scholarly journals Lentiviral Vectors for Delivery of Gene-Editing Systems Based on CRISPR/Cas: Current State and Perspectives

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1288
Author(s):  
Wendy Dong ◽  
Boris Kantor
Keyword(s):  
Large Scale ◽  
Lentiviral Vector ◽  
Clinical Applications ◽  
Scale Production ◽  
Base Editing ◽  
Epigenome Editing ◽  
Vector Systems ◽  
Dividing Cells

CRISPR/Cas technology has revolutionized the fields of the genome- and epigenome-editing by supplying unparalleled control over genomic sequences and expression. Lentiviral vector (LV) systems are one of the main delivery vehicles for the CRISPR/Cas systems due to (i) its ability to carry bulky and complex transgenes and (ii) sustain robust and long-term expression in a broad range of dividing and non-dividing cells in vitro and in vivo. It is thus reasonable that substantial effort has been allocated towards the development of the improved and optimized LV systems for effective and accurate gene-to-cell transfer of CRISPR/Cas tools. The main effort on that end has been put towards the improvement and optimization of the vector’s expression, development of integrase-deficient lentiviral vector (IDLV), aiming to minimize the risk of oncogenicity, toxicity, and pathogenicity, and enhancing manufacturing protocols for clinical applications required large-scale production. In this review, we will devote attention to (i) the basic biology of lentiviruses, and (ii) recent advances in the development of safer and more efficient CRISPR/Cas vector systems towards their use in preclinical and clinical applications. In addition, we will discuss in detail the recent progress in the repurposing of CRISPR/Cas systems related to base-editing and prime-editing applications.

Abstract 262: Derivation of Duchenne Muscular Dystrophy Disease Specific Cardiomyocytes From Patient Urine

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Xuan Guan ◽  
David L Mack ◽  
Claudia M Moreno ◽  
Fernando Santana ◽  
Charles E Murry ◽  
...  
Keyword(s):  
Stem Cells ◽  
Human Urine ◽  
Large Scale ◽  
Lentiviral Vector ◽  
Cellular Reprogramming ◽  
Urine Samples ◽  
Pcr Analysis ◽  
Scale Production ◽  
Mechanism Study

Introduction: Human somatic cells can be reprogrammed into primitive stem cells, termed induced pluripotent stem cells (iPSCs). These iPSCs can be extensively expanded in vitro and differentiated into multiple functional cell types, enabling faithful preservation of individual’s genotype and large scale production of disease targeted cellular components. These unique cellular reagents thus hold tremendous potential in disease mechanism study, drugs screening and cell replacement therapy. Due to the genetic mutation of the protein dystrophin, many DMD patients develop fatal cardiomyopathy with no effective treatment. The underlying pathogenesis has not been fully elucidated. Hypothesis: We tested the hypothesis that iPSCs could be generated from DMD patients’ urine samples and differentiated into cardiomyocytes, recapitulating the dystrophic phenotype. Methods: iPSCs generation was achieved by introducing a lentiviral vector expressing Oct4, Sox2, c-Myc and Klf4 into cells derived from patient’s (n=1) and healthy volunteers’ (n=3) urine. Cardiomyocytes were derived by sequentially treating iPSCs with GSK3 inhibitor CHIR99021 and Wnt inhibitor IWP4. Differentiated cardiomyocytes were subjected to calcium imaging, electrophysiology recording, Polymerase Chain Reaction (PCR) analysis, and immunostaining. Results: iPSCs were efficiently generated from human urine samples and further forced to differentiate into contracting cardiomyocytes. PCR analysis and immunostaining confirmed the expression of a panel of cardiac markers. Both normal and patient iPSC derived cardiomyocytes exhibited spontaneous and field stimulated calcium transients (up to 2Hz), as well as action potentials with ventricular-like and nodal-like characteristics. Anti-dystrophin antibodies stained normal iPSC-derived cardiomyocyte membranes but did not react against DMD iPSC-derived cardiomyocytes. Conclusions: Cardiomyocytes can be efficiently generated from human urine, through the cellular reprogramming technology. DMD cardiomyocytes retained the patient’s genetic information and manifested a dystrophin-null phenotype. Functional assessments are underway to determine differences that may exist between genotypes.

