scholarly journals Preclinical and clinical advances in transposon-based gene therapy

2017 ◽  
Vol 37 (6) ◽  
Author(s):  
Jaitip Tipanee ◽  
Yoke Chin Chai ◽  
Thierry VandenDriessche ◽  
Marinee K. Chuah
Keyword(s):  
Gene Therapy ◽  
Clinical Trials ◽  
Viral Vectors ◽  
Ex Vivo ◽  
Cell Types ◽  
Sleeping Beauty ◽  
Target Cells ◽  
Hematologic Cancer ◽  
Clinical Advances

Transposons derived from Sleeping Beauty (SB), piggyBac (PB), or Tol2 typically require cotransfection of transposon DNA with a transposase either as an expression plasmid or mRNA. Consequently, this results in genomic integration of the potentially therapeutic gene into chromosomes of the desired target cells, and thus conferring stable expression. Non-viral transfection methods are typically preferred to deliver the transposon components into the target cells. However, these methods do not match the efficacy typically attained with viral vectors and are sometimes associated with cellular toxicity evoked by the DNA itself. In recent years, the overall transposition efficacy has gradually increased by codon optimization of the transposase, generation of hyperactive transposases, and/or introduction of specific mutations in the transposon terminal repeats. Their versatility enabled the stable genetic engineering in many different primary cell types, including stem/progenitor cells and differentiated cell types. This prompted numerous preclinical proof-of-concept studies in disease models that demonstrated the potential of DNA transposons for ex vivo and in vivo gene therapy. One of the merits of transposon systems relates to their ability to deliver relatively large therapeutic transgenes that cannot readily be accommodated in viral vectors such as full-length dystrophin cDNA. These emerging insights paved the way toward the first transposon-based phase I/II clinical trials to treat hematologic cancer and other diseases. Though encouraging results were obtained, controlled pivotal clinical trials are needed to corroborate the efficacy and safety of transposon-based therapies.

scholarly journals Vector-mediated cancer gene therapy: A review

2020 ◽  
Vol 13 (2) ◽  
pp. 152-165
Author(s):  
Manisha. B. Shinde ◽  
Dr. Archana D. Kajale ◽  
Dr. Madhuri A. Channawar ◽  
Dr. Shilpa R. Gawande
Keyword(s):  
Gene Therapy ◽  
Viral Vectors ◽  
Nuclear Translocation ◽  
Ex Vivo ◽  
Gene Knockout ◽  
Genetic Material ◽  
Target Cells ◽  
Human Beings ◽  
Vector Systems

Gene therapy is the transfer of genetic material to cure a disease or at least to improve the clinical status of a patient. One of the basic concepts of gene therapy is to transform viruses into genetic shuttles, which will deliver the gene of interest into the target cells. Safe methods have been devised to do this, using several viral and non-viral vectors. Two main approaches emerged: in vivo modification and ex vivo modification. Retrovirus, adenovirus, adenoassociated virus are suitable for gene therapeutic approaches which are based on permanent expression of the therapeutic gene. Non-viral vectors are far less efficient than viral vectors, but they have advantages due to their low immunogenicity and their large capacity for therapeutic DNA. The most commonly used DNA virus vectors are based on adenoviruses and adeno-associated viruses. An example of gene-knockout mediated gene therapy is the knockout of the human CCR5 gene in T-cells in order to control HIV infection. To improve the function of non-viral vectors, the addition of viral functions such as receptor mediated uptake and nuclear translocation of DNA may finally lead to the development of an artificial virus. Gene transfer protocols have been approved for human use in inherited diseases, cancers and acquired disorders. Although the available vector systems are able to deliver genes in vivo into cells, the ideal delivery vehicle has not been found. Thus, the present viral vectors should be used only with great caution in human beings and further progress in vector development is necessary.

