scholarly journals Intravenous Lentiviral Gene Therapy for Hemophilia a: In Vitro and In Vivo Assessment of Universal Versus Tissue-Specific Promoters

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2941-2941
Author(s):  
Jie Gong ◽  
Tsai-Hua Chung ◽  
Min Zhou ◽  
Lung-Ji Chang
Keyword(s):  
Gene Therapy ◽  
Plasma Level ◽  
Hemophilia A ◽  
In Vivo Studies ◽  
Specific Expression ◽  
Tissue Specific ◽  
Ha Gene ◽  
Over Time

Abstract Background ：Hemophilia A (HA) is an X-linked monogenic coagulation disorder resulting from deficiency of the factor VIII (FVIII, F8) gene in the intrinsic coagulation cascade. The current treatment of HA is based on protein replacement therapy (PRT) through plasma-derived coagulation factors or recombinant proteins with limitations of short half-life, high cost, and life-time requirement of the treatment. Gene therapy has become a promising treatment for HA. Methods and Materials: We developed an advanced lentiviral vector (LV) system for intravenous (iv) F8 gene therapy. A selective codon optimized and B-domain deleted human F8 (hF8BDD) gene was synthesized, sequenced and functionally verified. LVs carrying a universal EF1α promoter, or several modified tissue-specific promoters including endothelial- (VEC), endothelial and epithelial- (KDR), and two megakaryocyte-specific (ITGA and Gp) promoters, were biologically and immunologically characterized in vitro using human endothelial and megakaryocytic cell lines, EA-hy926 and DAMI, and in vivo using F8 knockout (KO) mice. Results: We investigated the different LV promoter constructs and found that LV-VEC-F8BDD exhibited the highest virus packaging (1.3x10 9 transduction units/ml) and transduction efficiencies compared with the other LV constructs. Tissue-specific expression of the VEC, KDR, ITGA and Gp promoters was confirmed in EA-hy926 and DAMI cells by RT-PCR, Western blot and ELISA analyses. We detected F8 activities close to 6 folds and 4.5 folds above the normal plasma level from the EF1a LV-F8BDD transduced EA-hy926 cells and DAMI cells, respectively, whereas the VEC LV in EA-hy926 cells and the ITGA-LV in DAMI cells exhibited F8 activities at 1.5 folds and 5 folds above the normal plasma level, respectively. In vivo studies in F8 KO mice via iv injection of LVs after reduced radiation conditioning illustrated preferential vector expression in different cell lineages, with high expression of EF1a vector in CD11b, F4/80 and Ly-6G positive immune cells, and preferential expression of VEC vector in CD31 positive endothelial cells, and ITGA and Gp vectors in CD41 positive megakaryocytes. In addition, we detected variable phenotypic corrections as well as anti-F8 immune responses in the F8 KO mice treated with the different LVs. The iv deliveries of VEC and Gp F8BDD vectors illustrated therapeutic F8 activities over time, around 25% and 8%, respectively, in 60 days, which increased to high levels (80% and 25%, respectively) after 120 days (Figure A). Kinetic analyses of anti-F8 IgG and inhibitor titers (Bethesda assay) of the treated mice showed that the VEC vector exhibited the lowest F8 inhibitory immune response over time (Figure B). Conclusion: Based on the in vitro and in vivo studies, our results suggest that for HA gene therapy, optimal rather than high F8 expression is critical, and tissue-specific expression but not universal expression can reduce adverse inhibitor effect. We demonstrated that the LV-VEC-F8BDD vector displayed high tissue specificity in vivo, and high transgene delivery efficiency, high coagulation function and low immunogenicity. In addition, iv LV gene therapy could be a safe, convenient and effective HA gene therapy strategy. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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.

