scholarly journals Single-step cloning-screening method: a new tool for developing and studying high-titer viral vector producer cells

Gene Therapy ◽  
2015 ◽  
Vol 22 (9) ◽  
pp. 685-695 ◽  
Author(s):  
A F Rodrigues ◽  
A S Formas-Oliveira ◽  
M R Guerreiro ◽  
H A Tomás ◽  
P M Alves ◽  
...  
Keyword(s):  
Viral Vector ◽  
High Titer ◽  
Screening Method ◽  
Single Step

scholarly journals Fusogenics

2010 ◽  
Vol 16 (1) ◽  
pp. 90-100 ◽  
Author(s):  
Jeannick Cizeau ◽  
Marianne G. P. Torres ◽  
Sharla G. Cowling ◽  
Stacy Stibbard ◽  
Arjune Premsukh ◽  
...  
Keyword(s):  
Specific Antibody ◽  
Screening Method ◽  
Antibody Fragment ◽  
Vital Role ◽  
Single Step ◽  
Functional Assay ◽  
Plant Toxin ◽  
Promising Alternative ◽  
Pseudomonas Exotoxin A ◽  
Normal Tissues

Antibody-based therapeutics play a vital role in the treatment of certain cancers; however, despite commercial success, various strategies are being pursued to increase their potency and hence improve patient outcomes. The use of antibodies to deliver a cytotoxic payload offers a promising alternative for more efficacious therapies. Immunotoxins are composed of an internalizing antibody fragment linked to a bacterial or plant toxin. Once internalized, the payload, such as Pseudomonas exotoxin A (PE), blocks protein synthesis and induces apoptosis. Typically, immunotoxins are developed by first isolating a tumor-specific antibody, which is then either chemically linked to a toxin or reengineered as a fusion protein. Here, the authors describe the development of Fusogenics, an immunotoxin-based screening method that selects internalizing tumor-specific antibodies using a functional assay. Selected immune library clones were characterized and shown to be selective against normal tissues and specific to tumor tissues. In summary, the Fusogenics immunotoxin platform represents a unique, single-step selection approach combining specificity and functionality to isolate novel internalizing tumor-specific antibody fragments with potential for direct clinical application in the treatment of cancer.

scholarly journals Differences in Stability of Viral and Viral-Cellular Fusion Transcripts in HPV-Induced Cervical Cancers

2019 ◽  
Vol 21 (1) ◽  
pp. 112 ◽  
Author(s):  
Franziska Ehrig ◽  
Norman Häfner ◽  
Corina Driesch ◽  
Irene Kraus Christiansen ◽  
Katrin Beer ◽  
...  
Keyword(s):  
Viral Vector ◽  
High Titer ◽  
Rna Stability ◽  
Formal Proof ◽  
Core Motif ◽  
Hpv Dna ◽  
Fusion Transcripts ◽  
Significant Difference ◽  
The Stability ◽  
Cellular Fusion

HPV-DNA integration results in dysregulation of viral oncogene expression. Because viral-cellular fusion transcripts inherently lack the viral AU-rich elements of the 3’UTR, they are considered to be more stable than episome-derived transcripts. The aim of this study is to provide formal proof for this assumption by comparing the stability of viral early transcripts derived from episomal and integrated HPV16 DNA, respectively. Full-length cDNA of three fusion transcripts comprising viral and cellular sequences in sense orientation were amplified and cloned into the adeno-viral-vector pAd/CMV/V5-DEST. The most abundant HPV16 oncogene transcript E6*I-E7-E1vE4-E5 with and without 3’UTR, served as reference and control, respectively. Human primary keratinocytes were transduced using high titer virus stocks. qRT-PCR was performed to determine mRNA stability in relation to GAPDH in the presence of actinomycin-D. In four independent transduction experiments, all three viral-cellular fusion transcripts were significantly more stable compared to the episome-derived reference. Among the three viral-cellular fusion transcripts the most stable transcript was devoid of the instability core motif “AUUUA”. Unexpectedly, there was no significant difference in the stability between the episome-derived transcripts either with or without 3’UTR, indicating that the AU-rich elements of the 3’UTR are not contributing to RNA stability. Instead, the three “AUUUA” motifs located in the untranslated region between the viral E4 and E5 genes may be responsible for the instability. This is the first report showing that authentic viral-cellular fusion transcripts are more stable than episome-derived transcripts. The longer half-life of the fusion transcripts may result in increased levels of viral oncoproteins and thereby drive the carcinogenic process.