Non-Viral Vectors for Gene Delivery

2018 ◽  
Vol 9 (1) ◽  
pp. 4-11 ◽  
Author(s):  
Aparna Bansal ◽  
Himanshu
Keyword(s):  
Gene Therapy ◽  
Viral Vectors ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Expression System ◽  
Target Cells ◽  
Specific Expression ◽  
Naked Dna

Introduction: Gene therapy has emerged out as a promising therapeutic pave for the treatment of genetic and acquired diseases. Gene transfection into target cells using naked DNA is a simple and safe approach which has been further improved by combining vectors or gene carriers. Both viral and non-viral approaches have achieved a milestone to establish this technique, but non-viral approaches have attained a significant attention because of their favourable properties like less immunotoxicity and biosafety, easy to produce with versatile surface modifications, etc. Literature is rich in evidences which revealed that undoubtedly, non–viral vectors have acquired a unique place in gene therapy but still there are number of challenges which are to be overcome to increase their effectiveness and prove them ideal gene vectors. Conclusion: To date, tissue specific expression, long lasting gene expression system, enhanced gene transfection efficiency has been achieved with improvement in delivery methods using non-viral vectors. This review mainly summarizes the various physical and chemical methods for gene transfer in vitro and in vivo.

Gene therapy for critical limb ischemia: Per aspera ad astra

2021 ◽  
Vol 21 ◽  
Author(s):  
Vyacheslav Z. Tarantul ◽  
Alexander V. Gavrilenko
Keyword(s):  
Gene Therapy ◽  
Critical Limb Ischemia ◽  
Viral Vectors ◽  
Ex Vivo ◽  
Single Gene ◽  
Public Health Problem ◽  
Limb Ischemia ◽  
Therapeutic Angiogenesis ◽  
Effective Treatment Option

: Peripheral artery diseases remain a serious public health problem. Although there are many traditional methods for their treatment using conservative therapeutic techniques and surgery, gene therapy is an alternative and potentially more effective treatment option especially for “no option” patients. This review treats the results of many years of research and application of gene therapy as an example of treatment of patients with critical limb ischemia. Data on successful and unsuccessful attempts to use this technology for treating this disease are presented. Trends in changing the paradigm of approaches to therapeutic angiogenesis are noted: from viral vectors to non-viral vectors, from gene transfer to the whole organism to targeted transfer to cells and tissues, from single gene use to combination of genes; from DNA therapy to RNA therapy, from in vivo therapy to ex vivo therapy.

scholarly journals Towards Physiologically and Tightly Regulated Vectored Antibody Therapies

Cancers ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 962 ◽  
Author(s):  
Audrey Page ◽  
Floriane Fusil ◽  
François-Loïc Cosset
Keyword(s):  
B Cells ◽  
Viral Vectors ◽  
Checkpoint Inhibitors ◽  
Ex Vivo ◽  
Passive Immunization ◽  
Cell Types ◽  
Cell Receptor ◽  
Short Period ◽  
Antibody Secretion

Cancers represent highly significant health issues and the options for their treatment are often not efficient to cure the disease. Immunotherapy strategies have been developed to modulate the patient’s immune system in order to eradicate cancerous cells. For instance, passive immunization consists in the administration at high doses of exogenously produced monoclonal antibodies directed either against tumor antigen or against immune checkpoint inhibitors. Its main advantage is that it provides immediate immunity, though during a relatively short period, which consequently requires frequent injections. To circumvent this limitation, several approaches, reviewed here, have emerged to induce in vivo antibody secretion at physiological doses. Gene delivery vectors, such as adenoviral vectors or adeno-associated vectors, have been designed to induce antibody secretion in vivo after in situ cell modification, and have driven significant improvements in several cancer models. However, anti-idiotypic antibodies and escape mutants have been detected, probably because of both the continuous expression of antibodies and their expression by unspecialized cell types. To overcome these hurdles, adoptive transfer of genetically modified B cells that secrete antibodies either constitutively or in a regulated manner have been developed by ex vivo transgene insertion with viral vectors. Recently, with the emergence of gene editing technologies, the endogenous B cell receptor loci of B cells have been modified with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease (Cas-9) system to change their specificity in order to target a given antigen. The expression of the modified BCR gene hence follows the endogenous regulation mechanisms, which may prevent or at least reduce side effects. Although these approaches seem promising for cancer treatments, major questions, such as the persistence and the re-activation potential of these engineered cells, remain to be addressed in clinically relevant animal models before translation to humans.

scholarly journals Thalassemia Gene Therapy By In Vivo Transduction of Mobilized Hematopoietic Stem Cells (HSCs) with an Integrating Hybrid Adenovirus Vector System

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2193-2193
Author(s):  
Afrodite Georgakopoulou ◽  
Hongjie Wang ◽  
Chrysi Kapsali ◽  
Nikoleta Psatha ◽  
Angeliki Koufali ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Hematological Parameters ◽  
Sleeping Beauty ◽  
Vector System ◽  
Good Tolerability ◽  
Hematopoietic Stem

