scholarly journals Improving germline transmission efficiency in chimeric chickens using a multi-stage injection approach

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0247471
Author(s):  
Danial Naseri ◽  
Kianoush Dormiani ◽  
Mehdi Hajian ◽  
Farnoosh Jafarpour ◽  
Mahboobeh Forouzanfar ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Viral Vectors ◽  
Viral Vector ◽  
Lentiviral Vector ◽  
Chorioallantoic Membrane ◽  
Transmission Efficiency ◽  
Vector System ◽  
Germline Transmission ◽  
Multi Stage ◽  
Low Efficiency

Although different strategies have been developed to generate transgenic poultry, low efficiency of germline transgene transmission has remained a challenge in poultry transgenesis. Herein, we developed an efficient germline transgenesis method using a lentiviral vector system in chickens through multiple injections of transgenes into embryos at different stages of development. The embryo chorioallantoic membrane (CAM) vasculature was successfully used as a novel route of gene transfer into germline tissues. Compared to the other routes of viral vector administration, the embryo’s bloodstream at Hamburger-Hamilton (HH) stages 14–15 achieved the highest rate of germline transmission (GT), 7.7%. Single injection of viral vectors into the CAM vasculature resulted in a GT efficiency of 2.7%, which was significantly higher than the 0.4% obtained by injection into embryos at the blastoderm stage. Double injection of viral vectors into the bloodstream at HH stages 14–15 and through CAM was the most efficient method for producing germline chimeras, giving a GT rate of 13.6%. The authors suggest that the new method described in this study could be efficiently used to produce transgenic poultry in virus-mediated gene transfer systems.

scholarly journals Improving germline transmission efficiency in chimeric chickens using a multi-stage injection approach

2021 ◽  
Author(s):  
Danial Naseri ◽  
Kianoush Dormiani ◽  
Mehdi Hajian ◽  
Farnoosh Jafarpour ◽  
Mahboobeh Forouzanfar ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Viral Vectors ◽  
Developmental Stages ◽  
Viral Vector ◽  
Lentiviral Vector ◽  
Chorioallantoic Membrane ◽  
New Method ◽  
Vector System ◽  
Germline Transmission ◽  
Low Efficiency

AbstractAlthough different strategies have been developed to generate transgenic poultry, low efficiency of germline transgene transmission has remained a challenge in poultry transgenesis. Herein, we developed an efficient germline transgenesis method using a lentiviral vector system in chickens through multiple injections of transgenes into embryos at different stages of development. The embryo chorioallantoic membrane (CAM) vasculature was successfully used as a novel route of gene transfer into germline tissues. We established a new method of eggshell windowing for embryo manipulation at different developmental stages. Compared to the other routes of viral vector administration, the embryo’s bloodstream at Hamburger-Hamilton (HH) stages 14-15 achieved the highest rate of germline transmission (GT), 7.7%. Single injection of viral vectors into the CAM vasculature resulted in a GT efficiency of 2.7%, which was significantly higher than the 0.4% obtained by injection into embryos at the blastoderm stage. Double injection of viral vectors into the bloodstream at HH stages 14-15 and through CAM was the most efficient method for producing germline chimeras, giving a GT rate of 13.6%. The authors suggest that the new method described in this study could be efficiently used to produce transgenic poultry in virus-mediated gene transfer systems.

Comparison of Viral Vectors for Gene Transfer into CLL Cells: Efficient Transduction with Adeno-Associated Virus-8 (AAV-8).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2985-2985 ◽  
Author(s):  
Andrew P. Jewell ◽  
Melanie Cochrane ◽  
Jenny McIntosh ◽  
Reuben Benjamin ◽  
Amit Nathwani
Keyword(s):  
Gene Transfer ◽  
Viral Vectors ◽  
Fluorescent Protein ◽  
Viral Vector ◽  
Lentiviral Vector ◽  
Dependent Manner ◽  
Gfp Expression ◽  
Cmv Promoter ◽  
Adeno Associated Virus ◽  
Green Fluorescent

