scholarly journals Targeting site-specific chromosome integration.

2005 ◽  
Vol 52 (2) ◽  
pp. 285-291
Author(s):  
Patricia Nuno-Gonzalez ◽  
Hsu Chao ◽  
Kazuhiro Oka
Keyword(s):  
Gene Therapy ◽  
Great Promise ◽  
Target Cells ◽  
Site Specific ◽  
High Expectations ◽  
Hybrid Vectors ◽  
Site Specific Integration ◽  
Phage Integrases ◽  
Efficient Gene ◽  
Mammalian Genomes

The concept of gene therapy was introduced with great promise and high expectations. However, what appeared simple in theory has not translated into practice. Despite some success in clinical trials, the research community is still facing an old problem: namely, the need for a vector that can deliver a gene to target cells without adverse events while maintaining a long-term therapeutic effect. Some of these challenges are being addressed by the development of hybrid vectors which meld two different viral systems to incorporate efficient gene delivery and large cloning capacity with site-specific integration. The two known systems that integrate genes into specific sites in mammalian genomes are the adeno-associated virus and phage integrases. Recent experiments with hybrid vectors incorporating both of these systems are encouraging. However, extensive research should be directed towards the safety and efficacy of this approach before it will be available for gene therapy.

scholarly journals Site-Specific Integration of Functional Transgenes into the Human Genome by Adeno/AAV Hybrid Vectors

2004 ◽  
Vol 10 (4) ◽  
pp. 660-670 ◽  
Author(s):  
Alessandra Recchia ◽  
Laura Perani ◽  
Daniela Sartori ◽  
Clelia Olgiati ◽  
Fulvio Mavilio
Keyword(s):  
Human Genome ◽  
Site Specific ◽  
Hybrid Vectors ◽  
Site Specific Integration

scholarly journals Gene therapy: advances, challenges and perspectives

2017 ◽  
Vol 15 (3) ◽  
pp. 369-375 ◽  
Author(s):  
Giulliana Augusta Rangel Gonçalves ◽  
Raquel de Melo Alves Paiva
Keyword(s):  
Gene Therapy ◽  
Germ Line ◽  
Genetic Material ◽  
The United States ◽  
In Vivo Studies ◽  
Basic Unit ◽  
Target Cells ◽  
Site Specific ◽  
Genomic Editing

ABSTRACT The ability to make site-specific modifications to the human genome has been an objective in medicine since the recognition of the gene as the basic unit of heredity. Thus, gene therapy is understood as the ability of genetic improvement through the correction of altered (mutated) genes or site-specific modifications that target therapeutic treatment. This therapy became possible through the advances of genetics and bioengineering that enabled manipulating vectors for delivery of extrachromosomal material to target cells. One of the main focuses of this technique is the optimization of delivery vehicles (vectors) that are mostly plasmids, nanostructured or viruses. The viruses are more often investigated due to their excellence of invading cells and inserting their genetic material. However, there is great concern regarding exacerbated immune responses and genome manipulation, especially in germ line cells. In vivo studies in in somatic cell showed satisfactory results with approved protocols in clinical trials. These trials have been conducted in the United States, Europe, Australia and China. Recent biotechnological advances, such as induced pluripotent stem cells in patients with liver diseases, chimeric antigen receptor T-cell immunotherapy, and genomic editing by CRISPR/Cas9, are addressed in this review.

IL-7 surface-engineered lentiviral vectors promote survival and efficient gene transfer in resting primary T lymphocytes

Blood ◽  
2003 ◽  
Vol 101 (6) ◽  
pp. 2167-2174 ◽  
Author(s):  
Els Verhoeyen ◽  
Valerie Dardalhon ◽  
Odile Ducrey-Rundquist ◽  
Didier Trono ◽  
Naomi Taylor ◽  
...  
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
T Cell ◽  
Gene Transfer ◽  
Lentiviral Vectors ◽  
Target Cells ◽  
Interleukin 7 ◽  
Efficient Gene ◽  
Cognate Antigen ◽  
Therapy Target

Important gene therapy target cells such as resting human T cells are refractory to transduction with lentiviral vectors. Completion of reverse transcription, nuclear import, and subsequent integration of the lentiviral genome occur in these cells only if they have been activated. In T-cell–based gene therapy trials performed to date, cells have been activated via their cognate antigen receptor. To couple activation with gene transfer, we previously generated lentiviral vectors displaying an anti-CD3 scFv fragment that allowed up to 48% transduction of freshly isolated T cells. However, transduction of highly purified resting T cells with these anti-CD3–displaying lentiviral vectors was inefficient and shifted the T cells from the naive to the memory phenotype. Here, we describe interleukin-7 (IL-7)–displaying HIV-1–derived vectors. Like recombinant IL-7, these modified particles could promote the survival of primary T cells placed in culture without inducing a naive-to-memory phenotypic switch. Furthermore, a single exposure to the IL-7–displaying vectors resulted in efficient gene transfer in both resting memory adult T cells and naive cord blood T cells. With adult naive T cells, preactivation with recombinant IL-7 was necessary for efficient gene transfer. Altogether, these results suggest that IL-7–displaying vectors could constitute interesting tools for T-cell–targeted gene therapy.

