Gene transfer to the mammalian reproductive tract

Zygote ◽  
2010 ◽  
Vol 19 (4) ◽  
pp. 287-295 ◽  
Author(s):  
Pedro Esponda
Keyword(s):  
Gene Transfer ◽  
Reproductive Tract ◽  
Vas Deferens ◽  
Ex Vivo ◽  
Transfection Efficiency ◽  
Reproductive Organs ◽  
Secretory Proteins ◽  
Mouse Vas Deferens

SummaryThis review summarizes the results of research on gene transfer to the mammalian genital tract. Gene transfer experiments have been developed during the last 2 decades and have been applied using in vitro, ex vivo and in vivo procedures. (i) In vitro methods have been applied to the uterine epithelial cells with the principal purpose of analysing some pathological change occurring in the uterus. In the male tract, epididymal cell lines have been used to evaluate the expression of particular genes and the function of specific proteins. (ii) Ex vivo methods have been applied to both the uterus and the vas deferens in humans, and good transgene expression has been recorded. (iii) In vivo gene transfer in the female tract has been employed in the uterus and oviduct using gene injections or electroporation methods. The glandular epithelium of both organs can be transfected efficiently, and transfection efficiency depends on the hormonal stage of the animal. The best expression occurred during pseudopregnancy and meta-estrus periods, when high progesterone and low estradiol concentrations occur. In the male tract, in vivo methods have been applied to mouse vas deferens and epididymis. In both organs, patches of epithelial regions appeared to express the transgenes. Furthermore, the secretions of both organs were also modified using gene constructions that led to the expression of some secretory proteins. In summary, gene modifications in the epithelium of the mammalian reproductive tract have been successful employing different technologies. Further improvements in transfection efficiency would help provide new insights into the physiology of these reproductive organs. Furthermore, the use of these methods could also be used to modify the fertility of mammals.

Clonal Analyses of Retroviral Vector Integrations in ADA-SCID Patients Treated with Stem Cell Gene Therapy.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3249-3249
Author(s):  
Barbara Cassani ◽  
Grazia Andolfi ◽  
Massimiliano Mirolo ◽  
Luca Biasco ◽  
Alessandra Recchia ◽  
...  
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
T Cell ◽  
Gene Transfer ◽  
Human Genome ◽  
Ex Vivo ◽  
Cd34 Cells

Abstract Gene transfer into hematopoietic stem/progenitor cells (HSC) by gammaretroviral vectors is an effective treatment for patients affected by severe combined immunodeficiency (SCID) due to adenosine deaminase (ADA)-deficiency. Recent studied have indicated that gammaretroviral vectors integrate in a non-random fashion in their host genome, but there is still limited information on the distribution of retroviral insertion sites (RIS) in human long-term reconstituting HSC following therapeutic gene transfer. We performed a genome-wide analysis of RIS in transduced bone marrow-derived CD34+ cells before transplantation (in vitro) and in hematopoietic cell subsets (ex vivo) from five ADA-SCID patients treated with gene therapy combined to low-dose busulfan. Vector-genome junctions were cloned by inverse or linker-mediated PCR, sequenced, mapped onto the human genome, and compared to a library of randomly cloned human genome fragments or to the expected distribution for the NCBI annotation. Both in vitro (n=212) and ex vivo (n=496) RIS showed a non-random distribution, with strong preference for a 5-kb window around transcription start sites (23.6% and 28.8%, respectively) and for gene-dense regions. Integrations occurring inside the transcribed portion of a RefSeq genes were more represented in vitro than ex vivo (50.9 vs 41.3%), while RIS <30kb upstream from the start site were more frequent in the ex vivo sample (25.6% vs 19.4%). Among recurrently hit loci (n=50), LMO2 was the most represented, with one integration cloned from pre-infusion CD34+ cells and five from post-gene therapy samples (2 in granulocytes, 3 in T cells). Clone-specific Q-PCR showed no in vivo expansion of LMO2-carrying clones while LMO2 gene overexpression at the bulk level was excluded by RT-PCR. Gene expression profiling revealed a preference for integration into genes transcriptionally active in CD34+ cells at the time of transduction as well as genes expressed in T cells. Functional clustering analysis of genes hit by retroviral vectors in pre- and post-transplant cells showed no in vivo skewing towards genes controlling self-renewal or survival of HSC (i.e. cell cycle, transcription, signal transduction). Clonal analysis of long-term repopulating cells (>=6 months) revealed a high number of distinct RIS (range 42–121) in the T-cell compartment, in agreement with the complexity of the T-cell repertoire, while fewer RIS were retrieved from granulocytes. The presence of shared integrants among multiple lineages confirmed that the gene transfer protocol was adequate to allow stable engraftment of multipotent HSC. Taken together, our data show that transplantation of ADA-transduced HSC does not result in skewing or expansion of malignant clones in vivo, despite the occurrence of insertions near potentially oncogenic genomic sites. These results, combined to the relatively long-term follow-up of patients, indicate that retroviral-mediated gene transfer for ADA-SCID has a favorable safety profile.