Abstract Chronic Lymphocytic Leukaemia (CLL) remains largely incurable despite recent advances in therapy, and therefore alternative strategies are of interest in treating this disease. One such alternative is the use of gene therapy, but this relies on developing efficient gene transfer technologies. We have compared several viral vectors coding for green fluorescent protein (GFP) for their ability to transduce CLL cells. Three serotypes of adeno-associated virus (AAV) were used, AAV-2, AAV-5 and a relatively new isolate AAV-8, an EI-EIII deleted adenoviral 5 based vector, AV-5, all with GFP regulated by the CMV promoter, and a VSVG pseudotyped lentiviral vector in which GFP expression is controlled by EF1a promotor/enhancer complex. AV-5 resulted in variable GFP expression, 24.1±3.4%, n=10 but caused cell death at high multiplicities of infection (MOI). The lentiviral vector resulted in GFP expression of 23.5±2.6%, n=12, at the highest titre used, and expression declined in a distinct dose-dependent manner as titres were reduced. Of the AAV vectors, AAV-8 was the most efficient with GFP expression at 41.3±1.0% n=14. We conclude that AAV-8 is a promising viral vector for efficient transduction of CLL cells. Figure 1. Percentage GFP expression for three viral vectors. Three different MOI’s were used at log dilutions. Figure 1. Percentage GFP expression for three viral vectors. Three different MOI’s were used at log dilutions.

scholarly journals Nonviral Gene Therapy for Cancer: A Review

Diseases ◽  
2018 ◽  
Vol 6 (3) ◽  
pp. 57 ◽  
Author(s):  
Chiaki Hidai ◽  
Hisataka Kitano
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Viral Vectors ◽  
Viral Vector ◽  
Transfer Efficiency ◽  
Nonviral Vectors ◽  
Apparent Lack ◽  
Gene Transfer Efficiency ◽  
Relative Safety

Although the development of effective viral vectors put gene therapy on the road to commercialization, nonviral vectors show promise for practical use because of their relative safety and lower cost. A significant barrier to the use of nonviral vectors, however, is that they have not yet proven effective. This apparent lack of interest can be attributed to the problem of the low gene transfer efficiency associated with nonviral vectors. The efficiency of gene transfer via nonviral vectors has been reported to be 1/10th to 1/1000th that of viral vectors. Despite the fact that new gene transfer methods and nonviral vectors have been developed, no significant improvements in gene transfer efficiency have been achieved. Nevertheless, some notable progress has been made. In this review, we discuss studies that report good results using nonviral vectors in vivo in animal models, with a particular focus on studies aimed at in vivo gene therapy to treat cancer, as this disease has attracted the interest of researchers developing nonviral vectors. We describe the conditions in which nonviral vectors work more efficiently for gene therapy and discuss how the goals might differ for nonviral versus viral vector development and use.

Piggybac Mediated Gene Transfer To Correct Hemophilia A

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2900-2900
Author(s):  
Janice M Staber ◽  
Molly Pollpeter ◽  
Angela Arensdorf ◽  
Patrick L Sinn ◽  
Thomas D Rutkowski ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Factor Viii ◽  
Hemophilia A ◽  
Viral Vector ◽  
Conflicts Of Interest ◽  
Vector System ◽  
Expression Vectors ◽  
Dna Repeats ◽  
Tail Clip

Abstract Hemophilia A, caused by a deficiency in factor VIII (FVIII), is the most severe inherited bleeding disorder, affecting about 1 out of 5,000 males; those affected suffer disabling joint and muscle hemorrhages. Hemophilia A is an attractive gene therapy candidate, because even small increases in FVIII levels (5-10%) will alter the phenotype. Non-viral vector systems are used increasingly in gene targeting technologies and as tools for gene transfer applications. Nonviral DNA transposons are genetic elements consisting of inverted terminal DNA repeats which in their naturally occurring configuration flank a transposase coding sequence. The transposase follows a “cut and paste” mechanism to excise the transposon from its original genomic location and insert it into a new locus. The insect derived piggyBac (PB) can be engineered to carry a therapeutic transgene between the inverted terminal repeats. Advantages of this novel nonviral vector system include a large transgene cassette capacity, ease of production and purification, and potential for site-specific integration. We hypothesize that a PB transposon vector carrying a codon-optimized human FVIII cDNA along with a hyperactive transposase (iPB7) will confer persistent gene expression and correction of the hemophilia A bleeding phenotype. We engineered PB transposon to carry a codon-optimized human FVIII B-domain deleted cDNA (coFVIII-BDD). We evaluated the in vivo gene transfer efficiency in hemophilia A mice by hydrodynamic tail-vein injection using PB coFVIII-BDD driven by the murine albumin enhancer/human alpha anti-trypsin promoter. Factor VIII null mice received 25 micrograms each of the PB coFVIII-BDD transposon and iPB7 to determine long term expression and phenotypic correction. FVIII activity and antigen levels were measured prior to injection and then every 4 weeks for 24 weeks. Results revealed therapeutic levels (50-225%) of factor VIII activity and antigen post gene transfer with stable expression for 24 weeks in most mice. A goal of gene transfer based therapies is to develop the most efficacious expression vectors with the least toxicity. To assess endoplasmic reticulum stress in the livers of treated and untreated mice, we evaluated BiP, CHOP, and EDEM levels via q-PCR. All experimental mice, null mice, and transposon treated mice without the coFVIII-BDD cassette revealed no evidence of cell stress. These data indicate codon-optimized FVIII and the piggyBac transposon vector system may provide a safe long term gene transfer strategy. To evaluate phenotypic correction, a tail clip assay was performed at the end of the study. More than 75% of mice receiving PB coFVIII-BDD transposon and iPB7 demonstrated functional correction via tail clip. These data show that the PB vector can be used to deliver transgene expression to the liver and achieve long term expression and phenotypic correction. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Gene Therapy Applications of Non-Human Lentiviral Vectors

