journal
MENU ▼
Read by QxMD icon Read
search

Human Gene Therapy Methods

journal
https://www.readbyqxmd.com/read/30280937/cryopreservation-of-human-adipose-derived-stem-cells-for-use-in-ex-vivo-regional-gene-therapy-for-bone-repair
#1
Venus Vakhshori, Sofia Bougioukli, Osamu Sugiyama, Amy Tang, Robert Yoho, Jay R Lieberman
The development of an ex vivo regional gene therapy clinical pathway using adipose-derived stem cells (ASCs) may require cryopreservation for cell culture, storage, and transport prior to clinical use. ASCs isolated from five donors were transduced with a lentiviral vector containing BMP-2. Three groups were assessed: transduction without cell freezing (group 1), freezing of cells for 3 weeks followed by transduction (group 2), and cell transduction prior to freezing (group 3). Nontransduced cells were used as a control...
October 25, 2018: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/30351228/development-of-a-chemiluminescent-elisa-method-for-the-detection-of-total-anti-adeno-associated-virus-serotype-9-aav9-antibodies
#2
Uma Kavita, Yanshan Dai, Lisa Salvador, Wendy Miller, Leonard P Adam, Paul C Levesque, Yan J Zhang, Qin C Ji, Renuka C Pillutla
Recombinant adeno associated viruses (rAAV) have become an important tool for the delivery of gene therapeutics due to long-standing safety and success in clinical trials. Since humans often become exposed to AAVs and develop anti-AAV antibodies (Abs), a potential impediment to the success of gene therapeutics is neutralization of the viral particle before it has had a chance to bind and enter target cells to release the transgene. Identification of subjects with pre-existing Abs having neutralizing potential, and exclusion of such subjects from clinical studies is expected to enhance drug efficacy...
October 23, 2018: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/30079761/the-functional-effect-of-repeated-cryopreservation-on-transduced-cd34-cells-from-patients-with-thalassemia
#3
Garyfalia Karponi, Penelope-Georgia Papayanni, Fani Zervou, Asimina Bouinta, Achilles Anagnostopoulos, Evangelia Yannaki
Stable gene marking and effective engraftment of gene-modified CD34+ hematopoietic stem cells is a prerequisite for gene therapy success but may be challenged by the inevitable cryopreservation of the final product prior to extensive quality assurance testing. We investigated the β-globin gene transfer potency in fresh and cryopreserved CD34+ cells from mobilized patients with β-thalassemia, as well as the qualitative impact of repeated freeze/thaw cycles on the functionality of cultured and unmanipulated CD34+ cells in terms of engrafting capacity in a xenotransplantation model, under partial myeloablation...
October 2018: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/30051733/accurate-and-rapid-sequence-analysis-of-adeno-associated-virus-plasmids-by-illumina-next-generation-sequencing
#4
Alexei Saveliev, Juan Liu, Mingyao Li, Lee Hirata, Caitlin Latshaw, Jia Zhang, James M Wilson
Sequence validation of plasmid DNA is a crucial quality control step that must occur prior to adeno-associated virus (AAV) vector packaging through plasmid transfection. AAV cis-plasmids present unique challenges to sequence analysis, as they contain inverted terminal repeats and are prone to sequence rearrangements. An accurate and rapid next-generation sequencing approach has been established to analyze full-length sequences of AAV cis-plasmids within 3.5 days. Here, a step-by-step protocol is described that can reliably detect and identify the location and frequency of sequence variants commonly observed in AAV cis-plasmids...
October 2018: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/30032644/standardized-method-for-intra-cisterna-magna-delivery-under-fluoroscopic-guidance-in-nonhuman-primates
#5
Nathan Katz, Tamara Goode, Christian Hinderer, Juliette Hordeaux, James M Wilson
Intrathecal delivery of adeno-associated virus vectors and other therapeutics are currently being evaluated for the treatment of central nervous system sequelae of lysosomal storage diseases, motor neuron diseases, and neurodegenerative diseases. As products transition from preclinical to clinical studies, a standardized and clinically relevant method of intrathecal delivery is increasingly germane. Here, we describe a method of intrathecal delivery via suboccipital puncture into the cisterna magna under fluoroscopic guidance in nonhuman primates...
October 2018: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/29993287/lobe-specific-gene-vector-delivery-to-rat-lungs-using-a-miniature-bronchoscope
