal FVIII delivery vehicle, as FVIII, stored in α-granules, is protected from neutralizing antibodies and, during bleeding, activated platelets locally excrete FVIII to promote clot formation. Overall, a single IO infusion of G-F8-LV was sufficient to correct hemophilia phenotype for long term, indicating that this approach may provide an effective means to permanently treat FVIII deficiency.

Novel fluorescent genome editing reporters for monitoring DNA repair pathway utilization at endonuclease-induced breaks.

The creation of a DNA break at a specific locus by a designer endonuclease can be harnessed to edit a genome. However, DNA breaks may engage one of several competing repair pathways that lead to distinct types of genomic alterations. Therefore, understanding the contribution of different repair pathways following the introduction of a targeted DNA break is essential to further advance the safety and efficiency of nuclease-induced genome modification. To gain insight into the role of different DNA repair pathways in resolving nuclease-induced DNA breaks into genome editing outcomes, we previously developed a fluorescent-based reporter system, designated the Traffic Light Reporter, which provides a readout of gene targeting and gene disruption downstream of a targeted DNA double-strand break. Here we describe two related but novel reporters that extend this technology: one that allows monitoring of the transcriptional activity at the reporter locus, and thus can be applied to interrogate break resolution at active and repressed loci; and a second that reads out single-strand annealing in addition to gene targeting and gene disruption. Application of these reporters to assess repair pathway usage in several common gene editing contexts confirms the importance that chromatin status and initiation of end resection have on the resolution of nuclease-induced breaks.

Progressive engineering of a homing endonuclease genome editing reagent for the murine X-linked immunodeficiency locus.

LAGLIDADG homing endonucleases (LHEs) are compact endonucleases with 20-22 bp recognition sites, and thus are ideal scaffolds for engineering site-specific DNA cleavage enzymes for genome editing applications. Here, we describe a general approach to LHE engineering that combines rational design with directed evolution, using a yeast surface display high-throughput cleavage selection. This approach was employed to alter the binding and cleavage specificity of the I-Anil LHE to recognize a mutation in the mouse Bruton tyrosine kinase (Btk) gene causative for mouse X-linked immunodeficiency (XID)-a model of human X-linked agammaglobulinemia (XLA). The required re-targeting of I-AniI involved progressive resculpting of the DNA contact interface to accommodate nine base differences from the native cleavage sequence. The enzyme emerging from the progressive engineering process was specific for the XID mutant allele versus the wild-type (WT) allele, and exhibited activity equivalent to WT I-AniI in vitro and in cellulo reporter assays. Fusion of the enzyme to a site-specific DNA binding domain of transcription activator-like effector (TALE) resulted in a further enhancement of gene editing efficiency. These results illustrate the potential of LHE enzymes as specific and efficient tools for therapeutic genome engineering.

Efficient and sustained FOXP3 locus editing in hematopoietic stem cells as a therapeutic approach for IPEX syndrome.

Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is a monogenic disorder caused by mutations in the FOXP3 gene, required for generation of regulatory T (Treg) cells. Loss of Treg cells leads to immune dysregulation characterized by multi-organ autoimmunity and early mortality. Hematopoietic stem cell (HSC) transplantation can be curative, but success is limited by autoimmune complications, donor availability and/or graft-vs.-host disease. Correction of FOXP3 in autologous HSC utilizing a homology-directed repair (HDR)-based platform may provide a safer alternative therapy. Here, we demonstrate efficient editing of FOXP3 utilizing co-delivery of Cas9 ribonucleoprotein complexes and adeno-associated viral vectors to achieve HDR rates of >40% in vitro using mobilized CD34+ cells from multiple donors. Using this approach to deliver either a GFP or a FOXP3 cDNA donor cassette, we demonstrate sustained bone marrow engraftment of approximately 10% of HDR-edited cells in immune-deficient recipient mice at 16 weeks post-transplant. Further, we show targeted integration of FOXP3 cDNA in CD34+ cells from an IPEX patient and expression of the introduced FOXP3 transcript in gene-edited primary T cells from both healthy individuals and IPEX patients. Our combined findings suggest that refinement of this approach is likely to provide future clinical benefit in IPEX.

Normal collagen and bone production by gene-targeted human osteogenesis imperfecta iPSCs.