Viruses ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 1106
Author(s):  
Altar M. Munis
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Transgene Expression ◽  
Viral Vector ◽  
Lentiviral Vector ◽  
Cell Therapies ◽  
Dividing Cells ◽  
Cell Genome ◽  
Hiv 1

Recent commercialization of lentiviral vector (LV)-based cell therapies and successful reports of clinical studies have demonstrated the untapped potential of LVs to treat diseases and benefit patients. LVs hold notable and inherent advantages over other gene transfer agents based on their ability to transduce non-dividing cells, permanently transform target cell genome, and allow stable, long-term transgene expression. LV systems based on non-human lentiviruses are attractive alternatives to conventional HIV-1-based LVs due to their lack of pathogenicity in humans. This article reviews non-human lentiviruses and highlights their unique characteristics regarding virology and molecular biology. The LV systems developed based on these lentiviruses, as well as their successes and shortcomings, are also discussed. As the field of gene therapy is advancing rapidly, the use of LVs uncovers further challenges and possibilities. Advances in virology and an improved understanding of lentiviral biology will aid in the creation of recombinant viral vector variants suitable for translational applications from a variety of lentiviruses.

scholarly journals Gene therapy and viral vectors. Gene transfer into non-dividing cells by a lentiviral vector.

Uirusu ◽  
1997 ◽  
Vol 47 (2) ◽  
pp. 213-219
Author(s):  
Koichi Miyake ◽  
Takashi Shimada
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Viral Vectors ◽  
Lentiviral Vector ◽  
Dividing Cells

scholarly journals Sex significantly influences transduction of murine liver by recombinant adeno-associated viral vectors through an androgen-dependent pathway

Blood ◽  
2003 ◽  
Vol 102 (2) ◽  
pp. 480-488 ◽  
Author(s):  
Andrew M. Davidoff ◽  
Catherine Y. C. Ng ◽  
Junfang Zhou ◽  
Yunyu Spence ◽  
Amit C. Nathwani
Keyword(s):  
Gene Transfer ◽  
Targeted Delivery ◽  
Viral Vectors ◽  
Nuclear Protein ◽  
Viral Vector ◽  
Systematic Evaluation ◽  
Transduction Efficiency ◽  
Inverted Terminal Repeat ◽  
Male Mice ◽  
Receptor Complexes

AbstractA systematic evaluation of the influence of sex on transduction by recombinant adeno-associated viral vector (rAAV) indicated that transgene expression after liver-targeted delivery of vector particles was between 5- to 13-fold higher in male mice compared with female mice, irrespective of the proviral promoter or cDNA and mouse strain. Molecular analysis revealed that the rAAV genome was stably retained in male liver at levels that were 7-fold higher than those observed in females. Further, the sex difference in transduction was observed with AAV-2– and AAV-5–based vectors, which use distinct receptor complexes for infection. In concordance with the differences in AAV transduction, gel shift analysis with nuclear extracts derived from the liver of mice and humans revealed substantially higher binding of host nuclear protein to the rep-binding site (RBS) of AAV inverted terminal repeat (ITR) in males compared with females. Transduction efficiency and binding of nuclear protein to RBS was dramatically reduced in male mice by castration. In contrast, although oophorectomy did not significantly influence rAAV transduction, administration of 5α dihydrotestosterone, prior to gene transfer, increased stable hepatocyte gene transfer in females to levels observed in male mice, implying that androgens significantly influence hepatocyte gene transfer. Interestingly, sex did not have a significant effect on AAV gene transfer into nonhepatic tissue, indicating that there are distinct tissue- and sex-specific differences in the mechanisms responsible for efficient transduction with this vector. These results have significant implications for gene therapy of autosomal and acquired disorders affecting the liver.