scholarly journals Production of Phytotoxic Metabolite Using Biphasic Fermentation System from Strain C1136 of Lasiodiplodia pseudotheobromae, a Potential Bioherbicidal Agent

2017 ◽  
Vol 9 (3) ◽  
pp. 371-377
Author(s):  
Charles Oluwaseun ADETUNJI ◽  
Julius Kola OLOKE ◽  
Gandham PRASAD ◽  
Moses ABALAKA ◽  
Emenike Onyebum IROKANULO
Keyword(s):  
Large Scale ◽  
Wild Strain ◽  
Fermentation Medium ◽  
Scale Production ◽  
Conventional Farming ◽  
Large Scale Production ◽  
Phytotoxic Metabolite ◽  
Biphasic Fermentation

Formulation of effective and environmental friendly bioherbicides depends on the type of fermentation medium used for the production of phytotoxic metabolites. The effect of biomass, colony forming unit and the phytotoxic metabolite produced from the biphasic fermentation was carried out, while the phytotoxic metabolite was  tested in vivo and in-vitro on Echinochola crus-galli and dicotyledonous Chromolaena odorata. The mutant strain of Lasiodiplodia pseudotheobromae C1136 (Lp90) produced the highest amount of conidia and the largest necrotic area on the two tested weeds when compared to its wild strain in the different biphasic media combinations. The study revealed that the biphasic system containing PDB + rice produced the highest bioherbicidal activities. Therefore, the phytotoxic metabolites from strain C1136 are suggested for large scale production of bioherbicides for the management of weeds in conventional farming to improve yield and enhance food security.

MITOCHONDRIAL FUNCTION AND TISSUE VIABILITY IN VIVO: FROM ANIMAL EXPERIMENTS TO CLINICAL APPLICATIONS. FORTY YEARS OF FRUITFUL COLLABORATION WITH BRITTON CHANCE

2011 ◽  
Vol 04 (04) ◽  
pp. 337-359 ◽  
Author(s):  
AVRAHAM MAYEVSKY
Keyword(s):  
Mitochondrial Function ◽  
Large Scale ◽  
Technology Development ◽  
Animal Experiments ◽  
Clinical Applications ◽  
Experimental Systems ◽  
Britton Chance ◽  
Major Interest

The involvement of mitochondrial dysfunction in many pathophysiological conditions and human diseases is well documented. In order to evaluate mitochondrial function in vitro, many experimental systems have been developed. Nevertheless the number of in vivo monitoring systems for the evaluation of mitochondrial activities in intact animals and patients is relatively limited. The pioneering development of the conceptual and technological aspects of mitochondrial monitoring, in vitro and in vivo, was done by the late Prof. Britton Chance (July 24, 1913–November 16, 2010) since the early 1950s. It was my privilege to join his laboratory in 1972 and collaborate with him for almost four decades. The main achievements of our collaboration are presented in this paper. Our activities included cycles of technology development, followed by its applications to study various pathophysiological conditions. In the initial stage, the first fiber-optic–based NADH fluorometer was developed. This device enabled us to monitor various organs in anesthetized animals as well as the brain of nonanesthetized small animals. Later on, the addition of various physiological parameters to NADH monitoring enabled us to correlate mitochondrial function with other cellular functions. The application of the developed technology to clinical situations was a major interest of Prof. Chance and indeed this goal was achieved in the last decade. As of today, the basic tool for NADH monitoring and the large database of results are available for large-scale experimental and clinical applications.

scholarly journals Evaluation of the Skin Sensitization Potential of Carbon Nanotubes Using Alternative In Vitro and In Vivo Assays