Liposome-Mediated Gene Transfer into Established CNS Cell Lines, Primary Glial Cells, and in Vivo

1998 ◽  
Vol 7 (2) ◽  
pp. 175-185 ◽  
Author(s):  
Patrick Kofler ◽  
Bettina Wiesenhofer ◽  
Christine Rehrl ◽  
Gottfried Baier ◽  
Günter Stockhammer ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Glial Cells ◽  
Transfection Efficiency ◽  
Primary Cultures ◽  
C6 Glioma Cells ◽  
C6 Cells ◽  
Adult Rats ◽  
Neuronal Precursor Cells

Sufficient gene transfer into CNS-derived cells is the most crucial step to develop strategies for gene therapy. In this study liposome-mediated gene transfer using a β-galactosidase (β-GAL) reporter gene was performed in vitro (C6 glioma cells, NT2 neuronal precursor cells, 3T3 fibroblasts, primary glial cells) and in vivo. Using Trypan blue exclusion staining, optimal lipid concentration was observed in the range of 10-12 μg/mL. Under optimal conditions (80,000 cells/16 mm well, incubation overnight, lipid/DNA ratio = 1:18) a high transfection rate was achieved (<9% for C6 cells; <1% for NT2 cells). In primary cultures of glial cells a fair amount of positive stained cells (glial cell) was found, but the transfection efficiency was lower (<0.1%). A “boost-lipofection” markedly increased (twice) lipofection efficiency in C6 cells. Expression of β-GAL reached a maximum after 3-5 days. When the liposome–DNA complexes were injected/infused directly into the brains of adult rats, several weakly stained cells could be observed in the brain region adjacent to the injection site. It is concluded that liposome-mediated gene transfer is an efficient method for gene transfer into CNS cells in vitro, but the transfection efficiency into the rat brain in vivo is far too low and therefore not applicable.

scholarly journals Foreign Gene Expression in the Mouse Cauda Epididymis is Regulated by Androgens

2018 ◽  
Vol 2 (4) ◽  
pp. 671-678
Author(s):  
Pedro Esponda
Keyword(s):  
Gene Transfer ◽  
Reproductive Tract ◽  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Female Reproductive Tract ◽  
Foreign Gene ◽  
Total Period ◽  
Green Fluorescent ◽  
In Vivo Gene Transfer

This paper deals with the efficiency of in vivo gene transfer to the mouse cauda epididymis and its relation to androgens. Previous experiments in the female reproductive tract have indicated that the efficiency of transfection is related to the hormonal stage of the animal, nevertheless no analysis have been done in the male tract. We used in vivo gene transfer to the mouse cauda epididymis employing a gene construction that expresses the Green Fluorescent Protein (GFP). Untreated and Testosterone treated males were employed. Testosterone injections (5 μg/g weight) were done from 2 days before the gene transfer, and treatment continued each day during a total period of 15 days. Fluorescence microscopy observations showed the expression of GFP in the cytoplasm of the principal cells in the epididymal tubules. The application of the QWin Program that measures the percentage of fluorescent areas showed that they are increased in the epididymis of treated males. This increase was particularly observed two days after gene injections (from 32.24 % in untreated animals to 47.62 % in testosterone treated males) and after seven days (from 29.98 % to 43.05 %). The possibility to improve transfection efficiency would increase the knowledge on epididymal physiology and would permit to modify the fertilizing capacity in mammals.

scholarly journals Liver-Directed Gamma Interferon Gene Delivery in Chronic Hepatitis C

2005 ◽  
Vol 79 (21) ◽  
pp. 13412-13420 ◽  
Author(s):  
Eui-Cheol Shin ◽  
Ulrike Protzer ◽  
Andreas Untergasser ◽  
Stephen M. Feinstone ◽  
Charles M. Rice ◽  
...  
Keyword(s):  
T Cells ◽  
Chronic Hepatitis ◽  
Hepatitis C ◽  
Gene Transfer ◽  
Peripheral Blood ◽  
Ex Vivo ◽  
Ifn Γ ◽  
Cellular Immune