scholarly journals Gene Transfer to the Outflow Tract

2016 ◽  
Author(s):  
Yalong Dang ◽  
Ralitsa Loewen ◽  
Hardik Parikh ◽  
Pritha Roy ◽  
Nils Loewen
Keyword(s):  
Intraocular Pressure ◽  
Gene Transfer ◽  
Trabecular Meshwork ◽  
Viral Vectors ◽  
Lentiviral Vector ◽  
Open Angle Glaucoma ◽  
Outflow Tract ◽  
Human Pathogens ◽  
Vector Systems

Elevated intraocular pressure is the primary cause of open angle glaucoma. Outflow resistance exists within the trabecular meshwork but also at the level of Schlemm′s canal and further downstream within the outflow system. Viral vectors allow to take advantage of naturally evolved, highly efficient mechanisms of gene transfer, a process that is termed transduction. They can be produced at biosafety level 2 in the lab using protocols that have evolved considerably over the last 15 to 20 years. Applied by an intracameral bolus, vectors follow conventional as well as uveoscleral outflow pathways. They may affect other structures in the anterior chamber depending on their transduction kinetics which can vary among species when using the same vector. Not all vectors can express long-term, a desirable feature to address the chronicity of glaucoma. Vectors that integrate into the genome of the target cell can achieve transgene function for the life of the transduced cell but are mutagenic by definition. The most prominent long-term expressing vector systems are based on lentiviruses that are derived from HIV, FIV, or EIAV. Safety considerations make non-primate lentiviral vector systems easier to work with as they are not derived from human pathogens. Non-integrating vectors are subject to degradation and attritional dilution during cell division. Lentiviral vectors have to integrate in order to express while adeno-associated viral vectors (AAV) often persist as intracellular concatemers but may also integrate. Adeno- and herpes viral vectors do not integrate and earlier generation systems might be relatively immunogenic. Nonviral methods of gene transfer are termed transfection with few restrictions of transgene size and type but often a much less efficient gene transfer that is also short-lived. Traditional gene transfer delivers exons while some vectors (lentiviral, herpes and adenoviral) allow transfer of entire genes that include introns. Recent insights have highlighted the role of non-coding RNA, most prominently, siRNA, miRNA and lncRNA. SiRNA is highly specific, miRNA is less specific, while lncRNA uses highly complex mechanisms that involve secondary structures and intergenic, intronic, overlapping, antisense, and bidirectional location. Several promising preclinical studies have targeted the RhoA or the prostaglandin pathway or modified the extracellular matrix. TGF-β and glaucoma myocilin mutants have been transduced to elevate the intraocular pressure in glaucoma models. Cell based therapies have started to show first promise. Past approaches have focused on the trabecular meshwork and the inner wall of Schlemm′s canal while new strategies are concerned with modification of outflow tract elements that are downstream of the trabecular meshwork.

scholarly journals Spatiotemporally confined red light-controlled gene delivery at single-cell resolution using adeno-associated viral vectors

2021 ◽  
Vol 7 (25) ◽  
pp. eabf0797
Author(s):  
Maximilian Hörner ◽  
Carolina Jerez-Longres ◽  
Anna Hudek ◽  
Sebastian Hook ◽  
O. Sascha Yousefi ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Single Cell ◽  
Genetic Information ◽  
Viral Vectors ◽  
Single Cells ◽  
Red Light ◽  
Genetic Research ◽  
Vector System ◽  
Target Cells ◽  
Spatially Resolved

Methodologies for the controlled delivery of genetic information into target cells are of utmost importance for genetic engineering in both fundamental and applied research. However, available methods for efficient gene transfer into user-selected or even single cells suffer from low throughput, the need for complicated equipment, high invasiveness, or side effects by off-target viral uptake. Here, we engineer an adeno-associated viral (AAV) vector system that transfers genetic information into native target cells upon illumination with cell-compatible red light. This OptoAAV system allows adjustable and spatially resolved gene transfer down to single-cell resolution and is compatible with different cell lines and primary cells. Moreover, the sequential application of multiple OptoAAVs enables spatially resolved transduction with different transgenes. The approach presented is likely extendable to other classes of viral vectors and is expected to foster advances in basic and applied genetic research.

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