Abstract To overcome the cost and complexity of current thalassemia ex vivogene therapy protocols, we developed a minimally invasive and readily translatable approach for in vivo HSC gene delivery which abrogates the need for HSC leukapheresis, CD34+ cell selection, ex vivo HSC culture, myeloablation and ultimately, transplantation. Our approach involves HSC mobilization with G-CSF/AMD3100 andintravenous injections of a hybrid vector system consisting ofa CD46-targeting, helper-dependent adenoviral vector and the hyperactive Sleeping Beauty transposase (SB100x) that mediates integration of thevector-encoded γ-globin and mgmtP140K genes. Pretreatment with glucocorticoids before virus injectionsis used to blockthe release of pro-inflammatory cytokines andimmunosuppression is applied in order to avoid responses against human g-globin- and MGMT protein-expressing cells. We tested our approach in a mouse model recapitulating the phenotypeof human β-thalassemia intermedia (Hbbth-3/hCD46++ mice). At week 8 post transduction, hCD46+/+/Hbbth-3 mice expressed HbF in 31.2±2.7% of circulating erythrocytes. Due to a significant drop in HbF expression by week 16 (11.9±3.0%), a 4-dose O6BG/BCNU treatment was administeredin order to in vivo select forgene corrected hematopoietic progenitors, thus recovering the HbF expression in76.0±5.7% of the circulating erythrocytes, by week 29 post in vivo transduction. With an average vector copy number of 1.4/cell, the human γ-globin to mouse α-globin expression was ~10% by HPLC and the human γ-globin to mouse β-globin mRNA ratio ~10%, by qRT-PCR. Hematological parameters (RBCs, Ht, Hb, MCV, RDW, Reticulocytes) at week 29 post in vivo transduction, were significantly improved over baseline or were indistinguishable from normal values, suggesting near to complete phenotypic correction. Treated mice showed significant reduction of spleen size, extramedullary erythropoiesis and parenchymal hemosiderosis. After secondary transplantation and without in vivo selection, more than 90% of donor-derived erythrocytes (CD46+) were g-globin-positive, up to 20 weeks post-transplant. Safety was demonstrated by the good tolerability of treatment, the absence of alterations in hematopoiesis, the normal colony-forming potential of bone marrow cells and the random integration pattern of our vector system. Overall, we present a simplified platform for gene therapy of thalassemia, which can serve as a cost-efficient and "portable" approach to make gene therapy accessible even to resource-poor regions where thalassemia major is endemic but only minimally complex strategies could be adopted. Disclosures No relevant conflicts of interest to declare.

scholarly journals Cancer immunotherapy using cells modified with cytokine genes.

2003 ◽  
Vol 50 (3) ◽  
pp. 613-624 ◽  
Author(s):  
Dariusz W Kowalczyk ◽  
Piotr J Wysocki ◽  
Andrzej Mackiewicz
Keyword(s):  
Gene Therapy ◽  
Immune Responses ◽  
Antitumor Agents ◽  
Ex Vivo ◽  
Cell Types ◽  
Cancer Gene Therapy ◽  
Cytokine Gene ◽  
Cytokine Genes ◽  
Membrane Bound

The ability of various cytokines to hamper tumor growth or to induce anti-tumor immune response has resulted in their study as antitumor agents in gene therapy approaches. In this review we will concentrate on the costimulation of antitumor immune responses using modification of various cell types by cytokine genes. Several strategies have emerged such as (i). modification of tumor cells with cytokine genes ex vivo (whole tumor cell vaccines), (ii). ex vivo modification of other cell types for cytokine gene delivery, (iii). delivery of cytokine genes into tumor microenvironment in vivo, (iv). modification of dendritic cells with cytokine genes ex vivo. Originally single cytokine genes were used. Subsequently, multiple cytokine genes were applied simultaneously, or in combination with other factors such as chemokines, membrane bound co-stimulatory molecules, or tumor associated antigens. In this review we discuss these strategies and their use in cancer treatment as well as the promises and limitations of cytokine based cancer gene therapy. Clinical trials, including our own experience, employing the above strategies are discussed.

scholarly journals Gene Therapy Approaches

2021 ◽  
Vol 1 (1) ◽  
pp. 52-56
Author(s):  
Hogir Saadi
Keyword(s):  
Gene Therapy ◽  
Viral Vectors ◽  
Nuclear Translocation ◽  
Viral Vector ◽  
Ex Vivo ◽  
Genetic Material ◽  
Target Tissue ◽  
Human Beings ◽  
Vector Systems

