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Human Gene Therapy Methods

Anita Kruzik, Herwig Koppensteiner, Damir Fetahagic, Bettina Hartlieb, Sebastian Dorn, Stefan Romeder-Finger, Sogué Coulibaly, Alfred Weber, Werner Höllriegl, Frank M Horling, Friedrich Scheiflinger, Birgit M Reipert, Maurus de la Rosa
Patients with preexisting anti-adeno-associated virus 8 (AAV8) neutralizing antibodies (NAbs) are currently excluded from AAV8 gene therapy trials. Therefore, the assessment of biologically relevant AAV8-NAb titers is critical for product development in gene therapy. However, standardized assays have not been routinely used to determine anti-AAV8-NAb titers, contributing to a wide range of reported anti-AAV8 prevalences. Using a clinical in vitro NAb assay in a separate study, we found a higher than expected anti-AAV8-NAb prevalence in international cohorts of about 50%...
February 8, 2019: Human Gene Therapy Methods
Yan Liang, Xiaoyan Zhang, Xuejuan Bai, Yourong Yang, Wenping Gong, Tong Wang, Yanbo Ling, Junxian Zhang, Lan Wang, Jie Wang, Gaimei Li, Yi Chen, Xiaoyang Chen, Xueqiong Wu
In this study, the MTB latency-associated antigens Rv2660c, Rv1733c, Rv1813c, Rv2628, Rv2029c, Rv2659c were compared regarding their immunogenicity and potential therapeutic effects in an MTB reactivation mouse model. Normal mice or MTB reactivation mice were immunized intramuscularly three times at two-week intervals with saline, plasmid vector pVAX1, M. vaccae vaccine (a commercial inactivated vaccine), rv1813c DNA, rv2628 DNA, rv2029c DNA,rv2659c DNA, rv1733c DNA or rv2660c DNA. The normal mice immunized with rv2628 DNA or rv2659c DNA had low numbers of Th1 cells and a lower ratio of Th1/Th2 immune cells in whole blood (P<0...
February 6, 2019: Human Gene Therapy Methods
Guijun Sun, Yang Yang, Xiaoyan Lu, Qing Liu, Shengrong Wu, Jiajia Jin, Zijiao Huang, Xiangyi He
OBJECTIVE: The aim of this study was to construct the periodontal ligament cells overexpressing Human telomerase reverse transcriptase(hTERT) mediated by adenovirus vectors and lentivirus, and compare the osteogenic and proliferative abilities of the two cell lines, in order to establish an efficient and stable cell model that will be more appropriate for applying to the periodontal ligament regeneration. METHODS: After the construction of the recombinant adenovirus plasmid pAd-pshuttle-cmv-hTERT, then human periodontal ligament cells were infected by the successfully packaged adenovirus and lentivirus particles, by which the two periodontal ligament cell lines were established...
February 1, 2019: Human Gene Therapy Methods
Tao Wang, Wang Yin, Hadi AlShamaileh, Yumei Zhang, Phuong Tran, Tuong Nguyen, Yong Li, Kuisheng Chen, Miaomiao Sun, Yingchun Hou, Weihong Zhang, Qingxia Zhao, Changying Chen, Pei-Zhuo Zhang, Wei Duan
As a nucleic acid alternative to traditional antibody, aptamer holds great potential in various fields of biology and medicine such as targeted gene therapy, drug delivery, bio-sensing and laboratory medicine. Over the past decades, conventional SELEX method has undergone dramatic modifications and improvements owing to developments in material sciences and analytical techniques. However, many of the recently developed strategies either require complex materials and instruments or suffer from low efficiency and high failure rates in the selection of desired aptamers...
January 31, 2019: Human Gene Therapy Methods
Benjamin Strobel, Kai Zuckschwerdt, Gudrun Zimmermann, Christine Mayer, Ruth Eytner, Philipp Rechtsteiner, Sebastian Kreuz, Thorsten Lamla
Adeno-associated virus (AAV) vectors currently represent the most attractive platform for viral gene therapy and are also valuable research tools to study gene function or establish disease models. Consequently, many academic labs, core facilities and biotech/pharma companies meanwhile produce AAVs for research and early clinical development. Whereas fast, universal protocols for vector purification (downstream processing) are available, AAV production using adherent HEK-293 cells still requires time-consuming passaging and extensive culture expansion before transfection...
January 29, 2019: Human Gene Therapy Methods
Lise Pasquet, Sophie Chabot, Elisabeth Bellard, Marie-Pierre Rols, Justin Teissie, Muriel Golzio
The skin is considered as well suited for gene therapy and vaccination. DNA vaccines elicit both broad humoral and cellular immune responses when injected in the skin. Physical and chemical methods are needed to boost the expression. Gene electrotransfer (GET) is one of the most effective approaches. This step by step protocol describes the procedures to get an efficient GET targeted to the skin by using easy-to-use non-invasive electrodes after intradermal plasmid injection (ID GET). A specific pulse sequence is reported...
January 11, 2019: Human Gene Therapy Methods
Lucia Schoderboeck, Hollie Emma Wicky, Wickcliffe Carson Abraham, Stephanie Hughes
Our aim was to develop a method to silence a very specific set of cells in a spatially and temporally refined manner. Here we present an approach that combines the use of a transgenic mouse line, expressing cre recombinase under a nestin promoter, with lentiviral delivery of a floxed, ivermectin-gated chloride channel construct to the dentate gyrus. We used this approach to express an ivermectin-sensitive chloride channel in newly born granule cells in adult mouse brains, and tested its ability to silence neuronal activity by analysing the effect on immediate early gene expression in vitro, in cre-transgenic primary neuronal cultures...
December 7, 2018: Human Gene Therapy Methods
Xufeng Shen, Yuchen Xu, Zhengming Bai, Dongyue Ma, Qingsong Niu, Jialin Meng, Song Fan, Li Zhang, Zongyao Hao, Xiansheng Zhang, Chaozhao Liang
Gene therapy has great potential in treating human diseases, but little progress has been made in preclinical and clinical studies of renal diseases. To find an effective gene delivery approach in the kidney, we developed transparenchymal renal pelvis injection. Using AAV9 vectors, we evaluated the gene delivery efficiency and safety of this administration method. The results showed that the exogenous gene was expressed in the tubular epithelial cells of the injected kidney, with a much lower expression level in the contralateral kidney...
November 20, 2018: Human Gene Therapy Methods
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
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
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
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
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
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
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
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
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
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
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
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
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