Antitumoural hydroxyapatite nanoparticles-mediated hepatoma-targeted trans-arterial embolization gene therapy: in vitro and in vivo studies

2012 ◽  
Vol 32 (6) ◽  
pp. 998-1007 ◽  
Author(s):  
Gaopeng Li ◽  
Lu Ye ◽  
Jingsheng Pan ◽  
Miaoyun Long ◽  
Zizhuo Zhao ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Arterial Embolization ◽  
In Vivo Studies ◽  
Hydroxyapatite Nanoparticles

Codon optimization of human factor VIII cDNAs leads to high-level expression

Blood ◽  
2011 ◽  
Vol 117 (3) ◽  
pp. 798-807 ◽  
Author(s):  
Natalie J. Ward ◽  
Suzanne M. K. Buckley ◽  
Simon N. Waddington ◽  
Thierry VandenDriessche ◽  
Marinee K. L. Chuah ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Viral Vectors ◽  
Hemophilia A ◽  
Lentiviral Vector ◽  
Wild Type ◽  
Fviii Activity ◽  
Human Factor Viii

Abstract Gene therapy for hemophilia A would be facilitated by development of smaller expression cassettes encoding factor VIII (FVIII), which demonstrate improved biosynthesis and/or enhanced biologic properties. B domain deleted (BDD) FVIII retains full procoagulant function and is expressed at higher levels than wild-type FVIII. However, a partial BDD FVIII, leaving an N-terminal 226 amino acid stretch (N6), increases in vitro secretion of FVIII tenfold compared with BDD-FVIII. In this study, we tested various BDD constructs in the context of either wild-type or codon-optimized cDNA sequences expressed under control of the strong, ubiquitous Spleen Focus Forming Virus promoter within a self-inactivating HIV-based lentiviral vector. Transduced 293T cells in vitro demonstrated detectable FVIII activity. Hemophilic mice treated with lentiviral vectors showed expression of FVIII activity and phenotypic correction sustained over 250 days. Importantly, codon-optimized constructs achieved an unprecedented 29- to 44-fold increase in expression, yielding more than 200% normal human FVIII levels. Addition of B domain sequences to BDD-FVIII did not significantly increase in vivo expression. These significant findings demonstrate that shorter FVIII constructs that can be more easily accommodated in viral vectors can result in increased therapeutic efficacy and may deliver effective gene therapy for hemophilia A.

scholarly journals TALEN Mediated Gene Targeting for CF Gene Therapy

2018 ◽  
Author(s):  
Emily Xia ◽  
Yiqian Zhang ◽  
Huibi Cao ◽  
Jun Li ◽  
Rongqi Duan ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Gene Editing ◽  
Airway Epithelial Cells ◽  
In Vivo Studies ◽  
Lacz Reporter ◽  
Gene Correction ◽  
Transcription Activator ◽  
Stringent Requirement

Cystic Fibrosis (CF) is an inherited monogenic disorder, amenable to gene based therapies. Because CF lung disease is currently the major cause of mortality and morbidity, and lung airway is readily accessible to gene delivery, the major CF gene therapy effort at present is directed to the lung. Although airway epithelial cells are renewed slowly, permanent gene correction through gene editing or targeting in airway stem cells is needed to perpetuate the therapeutic effect. Transcription activator-like effector nuclease (TALEN) has been utilized widely for a variety of gene editing applications. The stringent requirement for nuclease binding target sites allows for gene editing with precision. In this study, we engineered helper-dependent adenoviral (HD-Ad) vectors to deliver a pair of TALENs together with donor DNA targeting the human AAVS1 locus. With homology arms of 4 kb in length, we demonstrated precise insertion of either a LacZ reporter gene or a human CFTR minigene into the target site. Using the LacZ reporter, we determined the efficiency of gene integration to be about 5%. In the CFTR vector transduced cells, we have detected both CFTR mRNA and protein expression by qPCR and Wetern analysis, respectively. We have also confirmed CFTR function correction by flurometric Image Plate Reader (FLIPR) and iodide efflux assays. Taking together, these findings suggest a new direction for future in vitro and in vivo studies in CF gene editing.

