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Human Gene Therapy Methods

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 (Ad5) and pre-decided vector tropism. It is highly demanded to develop novel AdVs originated from other types than Ad5. Here, we describe a method for direct cloning of adenovirus utilizing linear-linear homologous recombination (LLHR), followed by rapid adenoviral genome modification via linear-circular homologous recombination (LCHR)...
May 14, 2018: Human Gene Therapy Methods
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...
April 28, 2018: Human Gene Therapy Methods
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
Ilona Hauber, Niklas Beschorner, Silke Schrödel, Jan Chemnitz, Nikolaus 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 (PBSC)...
April 10, 2018: Human Gene Therapy Methods
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...
March 29, 2018: Human Gene Therapy Methods
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
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
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
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
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
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...
February 2018: Human Gene Therapy Methods
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
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
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
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
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