Gene therapy can be described broadly as the transfer of genetic material to control a disease or at least to enhance a patient's clinical status. The transformation of viruses into genetic shuttles is one of the core principles of gene therapy, which will introduce the gene of interest into the target tissue and cells. To do this, safe strategies have been invented, using many viral and non-viral vector delivery. Two major methods have emerged: modification in vivo and modification ex vivo. For gene therapeutic approaches which are focused on lifelong expression of the therapeutic gene, retrovirus, adenovirus, adeno-associated viruses are acceptable. Non-viral vectors are much less successful than viral vectors, but because of their low immune responses and their broad therapeutic DNA ability, they have advantages. The addition of viral functions such as receptor-mediated uptake and nuclear translocation of DNA may eventually lead to the development of an artificial virus in order to improve the role of non-viral vectors. For human use in genetic conditions, cancers and acquired illnesses, gene transfer techniques have been allowed. The ideal delivery vehicle has not been identified, although the accessible vector systems are capable of transporting genes in vivo into cells. Therefore, only with great caution can the present viral vectors be used in human beings and further progress in the production of vectors is required. Current progresses in our understanding of gene therapy approaches and their delivery technology, as well as the victors used to deliver therapeutic genes, are the primary goals of this review. For that reason, a literature search on PubMed and Google Scholar was carried out using different keywords.

scholarly journals Efficient and Highly Specific Gene Transfer Using Mutated Lentiviral Vectors Redirected with Bispecific Antibodies

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Christina L. Parker ◽  
Timothy M. Jacobs ◽  
Justin T. Huckaby ◽  
Dimple Harit ◽  
Samuel K. Lai
Keyword(s):  
Gene Therapy ◽  
Gene Delivery ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Endosomal Escape ◽  
Bispecific Antibodies ◽  
Target Cells ◽  
Specific Gene ◽  
Targeted Gene Delivery

ABSTRACT Despite their exceptional potencies, the broad tropism of most commonly used lentivirus (LV) vectors limits their use for targeted gene delivery in vivo. We hypothesized that we could improve the specificity of LV targeting by coupling (i) reduction of their binding to off-target cells with (ii) redirection of the vectors with a bispecific antibody (bsAb) that binds both LV and receptors on target cells. As a proof of concept, we pseudotyped nonreplicating LV using a mutated Sindbis envelope (mSindbis) with ablated binding to native receptors, while retaining the capacity to facilitate efficient fusion and endosomal escape. We then evaluated the transduction potencies of the mSindbis LV for HER2-positive (HER2+) (SKBR3) breast and HER2-negative (HER2−) (A2780) cells when redirected with different bsAbs. mSindbis LV alone failed to induce appreciable green fluorescent protein (GFP) expression in either cell. When mixed with HER2-targeting bsAb, mSindbis LV was exceptionally potent, transducing 12% to 16% of the SKBR3 cells at a multiplicity of infection (MOI [ratio of viral genome copies to target cells]) of 3. Transduction was highly specific, resulting in ∼50-fold-greater selectivity toward SKBR3 cells versus A2780 cells. Redirecting mSindbis LV led to a 10-fold improvement in cell-specific targeting compared to redirecting wild-type Sindbis LV with the same bsAb, underscoring the importance of ablating native virus tropism in order to maximize targeting specificity. The redirection of mutated LV using bsAb represents a potent and highly versatile platform for targeted gene therapy. IMPORTANCE The goal of gene therapy is specific delivery and expression of therapeutic genes to target cells and tissues. Common lentivirus (LV) vectors are efficient gene delivery vehicles but offer little specificity. Here, we report an effective and versatile strategy to redirect LV to target cells using bispecific antibodies (bsAbs) that bind both cell receptors and LV envelope domains. Importantly, we ablated the native receptor binding of LV to minimize off-target transduction. Coupling bsAb specificity and ablated native LV tropism synergistically enhanced the selectivity of our targeted gene delivery system. The modular nature of our bsAb-based redirection enables facile targeting of the same LV to diverse tissues/cells. By abrogating the native broad tropism of LV, our bsAb-LV redirection strategy may enable lentivirus-based gene delivery in vivo, expanding the current use of LV beyond ex vivo applications.