Osteogenesis imperfecta (OI) is caused by dominant mutations in the type I collagen genes. In principle, the skeletal abnormalities of OI could be treated by transplantation of patient-specific, bone-forming cells that no longer express the mutant gene. Here, we develop this approach by isolating mesenchymal cells from OI patients, inactivating their mutant collagen genes by adeno-associated virus (AAV)-mediated gene targeting, and deriving induced pluripotent stem cells (iPSCs) that were expanded and differentiated into mesenchymal stem cells (iMSCs). Gene-targeted iMSCs produced normal collagen and formed bone in vivo, but were less senescent and proliferated more than bone-derived MSCs. To generate iPSCs that would be more appropriate for clinical use, the reprogramming and selectable marker transgenes were removed by Cre recombinase. These results demonstrate that the combination of gene targeting and iPSC derivation can be used to produce potentially therapeutic cells from patients with genetic disease.

Filamentous Bacteria as Targets to Study Phagocytosis.

Filamentous targets are internalized via phagocytic cups that last for several minutes before closing to form a phagosome. This characteristic offers the possibility to study key events in phagocytosis with greater spatial and temporal resolution than is possible to achieve using spherical particles, for which the transition from a phagocytic cup to an enclosed phagosome occurs within a few seconds after particle attachment. In this chapter, we provide methodologies to prepare filamentous bacteria and describe how they can be used as targets to study different aspects of phagocytosis.

Raloxifene encapsulated spanlastic nanogel for the prevention of bone fracture risk via transdermal administration: Pharmacokinetic and efficacy study in animal model.

This research work is to evaluate spanlastic-loaded raloxifene (RLX) nanogel administration via the transdermal route to avoid its hepatic metabolism and to enhance the bioavailability for better management of osteoporosis. RLX-loaded spanlastic nanogel was prepared and characterized for its viscosity, pH, spreadability, and texture profile. The formulation was applied on the skin surface of the animal for pharmacokinetic evaluation, and later, the efficacy of the formulation was assessed in ovariectomized female Wistar rats. The nanogel was obtained with a viscosity (2552.66 ± 30.61 cP), pH (7.1 ± 0.1), and spreadability (7.1 ± 0.2 cm). The texture properties, cohesiveness, and adhesiveness of the nanogel showed its suitability for transdermal application. Nanogel showed no sign of edema and erythema in the skin irritation test which revealed its safety for transdermal application. The t1/2 obtained for RLX-spanlastic nanogel (37.02 ± 0.59 h) was much higher than that obtained for RLX-oral suspension (14.43 h). The relative bioavailability was found to be 215.96% for RLX-spanlastic nanogel, and the drug and formulation did not show any toxicity in any of the vital organs, as well as no hematological changes occurring in blood samples. In microarchitectural measurement, RLX-spanlastic nanogel exhibited no unambiguous deviations along with improved bone mineral density compared to the RLX suspension treated group. Transdermal administration of RLX-spanlastic nanogel showed significant improvement of drug bioavailability (approx. twice to oral administration) without any toxic effect in the treated rats. Hence, spanlastic nanogel could be a better approach to deliver RLX via transdermal route for the management of osteoporosis.

A lentiviral vector B cell gene therapy platform for the delivery of the anti-HIV-1 eCD4-Ig-knob-in-hole-reversed immunoadhesin.

Barriers to effective gene therapy for many diseases include the number of modified target cells required to achieve therapeutic outcomes and host immune responses to expressed therapeutic proteins. As long-lived cells specialized for protein secretion, antibody-secreting B cells are an attractive target for foreign protein expression in blood and tissue. To neutralize HIV-1, we developed a lentiviral vector (LV) gene therapy platform for delivery of the anti-HIV-1 immunoadhesin, eCD4-Ig, to B cells. The EµB29 enhancer/promoter in the LV limited gene expression in non-B cell lineages. By engineering a knob-in-hole-reversed (KiHR) modification in the CH3-Fc eCD4-Ig domain, we reduced interactions between eCD4-Ig and endogenous B cell immunoglobulin G proteins, which improved HIV-1 neutralization potency. Unlike previous approaches in non-lymphoid cells, eCD4-Ig-KiHR produced in B cells promoted HIV-1 neutralizing protection without requiring exogenous TPST2, a tyrosine sulfation enzyme required for eCD4-Ig-KiHR function. This finding indicated that B cell machinery is well suited to produce therapeutic proteins. Lastly, to overcome the inefficient transduction efficiency associated with VSV-G LV delivery to primary B cells, an optimized measles pseudotyped LV packaging methodology achieved up to 75% transduction efficiency. Overall, our findings support the utility of B cell gene therapy platforms for therapeutic protein delivery.

Detecting the Uncommon Imaging Manifestations of Posterior Reversible Encephalopathy Syndrome (PRES) in Adults: a Comprehensive Illustrated Guide for the Trainee Radiologist.

