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Human Gene Therapy Methods

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. qPCR and FACS methods are commonly used for determination of infectious titer. Here we introduce 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. In this paper we have defined the dynamic range, Limit of Quantification (LOQ) and data acceptance criteria for ddPCR against lentiviral sequence...
January 29, 2018: Human Gene Therapy Methods
Felix Ferdinand 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, like 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, we describe an easy-to-use laboratory protocol 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...
January 12, 2018: Human Gene Therapy Methods
Constantinos Christos Loucari, Petros Patsali, Thamar B van Dijk, Coralea Stephanou, Panayiota Papasavva, Maria Zanti, Ryo Kurita, Yukio Nakamura, Soteroulla Christou, Maria Sitarou, Sjaak Philipsen, Carsten Werner Lederer, Marina Kleanthous
The β-haemoglobinopathies sickle cell anaemia and β-thalassaemia are the focus of many gene-therapy studies. A key disease parameter is the abundance of globin chains, because it indicates the level of anaemia, 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 either from long run times, high sample requirements or inability to separate murine from human β-globin chains...
January 12, 2018: Human Gene Therapy Methods
Penghui Hu, Hongjie Chen, Eileen McGowan, Nina Ren, Ming Xu, Yiguang Lin
Lung cancer, mainly caused 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 the FGFR1 and its ligand basic FGF (bFGF) is a key step forward in developing new therapies for this crippling disease...
December 27, 2017: Human Gene Therapy Methods
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
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...
December 18, 2017: Human Gene Therapy Methods
Antonio Rodriguez, Guillermo Garaulet, Juan José Lazcano, Hernán Alarcón, Sergio de Frutos, Jorge Luis Martínez-Torrecuadrada
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. Here we have employed the streptococcal Albumin Binding Domain (ABD) to generate a construct to express ABD as a GPI-anchored protein. LV particles bearing ABD are able to bind bovine and human serum albumin in vitro...
November 21, 2017: Human Gene Therapy Methods
Andreas Jungmann, Barbara Leuchs, Jean Rommelaere, Hugo A Katus, Oliver J Müller
Adeno-associated virus vectors are a powerful tool for gene transfer approaches. We have established a simple and fast plasmid-based production system for achieving high adeno-associated virus titers within 6 working days. The same procedure can be used for all serotypes and thus allows direct comparability of different serotypes. In this protocol we describe a step-by-step procedure that results in well-characterized vectors suitable for both in vitro approaches and preclinical studies.
October 2017: Human Gene Therapy Methods
Adrien Savy, Yohann Dickx, Lucile Nauwynck, Delphine Bonnin, Otto-Wilhelm Merten, Lionel Galibert
Adeno-associated virus (AAV) inverted terminal repeats (ITRs) are key elements of AAV. These guanine-cytosine-rich structures are involved in the replication and encapsidation of the AAV genome, along with its integration in and excision from the host genome. These sequences are the only AAV-derived DNA sequences conserved in recombinant AAV (rAAV), as they allow its replication, encapsidation, and long-term maintenance and expression in target cells. Due to the original vector design, plasmids containing the gene of interest flanked by ITRs and used for rAAV production often present incomplete, truncated, or imperfect ITR sequences...
October 2017: Human Gene Therapy Methods
Adrian Hohl, Anne Sophie Ramms, Christian Dohmen, Klaus Mantwill, Andrea Bielmeier, Andreas Kolk, Andreas Ruppert, Roman Nawroth, Per Sonne Holm
Adenoviral vector production for therapeutic applications is a well-established routine process. However, current methods for measurement of adenovirus particle titers as a quality characteristic require highly purified virus preparations. While purified virus is typically obtained in the last step of downstream purification, rapid and reliable methods for adenovirus particle quantification in intermediate products and crude lysates to allow for optimization and validation of cell cultures and intermediate downstream processing steps are currently not at hand...
October 2017: Human Gene Therapy Methods
Xiaoying Jin, Lin Liu, Shelley Nass, Catherine O'Riordan, Eric Pastor, X Kate Zhang
The requirement for robust analytical methods to characterize adeno-associated virus (AAV) vectors is immediate, as the field advances more AAV gene therapies into the clinic and onto commercialization. AAV capsid proteins (VPs) are critical for viral infectivity and vector potency. Thus, complete characterization of the constituent viral capsid proteins of AAV vectors, including their sequences and post-translational modifications (PTMs), is highly recommended to ensure AAV product quality and consistency...
October 2017: Human Gene Therapy Methods
M B Kostina, A V Sass, E A Stukacheva, I V Korobko, E D Sverdlov
A set of vectors for Cre recombinase-dependent expression of the hybrid suicidal FCU1 transgene was constructed, including a two-plasmid system wherein the FCU1 and Cre transgenes reside in separate vectors, and single-plasmid variants in which a single plasmid bears both transgenes. To improve the safety profile and specificity in cancer gene therapy applications, as well as to ensure stable propagation of plasmids in bacterial cells, the Cre/LoxP system components were optimized. A bicistronic vector with the Cre expression cassette placed between the LoxP sites unidirectionally with FCU1 cDNA resulted in higher therapeutic efficiency compared with the double-plasmid system in an enzyme-prodrug suicide cancer gene therapy scheme...