#6
Chantelle McIntyre, Martin Donnelley, Nathan Rout-Pitt, David Parsons
For respiratory research utilizing gene vector delivery to the lung, the size of rodent models has typically necessitated relatively "blind" dosing via the nose, via an endotracheal tube, or through a surgical incision into the trachea. This commonly results in a limited ability to dose specific small regions of the lung reliably, and contributes to high levels of transduction variability between animals. The resultant poor reliability, reproducibility, and high variability compromises statistical capability, and so demands greater animal sample sizes than should be feasible...
October 2018: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/30064266/systemic-gene-delivery-by-single-dose-intracardiac-administration-of-scaav2-9-and-scaav2-rh10-variants-in-newborn-rats
#7
Lucie Chansel-Debordeaux, Mathieu Bourdenx, Nathalie Dutheil, Sandra Dovero, Marie-Helene Canron, Clement Jimenez, Erwan Bezard, Benjamin Dehay
Recombinant adeno-associated virus serotype 9 (rAAV2/9) and pseudotype rhesus-10 (rAAV2/rh10) are used for gene delivery, especially into the central nervous system. Both serotypes cross the blood-brain barrier and mediate stable long-term transduction in dividing and nondividing cells. Among possible routes of administration, intracardiac injection holds the potential for widespread vector diffusion associated with a relatively simple approach. In this study adopting the intracardiac route, we compare the cell-specific tropism and transfection efficacy of a panel of engineered rAAV2/9 and rAAV2/rh10 vectors encoding the enhanced green fluorescent protein...
August 2018: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/30032642/production-and-purification-of-supercoiled-minicircles-by-a-combination-of-in-vitro-endonuclease-nicking-and-hydrophobic-interaction-chromatography
#8
Cláudia P A Alves, Michaela Šimčíková, Liliana Brito, Gabriel A Monteiro, Duarte Miguel F Prazeres
A wider application of minicircle (MC) vectors in gene therapy research depends critically on the ability to purify supercoiled (sc) MC from related miniplasmid (MP) and parental plasmid (PP) impurities. This protocol describes a purification strategy that combines the in vitro enzymatic relaxation of sc MP and PP impurities by a nicking endonuclease, and topoisomer separation and RNA clearance by hydrophobic interaction chromatography. The time required to follow the full protocol, from production to isolation of sc MC, is approximately 50 h...
August 2018: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/29953259/delineating-the-cellular-mechanisms-associated-with-skin-electroporation
#9
Katherine Schultheis, Trevor R F Smith, William B Kiosses, Kimberly A Kraynyak, Amelia Wong, Janet Oh, Kate E Broderick
The immune responses elicited following delivery of DNA vaccines to the skin has previously been shown to be significantly enhanced by the addition of electroporation (EP) to the treatment protocol. Principally, EP increases the transfection of plasmid DNA (pDNA) into the resident skin cells. In addition to increasing the levels of in vivo transfection, the physical insult induced by EP is associated with activation of innate pathways which are believed to mediate an adjuvant effect, further enhancing DNA vaccine responses...
August 2018: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/29953257/targeted-delivery-and-tolerability-of-mri-guided-ced-infusion-into-the-cerebellum-of-nonhuman-primates
#10
Ernesto A Salegio, Michael V Campagna, Philip C Allen, Diane E Stockinger, Yuanquan Song, Granger G C Hwa
This study explored the feasibility of intraparenchymal delivery (gadoteridol and/or Serotype 5 Adeno-Associated Viral Vector-enhanced Green Fluorescent Protein [AAV5-eGFP]) into the cerebellum of nonhuman primates using real-time magnetic resonance imaging-guided convection enhanced delivery (MRI-CED) technology. All animals tolerated the neurosurgical procedure without any clinical sequela. Gene expression was detected within the cerebellar parenchyma at the site of infusion and resulted in transduction of neuronal cell bodies and fibers...
August 2018: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/29860898/efficiency-and-specificity-of-targeted-integration-mediated-by-the-adeno-associated-virus-serotype-2-rep-78-protein
#11
Pingjuan Li, Michael P Marino, Jizhong Zou, Takele Argaw, Michael T Morreale, Brian J Iaffaldano, Jakob Reiser