Posterior reversible encephalopathy syndrome (PRES) has traditionally been described as a reversible leukoencephalopathy with a distinct pattern of posteriorly distributed vasogenic oedema involving the subcortical regions of parietal and occipital lobes. PRES commonly occurs in the setting of hypertensive emergencies, pre-eclampsia/eclampsia, impaired renal function, and immunosuppressive therapy. The various clinical presentations of PRES include encephalopathy, seizures, headache, visual, and focal neurological deficits. As knowledge of this entity grows, the range of clinical, and radiological features is seen to be much broader than originally described. The brain oedema may not always be posteriorly distributed and the syndrome may not be uniformly reversible. Of special note are some uncommon imaging features (unilateral cerebral involvement, and isolated posterior fossa involvement) and also some uncommon complications (haemorrhage, cytotoxic oedema, and vasoconstriction). These red herrings may lead to potential diagnostic challenges and pitfalls especially for trainee radiologists, who often read these scans in an emergency setting. Early and accurate diagnosis is crucial for prompt optimum management, thereby avoiding residual morbidity. This review article focusses on the atypical radiological features of PRES in adults with extensive case-based imaging examples. A brief description of the pathophysiology, clinical, and classic radiological features of PRES has also been included. A tabulated summary of potential mimics with diagnostic pearls is provided to highlight pertinent take home points and to serve as an easy guide for day-to-day clinical practice.

Ex vivo engineered human plasma cells exhibit robust protein secretion and long-term engraftment in vivo.

Due to their unique longevity and capacity to secrete high levels of protein, plasma B cells have the potential to be used as a cell therapy for protein replacement. Here, we show that ex vivo engineered human plasma cells exhibit single-cell RNA profiles, scanning electron micrograph ultrastructural features, and in vivo homing capacity of long-lived plasma cells. After transferring human plasma cells to immunodeficient mice in the presence of the human cytokines BAFF and IL-6, we observe increases in retention of plasma cells in the bone marrow, with engraftment exceeding a year. The most profound in vivo effects of human IL-6 are observed within 20 days of transfer and could be explained by decreased apoptosis in newly differentiated plasma cells. Collectively, these results show that ex vivo engineered and differentiated human plasma cells have the potential for long-lived in vivo protein secretion, which can be modeled in small animals.

Brain targeted delivery of carmustine using chitosan coated nanoparticles via nasal route for glioblastoma treatment.

This study aims to develop chitosan-coated PLGA nanoparticles intended for nose-to-brain delivery of carmustine. Formulations were prepared by the double emulsion solvent evaporation method and optimized by using Box-Behnken Design. The optimized nanoparticles were obtained to satisfactory levels in terms of particle size, PDI, entrapment efficiency, and drug loading. In vitro drug release and ex-vivo permeation showed sustained release and enhanced permeability (approx. 2 fold) of carmustine compared to drug suspension. The AUC0-t of brain obtained with carmustine-loaded nanoparticles via nasal administration in Albino Wistar rats was 2.8 and 14.7 times that of intranasal carmustine suspension and intravenous carmustine, respectively. The MTT assay on U87 MG cell line showed a significant decrease (P < 0.05) in the IC50 value of the formulation (71.23 µg ml-1) as compared to drug suspension (90.02 µg ml-1).These findings suggest chitosan coated nanoparticles could be used to deliver carmustine via intranasal administration to treat Glioblastoma multiforme.

Pancreatic islet-specific engineered Tregs exhibit robust antigen-specific and bystander immune suppression in type 1 diabetes models.

Adoptive transfer of regulatory T cells (Tregs) is therapeutic in type 1 diabetes (T1D) mouse models. Tregs that are specific for pancreatic islets are more potent than polyclonal Tregs in preventing disease. However, the frequency of antigen-specific natural Tregs is extremely low, and ex vivo expansion may destabilize Tregs, leading to an effector phenotype. Here, we generated durable, antigen-specific engineered Tregs (EngTregs) from primary human CD4+ T cells by combining FOXP3 homology-directed repair editing and lentiviral T cell receptor (TCR) delivery. Using TCRs derived from clonally expanded CD4+ T cells isolated from patients with T1D, we generated islet-specific EngTregs that suppressed effector T cell (Teff) proliferation and cytokine production. EngTregs suppressed Teffs recognizing the same islet antigen in addition to bystander Teffs recognizing other islet antigens through production of soluble mediators and both direct and indirect mechanisms. Adoptively transferred murine islet-specific EngTregs homed to the pancreas and blocked diabetes triggered by islet-specific Teffs or diabetogenic polyclonal Teffs in recipient mice. These data demonstrate the potential of antigen-specific EngTregs as a targeted therapy for preventing T1D.