Toxics ◽  
2020 ◽  
Vol 8 (4) ◽  
pp. 122
Author(s):  
Sung-Hyun Kim ◽  
Dong Han Lee ◽  
Jin Hee Lee ◽  
Jun-Young Yang ◽  
Hyo-Sook Shin ◽  
...  
Keyword(s):  
Carbon Nanotubes ◽  
Large Scale ◽  
Test Method ◽  
Single Wall Carbon Nanotubes ◽  
Scale Production ◽  
Skin Sensitization ◽  
Alternative Test ◽  
Alternative Test Method

Carbon nanotubes (CNTs) are one of the major types of nanomaterials that have various industrial and biomedical applications. However, there is a risk of accidental exposure to CNTs in individuals involved in their large-scale production and in individuals who use products containing CNTs. This study aimed to evaluate the skin sensitization induced by CNTs using two alternative tests. We selected single-wall carbon nanotubes and multi-walled carbon nanotubes for this study. First, the physiochemical properties of the CNTs were measured, including the morphology, size, and zeta potential, under various conditions. Thereafter, we assessed the sensitization potential of the CNTs using the ARE-Nrf2 Luciferase KeratinoSens™ assay, an in vitro alternative test method. In addition, the CNTs were evaluated for their skin sensitization potential using the LLNA: BrdU-FCM in vivo alternative test method. In this study, we report for the first time the sensitization results of CNTs using the KeratinoSens™ and LLNA: BrdU-FCM test methods in this study. This study found that both CNTs do not induce skin sensitization. These results suggest that the KeratinoSens™ and LLNA: BrdU-FCM assay may be useful as alternative assays for evaluating the potential of some nanomaterials that can induce skin sensitization.

scholarly journals Biochemical assessment of in vitro and in vivo culture of Tylophora indica (Burm. F.) Merr

1970 ◽  
Vol 6 (2) ◽  
pp. 1-5
Author(s):  
Antaryami Kaushik ◽  
Chandra Gurnani ◽  
Shyam Sunder ◽  
Abha Dhingra ◽  
Vikram Chimpa
Keyword(s):  
Large Scale ◽  
Basal Medium ◽  
Nodal Segments ◽  
Scale Production ◽  
Tylophora Indica ◽  
Shoot Initiation ◽  
Large Scale Production ◽  
Chlorophyll Protein

Tylophora indica (Burm. F.) Merr is an endangered plant which can be protected from extinction by its large scale production. Nodal segments of healthy plants are used as explants and cultured on MS Basal medium fortified with different growth regulators. Optimum shoot induction conditions from explants were established. In vitro and in vivo phytochemical test were done by using standard methods for chlorophyll, carbohydrates, proteins, lipids and starch. 3mg/l 2, 4 D showed maximum and success full callus production. Shoot initiation started in 7 days and best shoot regeneration reported with 3 mg/ml BAP in Basal medium. Combination of IBA and NAA in concentration 2 and 4 mg/l respectively proved to be best for root initiation. Concentration of chlorophyll, protein, lipid, carbohydrate, and starch in vitro and in vivo culture are investigated. DOI: 10.3126/kuset.v6i2.4005Kathmandu University Journal of Science, Engineering and Technology Vol.6. No II, November, 2010, pp.1-5

scholarly journals HIF-1α and Pro-Inflammatory Signaling Improves the Immunomodulatory Activity of MSC-Derived Extracellular Vesicles

2021 ◽  
Vol 22 (7) ◽  
pp. 3416
Author(s):  
Marta Gómez-Ferrer ◽  
Estela Villanueva-Badenas ◽  
Rafael Sánchez-Sánchez ◽  
Christian M. Sánchez-López ◽  
Maria Carmen Baquero ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Extracellular Vesicles ◽  
Large Scale ◽  
Lentiviral Vector ◽  
Delayed Type Hypersensitivity ◽  
Cell Phenotype ◽  
Immunomodulatory Activity ◽  
Scale Production ◽  
Activated T Cells

