scholarly journals Adenovirus-mediated hypoxia-targeted gene therapy using HSV thymidine kinase and bacterial nitroreductase prodrug-activating genes in vitro and in vivo

2011 ◽  
Vol 18 (11) ◽  
pp. 773-784 ◽  
Author(s):  
T J Harvey ◽  
I M Hennig ◽  
S D Shnyder ◽  
P A Cooper ◽  
N Ingram ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Thymidine Kinase ◽  
Targeted Gene Therapy ◽  
Hsv Thymidine Kinase

scholarly journals Imaging Expression of Cytosine Deaminase-Herpes Virus Thymidine Kinase Fusion Gene (CD/TK) Expression with [124I]FIAU and PET

2002 ◽  
Vol 1 (1) ◽  
pp. 153535002002000 ◽  
Author(s):  
Trevor Hackman ◽  
Michail Doubrovin ◽  
Julius Balatoni ◽  
Tatiana Beresten ◽  
Vladimir Ponomarev ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Thymidine Kinase ◽  
Enzyme Assay ◽  
Fusion Gene ◽  
Cytosine Deaminase ◽  
Prodrug Activation ◽  
Wide Range ◽  
Different Levels

Double prodrug activation gene therapy using the Escherichia coli cytosine deaminase (CD)herpes simplex virus type 1 thymidine kinase ( HSV1-tk) fusion gene ( CD/TK) with 5-fluorocytosine (5FC), ganciclovir (GCV), and radiotherapy is currently under evaluation for treatment of different tumors. We assessed the efficacy of noninvasive imaging with [124I]FIAU (2′-fluoro-2′-deoxy-1-β-d-arabinofuranosyl-5-iodo-uracil) and positron emission tomography (PET) for monitoring expression of the CD/TK fusion gene. Walker-256 tumor cells were transduced with a retroviral vector bearing the CD/TK gene (W256CD/TK cells). The activity of HSV1-TK and CD subunits of the CD/TK gene product was assessed in different single cell-derived clones of W256CD/TK cells using the FIAU radiotracer accumulation assay in cells and a CD enzyme assay in cell homogenates, respectively. A linear relationship was observed between the levels of CD and HSV1-tk subunit expression in corresponding clones in vitro over a wide range of CD/TK expression levels. Several clones of W256CD/TK cells with significantly different levels of CD/TK expression were selected and used to produce multiple subcutaneous tumors in rats. PET imaging of HSV1-TK subunit activity with [124I]FIAU was performed on these animals and demonstrated that different levels of CD/TK expression in subcutaneous W256CD/TK tumors can be imaged quantitatively. CD expression in subcutaneous tumor sample homogenates was measured using a CD enzyme assay. A comparison of CD and HSV1-TK subunit enzymatic activity of the CD/TK fusion protein in vivo showed a significant correlation. Knowing this relationship, the parametric images of CD subunit activity were generated. Imaging with [124I]FIAU and PET could provide pre- and posttreatment assessments of CD/TK-based double prodrug activation in clinical gene therapy trials.

scholarly journals Restoring the natural tropism of AAV2 vectors for human liver

2020 ◽  
Vol 12 (560) ◽  
pp. eaba3312
Author(s):  
Marti Cabanes-Creus ◽  
Claus V. Hallwirth ◽  
Adrian Westhaus ◽  
Boaz H. Ng ◽  
Sophia H.Y. Liao ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Tissue Culture ◽  
Human Liver ◽  
Xenograft Model ◽  
Human Hepatocytes ◽  
Targeted Gene Therapy ◽  
Tissue Culture Propagation ◽  
Made In