scholarly journals Clinical Trials for Gene Therapy in Lysosomal Diseases With CNS Involvement

2021 ◽  
Vol 8 ◽  
Author(s):  
Caroline Sevin ◽  
Kumaran Deiva
Keyword(s):  
Gene Therapy ◽  
Central Nervous System ◽  
Clinical Trials ◽  
Nervous System ◽  
Ex Vivo ◽  
Central Nervous System Involvement ◽  
Large Animal ◽  
Hematopoietic Stem ◽  
The Brain

There are over 70 known lysosomal storage disorders (LSDs), most caused by mutations in genes encoding lysosomal hydrolases. Central nervous system involvement is a hallmark of the majority of LSDs and, if present, generally determines the prognosis of the disease. Nonetheless, brain disease is currently poorly targeted by available therapies, including systemic enzyme replacement therapy, mostly (but not only) due to the presence of the blood–brain barrier that restricts the access of orally or parenterally administered large molecules into the brain. Thus, one of the greatest and most exciting challenges over coming years will be to succeed in developing effective therapies for the treatment of central nervous system manifestations in LSDs. Over recent years, gene therapy (GT) has emerged as a promising therapeutic strategy for a variety of inherited neurodegenerative diseases. In LSDs, the ability of genetically corrected cells to cross-correct adjacent lysosomal enzyme-deficient cells in the brain after gene transfer might enhance the diffusion of the recombinant enzyme, making this group of diseases a strong candidate for such an approach. Both in vivo (using the administration of recombinant adeno-associated viral vectors) and ex vivo (auto-transplantation of lentiviral vector-modified hematopoietic stem cells-HSCs) strategies are feasible. Promising results have been obtained in an ever-increasing number of preclinical studies in rodents and large animal models of LSDs, and these give great hope of GT successfully correcting neurological defects, once translated to clinical practice. We are now at the stage of treating patients, and various clinical trials are underway, to assess the safety and efficacy of in vivo and ex vivo GT in several neuropathic LSDs. In this review, we summarize different approaches being developed and review the current clinical trials related to neuropathic LSDs, their results (if any), and their limitations. We will also discuss the pitfalls and the remaining challenges.

scholarly journals Keratinocyte Gene Transfer and Gene Therapy

1996 ◽  
Vol 7 (3) ◽  
pp. 204-221 ◽  
Author(s):  
J.A. Garlick ◽  
E.S. Fenjves
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Gene Transfer ◽  
Ex Vivo ◽  
Target Cells ◽  
Gene Products ◽  
Oral Keratinocytes ◽  
Systemic Delivery ◽  
Therapy Strategies

Gene therapy has moved beyond the pre-clinical stage to the treatment of a variety of inherited and acquired diseases. For such therapy to be successful, genes must be efficiently delivered to target cells and gene products must be expressed for prolonged periods of time without toxic effects to the host. This may be achieved by means of an in vivo strategy where genes are transferred directly into a host cell, or by means of an ex vivo approach through which cells are removed, cultured, targeted for gene delivery, and grafted back to the host. Several obstacles continue to delay safe and effective clinical application of gene therapy in a variety of target cells. The limited survival of transplanted cells, transient expression of transferred genes, and difficulties in targeting stem cells are technical issues requiring further investigation. Epidermal and oral keratinocytes are potential vehicles for gene therapy. Several features of these tissues can be utilized to achieve delivery of therapeutic gene products for local or systemic delivery. These qualities include: (1) the presence of stem cells; (2) the cell-, strata-, and site-specific regulation of keratinocyte gene expression; (3) tissue accessibility; and (4) secretory capacity. Such features can be exploited by the use of gene therapy strategies to facilitate: (1) identification, enrichment, and targeting of stem cells to ensure the continued presence of the transferred gene; (2) high-level and persistent transgene expression using keratinocyte-specific promoters; (3) tissue access needed for culture and grafting for ex vivo therapy and direct in vivo gene transfer; (4) secretion of transgene product for local or systemic delivery; and (5) monitoring of genetically modified tissue and removal if treatment termination is required. Optimal gene therapy strategies are being tested in a variety of tissues to treat dominant and recessive genetic disorders as well as acquired diseases such as neoplasia and infectious disease. This experience provides a basis for the application of such clinical studies to a spectrum of diseases effecting epidermal and oral keratinocytes. Gene therapy is in an early stage yet holds great promise for its ultimate clinical application.

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