Despite the strong evidence for the immunomodulatory activity of mesenchymal stromal cells (MSCs), clinical trials have so far failed to clearly show benefit, likely reflecting methodological shortcomings and lack of standardization. MSC-mediated tissue repair is commonly believed to occur in a paracrine manner, and it has been stated that extracellular vesicles (EVs) secreted by MSCs (EVMSC) are able to recapitulate the immunosuppressive properties of parental cells. As a next step, clinical trials to corroborate preclinical studies should be performed. However, effective dose in large mammals, including humans, is quite high and EVs industrial production is hindered by the proliferative senescence that affects MSCs during massive cell expansion. We generated a genetically modified MSC cell line overexpressing hypoxia-inducible factor 1-alpha and telomerase to increase the therapeutic potency of EVMSC and facilitate their large-scale production. We also developed a cytokine-based preconditioning culture medium to prime the immunomodulatory response of secreted EVs (EVMSC-T-HIFc). We tested the efficacy of this system in vitro and in a delayed-type hypersensitivity mouse model. MSC-T with an HIF-1α-GFP lentiviral vector (MSC-T-HIF) can be effectively expanded to obtain large amounts of EVs without major changes in cell phenotype and EVs composition. EVMSC-T-HIFc suppressed the proliferation of activated T-cells more effectively than did EVs from unmodified MSC in vitro, and significantly blunted the ear-swelling response in vivo by inhibiting cell infiltration and improving tissue integrity. We have developed a long-lived EV source that secretes high quantities of immunosuppressive EVs, facilitating a more standard and cost-effective therapeutic product.

scholarly journals Cigarette Mainstream Smoke: The Evolution of Methods and Devices for Generation, Exposure and Collection

2016 ◽  
Vol 27 (4) ◽  
pp. 137-274 ◽  
Author(s):  
Hubert Klus ◽  
Barbara Boenke-Nimphius ◽  
Lutz Müller
Keyword(s):  
Large Scale ◽  
Toxicity Testing ◽  
Scale Production ◽  
Mainstream Smoke ◽  
Technical Problems ◽  
Test Pieces ◽  
Artifact Formation ◽  
Cigarette Mainstream Smoke

SUMMARYThe objective of this review is to support tobacco scientists when evaluating information published on smoking machines, and on cigarette mainstream smoke (in vivoandin vitro) exposure systems and collection devices.The intriguing development of smoking machines (mainly for cigarettes) is followed for more than 170 years - from the first simple set-ups in the 1840s to the sophisticated and fully automated analytical smoking machines available today. Systems for the large-scale production of smoke (condensate) for preparative work are equally considered. The standardization of machine smoking methods and test pieces has solved several technical problems and produced sensible rules but, at the same time, given rise to new controversies like the compatibility of artificial and human smoking, and the implementation of more intense machine smoking regimes.Adequate space is allotted for the discussion of configurations forin vivosmoke exposure of rodent and non-rodent species and the machines generating the required smoke (condensate). Covered as well is the field ofin vitrotoxicity testing, including the increasingly informative new techniques of air-liquid interface exposure, which are becoming more and more refined with the use of organotypic cultures and genetic analyses.The review is completed by the examination of the considerable variety of mainstream smoke collection devices (filters and traps) developed over time - some for very specific purposes - and refers to the perpetual problem of artifact formation by aging.

scholarly journals Novel Strategy for Generation and Titration of Recombinant Adeno-Associated Virus Vectors

2005 ◽  
Vol 79 (1) ◽  
pp. 193-201 ◽  
Author(s):  
Ai-Li Shiau ◽  
Pu-Ste Liu ◽  
Chao-Liang Wu
Keyword(s):  
Large Scale ◽  
Pseudorabies Virus ◽  
High Titer ◽  
Helper Virus ◽  
Adeno Associated Virus ◽  
Prothymosin Α ◽  
Vector Systems ◽  
Novel Strategy