Recent clinical successes in gene therapy applications have intensified interest in using adeno-associated viruses (AAVs) as vectors for therapeutic gene delivery. Although prototypical AAV2 shows robust in vitro transduction of human hepatocyte–derived cell lines, it has not translated into an effective vector for liver-directed gene therapy in vivo. This is consistent with observations made in Fah−/−/Rag2−/−/Il2rg−/− (FRG) mice with humanized livers, showing that AAV2 functions poorly in this xenograft model. Here, we derived naturally hepatotropic AAV capsid sequences from primary human liver samples. We demonstrated that capsid mutations, likely acquired as an unintentional consequence of tissue culture propagation, attenuated the intrinsic human hepatic tropism of natural AAV2 and related human liver AAV isolates. These mutations resulted in amino acid changes that increased binding to heparan sulfate proteoglycan (HSPG), which has been regarded as the primary cellular receptor mediating AAV2 infection of human hepatocytes. Propagation of natural AAV variants in vitro showed tissue culture adaptation with resulting loss of tropism for human hepatocytes. In vivo readaptation of the prototypical AAV2 in FRG mice with a humanized liver resulted in restoration of the intrinsic hepatic tropism of AAV2 through decreased binding to HSPG. Our results challenge the notion that high affinity for HSPG is essential for AAV2 entry into human hepatocytes and suggest that natural AAV capsids of human liver origin are likely to be more effective for liver-targeted gene therapy applications than culture-adapted AAV2.

Limited efficacy of gene transfer in herpes simplex virus-thymidine kinase/ganciclovir gene therapy for brain tumors

2005 ◽  
Vol 102 (2) ◽  
pp. 328-335 ◽  
Author(s):  
Piotr Hadaczek ◽  
Hanna Mirek ◽  
Mitchel S. Berger ◽  
Krystof Bankiewicz
Keyword(s):  
Gene Therapy ◽  
Brain Tumor ◽  
Gene Transfer ◽  
Thymidine Kinase ◽  
Therapeutic Effects ◽  
Content Type ◽  
Over Time ◽  
Fine Print

Object. Low efficacy of gene transfer, transient gene expression, and toxicity of viral vectors are the major hurdles in successful anticancer gene therapy. The authors conducted in vitro (U87MG cell line) and in vivo (xenograft, tumor-bearing rodent model) studies to address the stability of transduction by using the adenoassociated virus serotype-2 (AAV2)—thymidine kinase (TK) vector over time. Methods. Standard methods for cell growth and a ganciclovir (GCV) cytotoxicity assay were applied. The AAV2-TK was infused into implanted tumors in athymic rats via convection-enhanced delivery (CED). Thymidine kinase expression was evaluated through immunohistochemical analysis, and the distribution volumes of the transduced tumors were calculated. Twenty-four hours following the viral infusions, animals were treated with GCV (50 mg/kg intraperitoneally every day for 10 days; six rats) or phosphate-buffered saline (six rats). A rapid decrease in TK expression over time was observed both in vitro and in vivo. A large volume of the tumor (up to 39%) was transduced with AAV2-TK following CED. Administration of GCV resulted in limited therapeutic effects (survival of 25.8 compared with 21.3 days). Conclusions. Rapid elimination of TK expression from dividing tumor cells and focal transduction of the brain tumor were most likely responsible for the limited bystander effect in this approach. Immediate administration of GCV is crucial to assure maximal efficacy in the elimination of cancer cells. In addition, the complete or diffused transduction of a brain tumor with TK may be required for its total eradication.

scholarly journals Retinoids augment the bystander effect in vitro and in vivo in herpes simplex virus thymidine kinase/ganciclovir-mediated gene therapy

Gene Therapy ◽  
1997 ◽  
Vol 4 (9) ◽  
pp. 909-917 ◽  
Author(s):  
JY Park ◽  
AA Elshami ◽  
K Amin ◽  
N Rizk ◽  
LR Kaiser ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Thymidine Kinase ◽  
Bystander Effect ◽  
Simplex Virus

SiRNA technology, the gene therapy of the future?

2008 ◽  
Vol 149 (4) ◽  
pp. 153-159 ◽  
Author(s):  
Zsuzsanna Rácz ◽  
Péter Hamar
Keyword(s):  
Gene Therapy ◽  
Short Interfering Rna ◽  
The Future ◽  
Interfering Rna

