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Human Gene Therapy Methods

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https://www.readbyqxmd.com/read/29993287/lobe-specific-gene-vector-delivery-to-rat-lungs-using-a-miniature-bronchoscope
#1
Chantelle McIntyre, Martin Donnelley, Nathan Rout-Pitt, David Parsons
For respiratory research utilising 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 reliably dose specific small regions of lung, 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...
July 11, 2018: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/29953259/delineating-the-cell-death-mechanisms-associated-with-skin-electroporation
#2
Katherine Schultheis, Trevor R F Smith, William B Kiosses, Kimberly A Kraynyak, Amelia Wong, Janet Oh, Kate Elizabeth 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 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...
June 28, 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
#3
Ernesto 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 AAV5-GFP) into the cerebellum of nonhuman primates using real-time MRI-guided 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. Histopathology indicated localized damage along the stem of the cannula tract. These findings demonstrate the potential of real-time MRI-guided CED to deliver therapeutics into the cerebellum, which has extensive reciprocal connections and may be used as a target for the treatment of neurological disorders...
June 28, 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
#4
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
#5
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
#6
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
#7
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
#8
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
#9
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
#10
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
#11
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
#12
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
#13
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
https://www.readbyqxmd.com/read/29325430/rapid-and-sensitive-assessment-of-globin-chains-for-gene-and-cell-therapy-of-hemoglobinopathies
#14
Constantinos C Loucari, Petros Patsali, Thamar B van Dijk, Coralea Stephanou, Panayiota Papasavva, Maria Zanti, Ryo Kurita, Yukio Nakamura, Soteroulla Christou, Maria Sitarou, Sjaak Philipsen, Carsten W Lederer, Marina Kleanthous
The β-hemoglobinopathies sickle cell anemia and β-thalassemia are the focus of many gene-therapy studies. A key disease parameter is the abundance of globin chains because it indicates the level of anemia, likely toxicity of excess or aberrant globins, and therapeutic potential of induced or exogenous β-like globins. Reversed-phase high-performance liquid chromatography (HPLC) allows versatile and inexpensive globin quantification, but commonly applied protocols suffer from long run times, high sample requirements, or inability to separate murine from human β-globin chains...
February 2018: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/29281903/assessment-of-fgfr1-over-expression-and-over-activity-in-lung-cancer-cells-a-toolkit-for-anti-fgfr1-drug-screening
#15
Penghui Hu, Hongjie Chen, Eileen M McGowan, Nina Ren, Meng Xu, Yiguang Lin
Lung cancer, caused mainly by smoking, is one of the most prevalent diseases in China, resulting in high mortality rates. The increasing incidence of chronic disease due to lung cancer places a huge burden on the welfare and cost to the Chinese society. Amplification of the fibroblast growth factor receptor 1 (FGFR1) is associated with high incidence and mortality in lung cancer patients. FGFR1 signaling is implicated in oncogenic traits such as proliferation, cell survival, angiogenesis, and migration. Targeting FGFR1 and its ligand basic FGF (bFGF) is a key step forward in developing new therapies for this crippling disease...
February 2018: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/29212357/manufacture-of-third-generation-lentivirus-for-preclinical-use-with-process-development-considerations-for-translation-to-good-manufacturing-practice
#16
Carolina Gándara, Valerie Affleck, Elizabeth Ann Stoll
Lentiviral vectors are used in laboratories around the world for in vivo and ex vivo delivery of gene therapies, and increasingly clinical investigation as well as preclinical applications. The third-generation lentiviral vector system has many advantages, including high packaging capacity, stable gene expression in both dividing and post-mitotic cells, and low immunogenicity in the recipient organism. Yet, the manufacture of these vectors is challenging, especially at high titers required for direct use in vivo, and further challenges are presented by the process of translating preclinical gene therapies toward manufacture of products for clinical investigation...
February 2018: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/29160102/improving-the-transduction-of-bone-marrow-derived-cells-with-an-integrase-defective-lentiviral-vector
#17
S Louise Pay, Xiaoping Qi, Jeffrey F Willard, Juliana Godoy, Kavya Sankhavaram, Ranier Horton, Sayak K Mitter, Judith L Quigley, Lung-Ji Chang, Maria B Grant, Michael E Boulton
In lentiviral vector (LV) applications where transient transgene expression is sufficient, integrase-defective lentiviral vectors (IDLVs) are beneficial for reducing the potential for off-target effects associated with insertional mutagenesis. It was previously demonstrated that human RPE65 mRNA expression from an integrating lentiviral vector (ILV) induces endogenous Rpe65 and Cralbp mRNA expression in murine bone marrow-derived cells (BMDCs), initiating programming of the cells to retinal pigment epithelium (RPE)-like cells...
February 2018: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/29160106/display-of-the-albumin-binding-domain-in-the-envelope-improves-lentiviral-vector-bioavailability
#18
Guillermo Garaulet, Juan José Lazcano, Hernán Alarcón, Sergio de Frutos, Jorge Luis Martínez-Torrecuadrada, Antonio Rodríguez
Vesicular stomatitis virus G glycoprotein (VSVg) is extensively used for retroviral and lentiviral vector (LV) pseudotyping. However, VSVg pseudotyped vectors are serum inactivated, blocking the in vivo gene delivery. Several strategies have been employed to prevent complement inactivation, including chemical and genetic envelope modifications. This study employed the streptococcal albumin-binding domain (ABD) to generate a construct to express ABD as a glycosylphosphatidylinositol-anchored protein. LV particles bearing ABD are able to bind bovine and human serum albumin in vitro...
December 2017: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/28870117/pseudotyped-lentiviral-vectors-one-vector-many-guises
#19
Alok V Joglekar, Salemiz Sandoval
Viruses have evolved specialized molecular mechanisms to transfer their genome efficiently into host cells. Viruses can be repurposed into viral vectors to achieve controlled gene transfer to desired cells. One of the most popular classes of vectors, lentiviral vectors (LVs), transduce mammalian cells efficiently. LVs are pseudotyped with various heterologous viral envelopes to alter their tropism. While the most common example is the envelope glycoprotein from vesicular stomatitis virus (VSVG), many other viral proteins have also been used...
December 2017: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/28854814/function-and-safety-of-lentivirus-mediated-gene-transfer-for-csf2ra-deficiency
#20
Miriam Hetzel, Takuji Suzuki, Anna Rafiei Hashtchin, Paritha Arumugam, Brenna Carey, Marc Schwabbauer, Alexandra Kuhn, Johann Meyer, Axel Schambach, Johannes Van Der Loo, Thomas Moritz, Bruce C Trapnell, Nico Lachmann
Hereditary pulmonary alveolar proteinosis (hPAP) is a rare disorder of pulmonary surfactant accumulation and hypoxemic respiratory failure caused by mutations in CSF2RA (encoding the granulocyte/macrophage colony-stimulating factor [GM-CSF] receptor α-chain [CD116]), which results in reduced GM-CSF-dependent pulmonary surfactant clearance by alveolar macrophages. While no pharmacologic therapy currently exists for hPAP, it was recently demonstrated that endotracheal instillation of wild-type or gene-corrected mononuclear phagocytes (pulmonary macrophage transplantation [PMT]) results in a significant and durable therapeutic efficacy in a validated murine model of hPAP...
December 2017: Human Gene Therapy Methods
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