Bioengineering of coagulation factor VIII for improved secretion

Blood ◽  
2004 ◽  
Vol 103 (9) ◽  
pp. 3412-3419 ◽  
Author(s):  
Hongzhi Z. Miao ◽  
Nongnuch Sirachainan ◽  
Lisa Palmer ◽  
Phillip Kucab ◽  
Michael A. Cunningham ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Hemophilia A ◽  
Coagulation Factor ◽  
Mrna Levels ◽  
Golgi Transport ◽  
Recombinant Fviii ◽  
Therapy Strategies

Abstract Factor VIII (FVIII) functions as a cofactor within the intrinsic pathway of blood coagulation. Quantitative or qualitative deficiencies of FVIII result in the inherited bleeding disorder hemophilia A. Expression of FVIII (domain structure A1-A2-B-A3-C1-C2) in heterologous mammalian systems is 2 to 3 orders of magnitude less efficient compared with other proteins of similar size compromising recombinant FVIII production and gene therapy strategies. FVIII expression is limited by unstable mRNA, interaction with endoplasmic reticulum (ER) chaperones, and a requirement for facilitated ER to Golgi transport through interaction with the mannose-binding lectin LMAN1. Bioengineering strategies can overcome each of these limitations. B-domain-deleted (BDD)-FVIII yields higher mRNA levels, and targeted point mutations within the A1 domain reduce interaction with the ER chaperone immunoglobulin-binding protein. In order to increase ER to Golgi transport we engineered several asparagine-linked oligosaccharides within a short B-domain spacer within BDD-FVIII. A bioengineered FVIII incorporating all of these elements was secreted 15- to 25-fold more efficiently than full-length FVIII both in vitro and in vivo. FVIII bioengineered for improved secretion will significantly increase potential for success in gene therapy strategies for hemophilia A as well as improve recombinant FVIII production in cell culture manufacturing or transgenic animals. (Blood. 2004;103: 3412-3419)

scholarly journals In vivo evaluation of GG2–GG1/A2 element activity in the insulin promoter region using the CRISPR–Cas9 system

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Hirofumi Noguchi ◽  
Chika Miyagi-Shiohira ◽  
Takao Kinjo ◽  
Issei Saitoh ◽  
Masami Watanabe
Keyword(s):  
Promoter Region ◽  
In Vivo Studies ◽  
Pancreatic Β Cell ◽  
Β Cell ◽  
In Vivo Evaluation ◽  
Specific Expression ◽  
Insulin Promoter ◽  
Element Activity

AbstractThe insulin promoter is regulated by ubiquitous as well as pancreatic β-cell-specific transcription factors. In the insulin promoter, GG2–GG1/A2–C1 (bases − 149 to − 116 in the human insulin promoter) play important roles in regulating β-cell-specific expression of the insulin gene. However, these events were identified through in vitro studies, and we are unaware of comparable in vivo studies. In this study, we evaluated the activity of GG2–GG1/A2 elements in the insulin promoter region in vivo. We generated homozygous mice with mutations in the GG2–GG1/A2 elements in each of the Ins1 and Ins2 promoters by CRISPR–Cas9 technology. The mice with homozygous mutations in the GG2–GG1/A2 elements in both Ins1 and Ins2 were diabetic. These data suggest that the GG2–GG1/A2 element in mice is important for Ins transcription in vivo.

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.

DDAVP-Induced Increase of Factor VIII Activity in Blood Is Likely Due To Release of Factor VIII That Is Synthesized by Endothelial Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 692-692 ◽  
Author(s):  
Lingfei Xu ◽  
Timothy C. Nichols ◽  
Stephanie McCorquodale ◽  
Aaron Dillow ◽  
Elizabeth Merricks ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Endothelial Cells ◽  
Factor Viii ◽  
Hemophilia A ◽  
Fold Increase ◽  
Von Willebrand Disease ◽  
Major Site ◽  
Von Willebrand