The adeno-associated virus serotype 2 (AAV2) Rep 78 protein, a strand-specific endonuclease (nickase) promotes site-specific integration of transgene sequences bearing homology arms corresponding to the AAVS1 safe harbor locus. To investigate the efficiency and specificity of this approach, plasmid-based donor vectors were tested in concert with nuclease encoding vectors, including an engineered version of the AAV2 Rep 78 protein, an AAVS1-specific zinc finger nuclease (ZFN), and the CRISPR-Cas9 components in HEK 293 cells...
June 2018: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/29848071/preparation-of-nonhuman-primate-eyes-for-histological-evaluation-after-retinal-gene-transfer
#12
Peter Bell, Hongwei Yu, Leah Kuntz, Omua Ahonkhai, Anna Tretiakova, Maria P Limberis, James M Wilson
To evaluate gene therapy for retinal disorders, appropriate models of the human eye are needed. Nonhuman primate eyes offer significant advantages over rodent eyes. However, current preparation methods have limitations. Here, a protocol is described for histological processing of nonhuman primate eyes after gene transfer. The user dissects unfixed eyes, flattens the globe parts within filter paper, and performs formalin fixation and paraffin embedding. This method obviates the need for harsh fixatives, allowing subsequent immunostaining or in situ hybridization while preserving tissue integrity for histopathological evaluation...
June 2018: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/29756505/novel-vector-construction-based-on-alternative-adenovirus-types-via-homologous-recombination
#13
Wenli Zhang, Jun Fu, Anja Ehrhardt
Adenoviral vector (AdV) is one of the most used vectors in gene therapy clinical trials. However the therapeutic effect of AdV is limited due to preexisting immunity to the currently used human adenovirus type 5 and pre-decided vector tropism. It is highly demanded to develop novel AdVs originated from other types than adenovirus type 5. Here, we describe a method for direct cloning of adenovirus utilizing linear-linear homologous recombination, followed by rapid adenoviral genome modification via linear-circular homologous recombination...
June 2018: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/29706115/in-vivo-potency-assay-for-adeno-associated-virus-based-gene-therapy-vectors-using-aavrh-10-as-an-example
#14
Bishnu P De, Alvin Chen, Christiana O Salami, Benjamin Van de Graaf, Jonathan B Rosenberg, Odelya E Pagovich, Dolan Sondhi, Ronald G Crystal, Stephen M Kaminsky
The development of a drug product requires rigorous methods of characterization and quality control to assure drug potency. Gene therapy products, a relatively new strategy for drug design with very few licensed examples, represent a unique challenge for the measure of potency. Unlike traditional drugs, potency for a gene therapeutic is a tally of the measures of multiple steps, including infectivity, transcription, translation, protein modifications, proper localization of the protein product, and protein function...
June 2018: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/29631443/assessment-of-humoral-innate-and-t-cell-immune-responses-to-adeno-associated-virus-vectors
#15
Roberto Calcedo, Jessica A Chichester, James M Wilson
Adeno-associated virus (AAV)-based gene therapy is being applied to treat a wide array of diseases. Pre-existing host immune responses to AAV and immune responses elicited by AAV vector administration remain a problem that needs to be further studied. Here, we present a series of protocols to assess immune responses before and after AAV vector administration that is applicable to multiple animal models and Phase I clinical trials. More specifically, to evaluate: 1) the humoral immune response, through levels of AAV-neutralizing and binding antibodies; 2) the innate immune response, through the acute induction of inflammatory cytokines; and 3) the T-cell immune response, through the activation of transgene- and vector-specific CD8 and CD4 T cells...
April 10, 2018: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/29668327/assessment-of-humoral-innate-and-t-cell-immune-responses-to-adeno-associated-virus-vectors
#16
Roberto Calcedo, Jessica A Chichester, James M Wilson
Adeno-associated virus (AAV)-based gene therapy is being applied to treat a wide array of diseases. Preexisting host immune responses to AAV and immune responses elicited by AAV vector administration remain a problem that needs to be further studied. Here we present a series of protocols to assess immune responses before and after AAV vector administration that are applicable to multiple animal models and phase 1 clinical trials. More specifically, they may be use to evaluate (1) the humoral immune response, through levels of AAV-neutralizing and binding antibodies; (2) the innate immune response, through the acute induction of inflammatory cytokines; and (3) the T-cell immune response, through the activation of transgene- and vector-specific CD8+ and CD4+ T cells...