A genetikában új korszak kezdődött 17 éve, amikor a petúniában felfedezték a koszuppressziót. Később a koszuppressziót azonosították a növényekben és alacsonyabb rendű eukariótákban megfigyelt RNS-interferenciával (RNSi). Bár a növényekben ez ősi vírusellenes gazdaszervezeti védekezőmechanizmus, emlősökben az RNSi élettani szerepe még nincs teljesen tisztázva. Az RNSi-t rövid kettős szálú interferáló RNS-ek (short interfering RNA, siRNS) irányítják. A jelen cikkben összefoglaljuk az RNSi történetét és mechanizmusát, az siRNS-ek szerkezete és hatékonysága közötti összefüggéseket, a célsejtbe való bejuttatás virális és nem virális módjait. Az siRNS-ek klinikai alkalmazásának legfontosabb akadálya az in vivo alkalmazás. Bár a hidrodinamikus kezelés állatokban hatékony, embereknél nem alkalmazható. Lehetőséget jelent viszont a szervspecifikus katéterezés. A szintetizált siRNS-ek ismert mellékhatásait szintén tárgyaljuk. Bár a génterápia ezen új területén számos problémával kell szembenézni, a sikeres in vitro és in vivo kísérletek reményt jelentenek emberi betegségek siRNS-sel történő kezelésére.

Exosome as a Natural Gene Delivery Vector for Cancer Treatment

2020 ◽  
Vol 20 (11) ◽  
pp. 821-830
Author(s):  
Prasad Pofali ◽  
Adrita Mondal ◽  
Vaishali Londhe
Keyword(s):  
Gene Therapy ◽  
Gene Delivery ◽  
Nucleic Acids ◽  
Cancer Treatment ◽  
Intestinal Barrier ◽  
Gene Carrier ◽  
Gene Delivery Vector ◽  
Delivery Vector

Background: Current gene therapy vectors such as viral, non-viral, and bacterial vectors, which are used for cancer treatment, but there are certain safety concerns and stability issues of these conventional vectors. Exosomes are the vesicles of size 40-100 nm secreted from multivesicular bodies into the extracellular environment by most of the cell types in-vivo and in-vitro. As a natural nanocarrier, exosomes are immunologically inert, biocompatible, and can cross biological barriers like the blood-brain barrier, intestinal barrier, and placental barrier. Objective: This review focusses on the role of exosome as a carrier to efficiently deliver a gene for cancer treatment and diagnosis. The methods for loading of nucleic acids onto the exosomes, advantages of exosomes as a smart intercellular shuttle for gene delivery and therapeutic applications as a gene delivery vector for siRNA, miRNA and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and also the limitations of exosomes as a gene carrier are all reviewed in this article. Methods: Mostly, electroporation and chemical transfection are used to prepare gene loaded exosomes. Results: Exosome-mediated delivery is highly promising and advantageous in comparison to the current delivery methods for systemic gene therapy. Targeted exosomes, loaded with therapeutic nucleic acids, can efficiently promote the reduction of tumor proliferation without any adverse effects. Conclusion: In the near future, exosomes can become an efficient gene carrier for delivery and a biomarker for the diagnosis and treatment of cancer.

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.

scholarly journals Preclinical Development of Autologous Hematopoietic Stem Cell-Based Gene Therapy for Immune Deficiencies: A Journey from Mouse Cage to Bed Side

Pharmaceutics ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 549
Author(s):  
Laura Garcia-Perez ◽  
Anita Ordas ◽  
Kirsten Canté-Barrett ◽  
Pauline Meij ◽  
Karin Pike-Overzet ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Severe Combined Immunodeficiency ◽  
Good Manufacturing Practice ◽  
Proof Of Concept ◽  
Preclinical Development ◽  
Hematopoietic Stem ◽  
Immune Deficiencies ◽  
Suitable Vector

Recent clinical trials using patient’s own corrected hematopoietic stem cells (HSCs), such as for primary immunodeficiencies (Adenosine deaminase (ADA) deficiency, X-linked Severe Combined Immunodeficiency (SCID), X-linked chronic granulomatous disease (CGD), Wiskott–Aldrich Syndrome (WAS)), have yielded promising results in the clinic; endorsing gene therapy to become standard therapy for a number of diseases. However, the journey to achieve such a successful therapy is not easy, and several challenges have to be overcome. In this review, we will address several different challenges in the development of gene therapy for immune deficiencies using our own experience with Recombinase-activating gene 1 (RAG1) SCID as an example. We will discuss product development (targeting of the therapeutic cells and choice of a suitable vector and delivery method), the proof-of-concept (in vitro and in vivo efficacy, toxicology, and safety), and the final release steps to the clinic (scaling up, good manufacturing practice (GMP) procedures/protocols and regulatory hurdles).

Sign in / Sign up

Export Citation Format

Share Document