Abstract Desmopressin (1-deamino-8-D-arginine vasopressin, DDAVP) is commonly used as a nonreplacement therapy for mild von Willebrand disease (VWD) and hemophilia A. In humans, IV injection of 0.3 μg/kg of DDAVP induces a rapid 2 to 5-fold increase in plasma levels of both von Willebrand factor (VWF) and Factor VIII (FVIII) within 30–60 minutes, which is due to release from Wiebel-Palade bodies (WPBs) in endothelial cells. The stored FVIII may be synthesized by endothelial cells, which express FVIII in vitro. However, hepatoma cells can also express FVIII in vitro, and liver transplantation can correct hemophilia A. Thus, the liver may be the major site of production of FVIII in vivo, thus, an alternative explanation is that endothelial cells take up FVIII from blood and store it in WPBs with VWF, which can be released after DDAVP. DDAVP is effective in humans and dogs, but not in mice. In this study, we tested the effect of DDAVP on hemophilia A dogs after neonatal hepatic gene therapy with a retroviral vector (RV) expressing canine FVIII (cFVIII). With this gene therapy approach, canine hepatocytes express high levels of a reporter gene from an RV, but no expression is observed in endothelial cells. Thus, the major site of FVIII synthesis is the hepatocyte in this model. Our hypothesis is that if DDAVP increases FVIII levels in this dog model, it would indicate that the FVIII increase is due to uptake from blood by endothelial cells. Alternatively, if no increase in FVIII occurs after DDAVP stimulation, it would suggest that the increase in normal dogs is due to synthesis of FVIII by endothelial cells. An RV that contains the liver-specific human α1-antitrypsin promoter and the canine B-domain deleted FVIII cDNA was generated. RV was given IV to two hemophilia A dogs at 8x109 transducing units (TU)/kg at 3 days after birth. The whole blood clotting time (WBCT) and APTT time in both dogs have been normalized, and the plasma cFVIII COATEST activity has been maintained at 100–200% of normal for 11 months to date. DDAVP was injected IV at 0.5 μg/kg into RV-treated hemophilia A dogs at 7 months of age. Two separate doses of DDAVP were given with an interval of one week. The same dose of DDAVP was given to normal dogs as controls (N=4). In normal dogs, both VWF and FVIII levels increased 40% and 50% between 15 to 60 minutes after DDAVP, respectively. However, FVIII levels were not changed in RV-treated dogs, although VWF levels increased 150% or 60%. Thus, our data suggest that the normal FVIII increase after DDAVP administration is due to release of FVIII that is synthesized by endothelial cells. These data also demonstrate that DDAVP will not be effective at increasing FVIII activity in patients that receive liver-directed gene therapy and only achieve partial correction. Such patients would need to be treated with factor replacement if bleeding episodes occur.

scholarly journals Tissue-specific regulation and function of Grb10 during growth and neuronal commitment

2014 ◽  
Vol 112 (22) ◽  
pp. 6841-6847 ◽  
Author(s):  
Robert N. Plasschaert ◽  
Marisa S. Bartolomei
Keyword(s):  
Motor Neurons ◽  
Cellular Proliferation ◽  
Neuronal Development ◽  
Embryonic Stem ◽  
Specific Expression ◽  
Tissue Specific ◽  
Specific Regulation ◽  
And Function

Growth-factor receptor bound protein 10 (Grb10) is a signal adapter protein encoded by an imprinted gene that has roles in growth control, cellular proliferation, and insulin signaling. Additionally, Grb10 is critical for the normal behavior of the adult mouse. These functions are paralleled by Grb10’s unique tissue-specific imprinted expression; the paternal copy of Grb10 is expressed in a subset of neurons whereas the maternal copy is expressed in most other adult tissues in the mouse. The mechanism that underlies this switch between maternal and paternal expression is still unclear, as is the role for paternally expressed Grb10 in neurons. Here, we review recent work and present complementary data that contribute to the understanding of Grb10 gene regulation and function, with specific emphasis on growth and neuronal development. Additionally, we show that in vitro differentiation of mouse embryonic stem cells into alpha motor neurons recapitulates the switch from maternal to paternal expression observed during neuronal development in vivo. We postulate that this switch in allele-specific expression is related to the functional role of Grb10 in motor neurons and other neuronal tissues.

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