scholarly journals Single-Step Scalable-Throughput Molecular Screening for Huntington Disease

2008 ◽  
Vol 54 (6) ◽  
pp. 964-972 ◽  
Author(s):  
Clara R L Teo ◽  
Wen Wang ◽  
Hai Yang Law ◽  
Caroline G Lee ◽  
Samuel S Chong
Keyword(s):  
Huntington Disease ◽  
Trinucleotide Repeat ◽  
Neurodegenerative Disorder ◽  
False Positive Rate ◽  
Screening Method ◽  
Melting Curve ◽  
Single Step ◽  
Cag Repeat ◽  
Clinical Samples ◽  
Melting Curve Analysis

Abstract Background: Huntington disease (HD) is a fatal autosomal dominant neurodegenerative disorder caused by an unstable expansion of the CAG trinucleotide repeat in exon 1 of the HTT (huntingtin) gene and typically has an adult onset. Molecular diagnosis and screening for HD currently involve separate amplification and detection steps. Methods: We evaluated a novel, rapid microplate-based screening method for HD that combines the amplification and detection procedures in a single-step, closed-tube format. We carried out both the PCR for the HTT CAG-repeat region and the subsequent automated melting-curve analysis of the amplicon in the same wells on the plate. To establish cutoff melting temperatures (Tms) for each allelic class, we used a panel of reference DNA samples of known CAG-repeat sizes that represent a range of HTT alleles [normal (≤26 repeats), intermediate (27–35 repeats), reduced penetrance expanded (36–39 repeats), and fully penetrant expanded (≥40 repeats)]. We also measured well-to-well variation in Tm across the thermal block and validated cutoff Tms with DNA samples from 5 different populations. We also conducted a blinded validation analysis of clinical samples from an additional 40 HD-affected and 30 unaffected individuals. Results: We observed a strong correlation between CAG-repeat size and amplicon Tm among the reference DNA samples. Use of the Tm cutoffs we established revealed that 5 samples from unaffected individuals had been misclassified as affected (1.1% false-positive rate). All samples from HD-affected and unaffected individuals were correctly identified in the blinded analysis. Conclusions: This simple and scalable homogeneous assay may serve as a convenient, rapid, and accurate screen to detect the presence of pathologic expanded HD alleles in symptomatic patients.

Synthesis and Characterization of Novel Crystalline Mesoporous Beta-Tricalcium Phosphate Nanoparticles

2018 ◽  
Vol 930 ◽  
pp. 63-66
Author(s):  
Flávia Rodrigues de Oliveira Silva ◽  
Walter Kenji Yoshito ◽  
Ivana Conte Cosentino ◽  
Ana Helena Almeida Bressiani ◽  
Nelson Batista de Lima
Keyword(s):  
Tricalcium Phosphate ◽  
Room Temperature ◽  
Viral Vector ◽  
High Specific Surface Area ◽  
Single Step ◽  
Precipitation Method ◽  
The Novel ◽  
Beta Tricalcium Phosphate ◽  
20 Nm

A nanosized magnesium substituted beta-tricalcium phosphate (Mg:β-TCP) was synthesized by an aqueous precipitation method, at room temperature, in one single step. In the present study, the novel and stable Mg:β-TCP resulted in a crystalline and spherical nanoparticles (diameter of approximately 20 nm) with mesoporous structures and a high specific surface area (about 574 m2/g). These special characteristics make this novel crystalline mesoporous Mg: β-TCP nanoparticles ideal candidates for drug delivery system and a promising non-viral vector for gene therapy.

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