ABSTRACT Recombinant adeno-associated virus (rAAV) vectors have many advantages for gene therapeutic applications compared with other vector systems. Several methods that use plasmids or helper viruses have been reported for the generation of rAAV vectors. Unfortunately, the preparation of large-scale rAAV stocks is labor-intensive. Moreover, the biological titration of rAAV is still difficult, which may limit its preclinical and clinical applications. For this study, we developed a novel strategy to generate and biologically titrate rAAV vectors. A recombinant pseudorabies virus (PrV) with defects in its gD, gE, and thymidine kinase genes was engineered to express the AAV rep and cap genes, yielding PS virus, which served as a packaging and helper virus for the generation of rAAV vectors. PS virus was useful not only for generating high-titer rAAV vectors by cotransfection with an rAAV vector plasmid, but also for amplifying rAAV stocks. Notably, the biological titration of rAAV vectors was also feasible when cells were coinfected with rAAV and PS virus. Based on this strategy, we produced an rAAV that expresses prothymosin α (ProT). Expression of the ProT protein in vitro and in vivo mediated by rAAV/ProT gene transfer was detected by immunohistochemistry and a bioassay. Taken together, our results demonstrate that the PrV vector-based system is useful for generating rAAV vectors carrying various transgenes.

High-yield amplification ofCryptosporidium parvumin interferon γ receptor knockout mice

Parasitology ◽  
2008 ◽  
Vol 135 (10) ◽  
pp. 1151-1156 ◽  
Author(s):  
J. von OETTINGEN ◽  
M. NATH-CHOWDHURY ◽  
B. J. WARD ◽  
A. C. RODLOFF ◽  
M. J. ARROWOOD ◽  
...  
Keyword(s):  
Large Scale ◽  
Interferon Γ ◽  
High Yield ◽  
Scale Production ◽  
Large Scale Production ◽  
Ether Extraction ◽  
External Sources ◽  
And Storage

SUMMARYTo date, large-scale production ofCryptosporidium parvumoocysts has only been achieved by amplification in neonatal calves and sheep. Many laboratories currently depend on supplies from external sources and store oocysts for prolonged periods which results in progressive loss of viability. Six to 8-week-old interferon γ receptor knockout (IFNγR-KO) mice on a C57BL/6 background were inoculated by gavage (2000 oocysts/animal). Fecal pellets were collected daily from 7 days post-infection (p.i.) up to 2 weeks p.i. Intestinal oocyst yield was assessed at days 11, 12 and 14 p.i. by homogenization of intestinal tissues. Ether extraction and one or more NaCl flotations were used to purify oocysts. Total recoveries averaged 2·6×106oocysts/mouse from fecal material and 3·8×107oocysts/mouse from intestinal tissues. Overall, 2·3×109purified oocysts were obtained from 60 mice. Recovered oocysts were capable of sporulation and were shown to be infectious bothin vitroandin vivo. Oocyst amplification was achieved in only 11–14 days with minimal expense. The simplicity of this method presents a practical alternative for the routine passage, maintenance and storage ofC. parvumin biomedical laboratories.

Fluorescent carbon dots as prospective nanoagents for imaging-assisted biomedical applications

2021 ◽  
Vol 28 ◽  
Author(s):  
Le Minh Tu Phan
Keyword(s):  
Carbon Dots ◽  
Biomedical Applications ◽  
Large Scale ◽  
Low Cost ◽  
Scale Production ◽  
Large Scale Production ◽  
Intense Exploitation ◽  
Fluorescent Carbon Dots

: Carbon dots (CDs), an emerging nanoagent providing an alternative to conventional fluorescent agents, are sparking the scientist’s interest in biomedical applications owing to their unique advantages, including ease of synthesis, large scale production, low cost, prominent photoluminescence, good photostability, easy functionalization, sufficient biocompatibility, good nanocarrier, and excellent ability to generate reactive oxygen species or heat. Herein, this perspective provides a viewpoint about imaging-assisted biomedical applications using fluorescent CDs regarding in vitro and in vivo bioimaging, imaging-assisted sensing, and imaging-guided therapy. The opinions about their potential and challenges in applicable biomedical applications are discussed to develop, further ameliorated CDs for their intense exploitation in diverse imaging-assisted biomedical applications.

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