April 2018: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/29631437/improving-lentiviral-transduction-of-cd34-hematopoietic-stem-and-progenitor-cells
#17
Ilona Hauber, Niklas Beschorner, Silke Schrödel, Jan Chemnitz, Nicolaus Kröger, Joachim Hauber, Christian Thirion
The delivery of therapeutic genes for treatment of inherited or infectious diseases frequently requires lentiviral transduction of CD34+ hematopoietic stem and progenitor cells (HSC). Optimized transduction protocols with a therapeutic goal aim to maximize the number of transduction-positive cells while limiting the vector copy number that reach each individual cell. Importantly, the transduced HSC should maintain their "stem-like" properties. Here, we analyzed LentiBOOST™ reagent, a membrane-sealing poloxamer, with respect to enhancing lentiviral transduction of CD34+ peripheral blood stem cells...
April 2018: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/29596011/slow-infusion-of-recombinant-adeno-associated-viruses-into-the-mouse-cerebrospinal-fluid-space
#18
Dan Wang, Jia Li, Karen Tran, Daniel R Burt, Li Zhong, Guangping Gao
Recombinant adeno-associated viruses (rAAVs) are the leading in vivo gene delivery platform, and have been extensively studied in gene therapy targeting various tissues, including the central nervous system (CNS). A single-bolus rAAV injection to the cerebrospinal fluid (CSF) space has been widely used to target the CNS, but it suffers from several drawbacks, such as leakage to peripheral tissues. Here, a protocol is described using an osmotic pump to infuse rAAV slowly into the mouse CSF space. Compared to the single-bolus injection technique, pump infusion can lead to higher CNS transduction and lower transduction in the peripheral tissues...
April 2018: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/29378428/determination-of-lentiviral-infectious-titer-by-a-novel-droplet-digital-pcr-method
#19
Yu Wang, Svetlana Bergelson, Marina Feschenko
Lentivirus is one of the best vehicles in delivering exogenous genes for therapeutics. Prior to application, it is very important to determine the infectious titer, which measures only mature virus capable of infecting target cells. Quantitative polymerase chain reaction (PCR) and fluorescence-activated cell sorting are commonly used for determination of infectious titer. This study introduces a new method based on Droplet Digital PCR (ddPCR), a recently developed PCR technology that quantifies the absolute amount of target DNA in the reaction...
April 2018: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/29325442/pooled-generation-of-lentiviral-tetracycline-regulated-microrna-embedded-short-hairpin-rna-libraries
#20
Felix F Adams, Thomas Hoffmann, Johannes Zuber, Dirk Heckl, Axel Schambach, Adrian Schwarzer
Short hairpin RNA (shRNA) screens are powerful tools to probe genetic dependencies in loss-of-function studies, such as the identification of therapeutic targets in cancer research. Lentivirally delivered shRNAs embedded in endogenous microRNA contexts (shRNAmiRs) mediate efficient long-term suppression of target genes suitable for numerous experimental contexts and clinical applications. Here, an easy-to-use laboratory protocol is described, covering the design and pooled assembly of focused shRNAmiR libraries into an optimized, Tet-inducible all-in-one lentiviral vector, packaging of viral particles, followed by retrieval and quantification of hairpin sequences after cellular DNA-recovery...
February 2018: Human Gene Therapy Methods
journal
journal
43838
1
2
Fetch more papers »
Fetching more papers... Fetching...
Read by QxMD. Sign in or create an account to discover new knowledge that matter to you.
Remove bar
Read by QxMD icon Read
×

Search Tips

Use Boolean operators: AND/OR

diabetic AND foot
diabetes OR diabetic

Exclude a word using the 'minus' sign

Virchow -triad

Use Parentheses

water AND (cup OR glass)

Add an asterisk (*) at end of a word to include word stems

Neuro* will search for Neurology, Neuroscientist, Neurological, and so on

Use quotes to search for an exact phrase

"primary prevention of cancer"
(heart or cardiac or cardio*) AND arrest -"American Heart Association"