October 2017: Human Gene Therapy Methods
Andreas Jungmann, Barbara Leuchs, Hugo A Katus, Jean Rommelaere, Oliver J Müller
AAV vectors are a powerful tool for gene transfer approaches. We have established a simple and fast plasmid-based production system for achieving high AAV titers within 6 working days. The same procedure can be used for all serotypes and thus allows direct comparability of different serotypes. In this protocol we describe a step-by-step procedure which results in well-characterized vectors suitable for both in vitro approaches and preclinical studies.
September 21, 2017: Human Gene Therapy Methods
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...
September 4, 2017: Human Gene Therapy Methods
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...
August 30, 2017: Human Gene Therapy Methods
Aziza P Manceur, Howard Kim, Vanja Misic, Nadejda Andreev, July Dorion-Thibaudeau, Stéphane Lanthier, Alice Bernier, Sonia Tremblay, Anne-Marie Gélinas, Sophie Broussau, Rénald Gilbert, Sven Ansorge
Lentiviral vectors (LV) represent a key tool for gene and cell therapy applications. The production of these vectors in sufficient quantities for clinical applications remains a hurdle, prompting the field towards developing suspension processes that are conducive to large scale production. We describe here a LV production strategy using a stable inducible producer cell line. The HEK293 cell line employed grows in suspension, thus offering direct scalability, and produces a Green Fluorescent Protein (GFP)-expressing lentiviral vector in the 10<sup>6</sup> Transduction Units (TU)/ml range without optimization...
August 21, 2017: Human Gene Therapy Methods
María Fernández-García, Cristina Mesa, Elena Almarza, Juan Bueren, Rosa Yañez
Transduction of hematopoietic stem and progenitor cells (HSPCs) with lentiviral vectors (LVs) constitutes a new therapeutic option for the treatment of various monogenic diseases affecting the lymphohematopoietic system. The development of detailed preclinical studies of gene therapy in animal disease models constitutes an essential step in expanding the application of gene therapy in a wide variety of inherited and acquired diseases. Here we describe an efficient protocol to transduce HSPCs from wild-type and Fanconi anemia mice with either gene-marking or therapeutic LVs...
August 17, 2017: Human Gene Therapy Methods
Melanie Pavlovic, Nina Koehler, Martina Anton, Anna Dinkelmeier, Maren Haase, Thorsten Stellberger, Ulrich Busch, Armin E Baiker
The purpose of the described method is the detection of and differentiation between RNA and DNA of human immunodeficiency virus (HIV)-derived lentiviral vectors (LV) in cell culture supernatants and swab samples. For the analytical surveillance of genetic engineering, operations methods for the detection of the HIV-1-based LV generations are required. Furthermore, for research issues, it is important to prove the absence of LV particles for downgrading experimental settings in terms of the biosafety level. Here, a quantitative polymerase chain reaction method targeting the long terminal repeat U5 subunit and the start sequence of the packaging signal ψ is described...
August 2017: Human Gene Therapy Methods
Michael White, Roger Whittaker, Carolina Gándara, Elizabeth A Stoll
Lentiviral vectors are increasingly the gene transfer tool of choice for gene or cell therapies, with multiple clinical investigations showing promise for this viral vector in terms of both safety and efficacy. The third-generation vector system is well characterized, effectively delivers genetic material and maintains long-term stable expression in target cells, delivers larger amounts of genetic material than other methods, is nonpathogenic, and does not cause an inflammatory response in the recipient. This report aims to help academic scientists and regulatory managers negotiate the governance framework to achieve successful translation of a lentiviral vector-based gene therapy...
August 2017: Human Gene Therapy Methods
Anne Louise Askou, Josephine Natalia Esther Benckendorff, Andreas Holmgaard, Tina Storm, Lars Aagaard, Toke Bek, Jacob Giehm Mikkelsen, Thomas Juhl Corydon
Lentivirus-based vectors have been used for the development of potent gene therapies. Here, application of a multigenic lentiviral vector (LV) producing multiple anti-angiogenic microRNAs following subretinal delivery in a laser-induced choroidal neovascularization (CNV) mouse model is presented. This versatile LV, carrying back-to-back RNApolII-driven expression cassettes, enables combined expression of microRNAs targeting vascular endothelial growth factor A (Vegfa) mRNA and fluorescent reporters. In addition, by including a vitelliform macular dystrophy 2 (VMD2) promoter, expression of microRNAs is restricted to the retinal pigment epithelial (RPE) cells...
August 2017: Human Gene Therapy Methods
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