Gene transfer therapy for heritable disease: cell and expression targeting

1993 ◽  
Vol 339 (1288) ◽  
pp. 217-224 ◽  
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Target Cells ◽  
Somatic Gene Therapy ◽  
Hematopoietic Stem ◽  
Large Size ◽  
Efficient Gene ◽  
Somatic Gene ◽  
Transfer Strategies ◽  
Therapy Model

Gene therapy is defined as the delivery of a functional gene for expression in somatic tissues with the intent to cure a disease. Different gene transfer strategies may be required to target different tissues. Adenosine deaminase (ADA) deficiency is a good gene therapy model for targeting a rare population of pluripotent hematopoietic stem cells capable of self-renewal. We present evidence for the highly efficient gene transfer and sustained expression of hum an ADA in human primitive hematopoietic progenitors using retroviral supernatant with a supportive stromal layer. A stem cell-enriched (CD34 + ) fraction was also successfully transduced. Duchenne muscular dystrophy (DMD) is also a good model for somatic gene therapy. Two of the challenges presented by this model are the large size of the gene and the large number of target cells. Germline gene transfer and correction of the phenotype has been demonstrated in transgenic mdx mice using both a full-length and a truncated form of the dystrophin cDNA. We present here a deletion mutagenesis strategy to truncate the dystrophin cDNA such that it can be accommodated by retroviral and adenoviral vectors useful for somatic gene therapy.

scholarly journals Lipofection of Purified Adeno-Associated Virus Rep68 Protein: toward a Chromosome-Targeting Nonviral Particle

1998 ◽  
Vol 72 (9) ◽  
pp. 7653-7658 ◽  
Author(s):  
Stefania Lamartina ◽  
Giuseppe Roscilli ◽  
Daniela Rinaudo ◽  
Paola Delmastro ◽  
Carlo Toniatti
Keyword(s):  
Gene Therapy ◽  
Chromosome 19 ◽  
Adeno Associated Virus ◽  
Site Specific ◽  
Dna Junctions ◽  
Site Specific Integration ◽  
Nonviral Delivery ◽  
Latently Infected ◽  
Infected Cells ◽  
Human Chromosome 19

ABSTRACT Adeno-associated virus (AAV) integrates very efficiently into a specific site (AAVS1) of human chromosome 19. Two elements of the AAV genome are sufficient: the inverted terminal repeats (ITRs) and the Rep78 or Rep68 protein. The incorporation of the AAV integration machinery in nonviral delivery systems is of great interest for gene therapy. We demonstrate that purified recombinant Rep68 protein is functionally active when directly delivered into human cells by using the polycationic liposome Lipofectamine, promoting the rescue-replication of a codelivered ITR-flanked cassette in adenovirus-infected cells and its site-specific integration in noninfected cells. The sequencing of cloned virus-host DNA junctions confirmed that lipofected Rep68 protein triggers site-specific integration at the same sites in chromosome 19 already characterized in cells latently infected with AAV.

scholarly journals Site-Specific Integration in Mammalian Cells Mediated by a New Hybrid Baculovirus–Adeno-Associated Virus Vector

1998 ◽  
Vol 72 (6) ◽  
pp. 5025-5034 ◽  
Author(s):  
Fabio Palombo ◽  
Andrea Monciotti ◽  
Alessandra Recchia ◽  
Riccardo Cortese ◽  
Gennaro Ciliberto ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Ex Vivo ◽  
High Titer ◽  
Human Fibroblasts ◽  
Pcr Amplification ◽  
Adeno Associated Virus ◽  
Site Specific ◽  
Hybrid Vectors ◽  
Site Specific Integration ◽  
Transgene Cassette

ABSTRACT Baculovirus can transiently transduce primary human and rat hepatocytes, as well as a subset of stable cell lines. To prolong transgene expression, we have developed new hybrid vectors which associate key elements from adeno-associated virus (AAV) with the elevated transducing capacity of baculovirus. The hybrid vectors contain a transgene cassette composed of the β-galactosidase (β-Gal) reporter gene and the hygromycin resistance (Hygr) gene flanked by the AAV inverted terminal repeats (ITRs), which are necessary for AAV replication and integration in the host genome. Constructs were derived both with and without the AAVrep gene under the p5 and p19 promoters cloned in different positions with respect to the baculovirus polyheidrin promoter. A high-titer preparation of baculovirus-AAV (Bac-AAV) chimeric virus containing the ITR–Hygr–β-Gal sequence was obtained with insect cells only when the rep gene was placed in an antisense orientation to the polyheidrin promoter. Infection of 293 cells with Bac-AAV virus expressing the rep gene results in a 10- to 50-fold increase in the number of Hygr stable cell clones. Additionally, rep expression determined the localization of the transgene cassette in the aavs1 site in approximately 41% of cases as detected by both Southern blotting and fluorescent in situ hybridization analysis. Moreover, site-specific integration of the ITR-flanked DNA was also detected by PCR amplification of the ITR-aavs1 junction in transduced human fibroblasts. These data indicate that Bac-AAV hybrid vectors can allow permanent, nontoxic gene delivery of DNA constructs for ex vivo treatment of primary human cells.

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