ABSTRACT Gamma interferon (IFN-γ) has been shown to inhibit replication of subgenomic and genomic hepatitis C virus (HCV) RNAs in vitro and to noncytolytically suppress hepatitis B virus (HBV) replication in vivo. IFN-γ is also known for its immunomodulatory effects and as a marker of a successful cellular immune response to HCV. Therapeutic expression of IFN-γ in the liver may therefore facilitate resolution of chronic hepatitis C, an infection that is rarely resolved spontaneously. To analyze immunomodulatory and antiviral effects of liver-specific IFN-γ expression in vivo, we intravenously injected two persistently HCV-infected chimpanzees twice with a recombinant, replication-deficient HBV vector and subsequently with a recombinant adenoviral vector. These vectors expressed human IFN-γ under control of HBV- and liver-specific promoters, respectively. Gene transfer resulted in a transient increase of intrahepatic IFN-γ mRNA, without increase in serum alanine aminotransferase levels. Ex vivo analysis of peripheral blood lymphocytes demonstrated enhanced CD16 expression on T cells and upregulation of the liver-homing marker CXCR3. Moreover, an increased frequency of HCV-specific T cells was detected ex vivo in the peripheral blood and in vitro in liver biopsy-derived, antigen-nonspecifically expanded T-cell lines. None of these immunologic effects were observed in the third chimpanzee injected with an HBV control vector. Despite these immunologic effects of the experimental vector, however, IFN-γ gene transfer did not result in a significant and long-lasting decrease of HCV titers. In conclusion, liver-directed IFN-γ gene delivery resulted in HCV-specific and nonspecific activation of cellular immune responses but did not result in effective control of HCV replication.

Functional Amphiphiles for Gene Delivery

MRS Bulletin ◽  
2005 ◽  
Vol 30 (9) ◽  
pp. 647-653 ◽  
Author(s):  
Philippe Barthélémy ◽  
Michel Camplo
Keyword(s):  
Gene Transfer ◽  
Gene Delivery ◽  
Viral Vectors ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Research Activity ◽  
Safety Issues ◽  
Significant Research

AbstractThe design of safe and efficient gene transfer vectors remains one of the key challenges in gene therapy. Despite their remarkable transfection efficiency, viral vectors suffer from known safety issues. Consequently, significant research activity has been undertaken to develop nonviral approaches to gene transfer during the last decade. Numerous academic and industrial research groups are investigating synthetic cationic vectors, such as cationic amphiphiles, with the objective of increasing the gene transfection activity. Within this area, the development of functional synthetic vectors that respond to local environmental effects have met with success. These synthetic vectors are based on mechanistic principles and represent a significant departure from earlier systems. Many of these systems for gene delivery in vitro and in vivo are discussed in this article.

scholarly journals Senile Octodon degus Reproductive Strategy: Morphometric and Histological Description of the Penis

2018 ◽  
Vol 2 (4) ◽  
pp. 679-688
Author(s):  
María Reyes ◽  
Eduardo Bustos-Obregón ◽  
Mariana Rojas
Keyword(s):  
Green Fluorescent Protein ◽  
Gene Transfer ◽  
Reproductive Tract ◽  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Female Reproductive Tract ◽  
Total Period ◽  
Green Fluorescent ◽  
In Vivo Gene Transfer

This paper deals with the efficiency of in vivo gene transfer to the mouse cauda epididymis and its relation to androgens. Previous experiments in the female reproductive tract have indicated that the efficiency of transfection is related to the hormonal stage of the animal, nevertheless no analysis have been done in the male tract. We used in vivo gene transfer to the mouse cauda epididymis employing a gene construction that expresses the Green Fluorescent Protein (GFP). Untreated and Testosterone treated males were employed. Testosterone injections (5 μg/g weight) were done from 2 days before the gene transfer, and treatment continued each day during a total period of 15 days. Fluorescence microscopy observations showed the expression of GFP in the cytoplasm of the principal cells in the epididymal tubules. The application of the QWin Program that measures the percentage of fluorescent areas showed that they are increased in the epididymis of treated males. This increase was particularly observed two days after gene injections (from 32.24 % in untreated animals to 47.62 % in testosterone treated males) and after seven days (from 29.98 % to 43.05 %). The possibility to improve transfection efficiency would increase the knowledge on epididymal physiology and would permit to modify the fertilizing capacity in mammals.

Reducing Stem Cell Loss and Ex Vivo Differentiation during Lentivirus Gene Transfer to Murine Stem Cells - an Ultra-Short Transduction Protocol.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2113-2113
Author(s):  
Peter Kurre ◽  
Ponni Anandakumar ◽  
Vladimir A. Lesnikov ◽  
Hans-Peter Kiem
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Gene Transfer ◽  
Ex Vivo ◽  
Lentivirus Vectors ◽  
Ex Vivo Culture ◽  
Marrow Cells

Abstract Most gene transfer models using Moloney murine leukemia virus (MLV) - derived vectors to target hematopoietic repopulating cells require progenitor cell enrichment and extended ex vivo culture for efficient long-term marking. Both may result in qualitative, and/or quantitative, loss of stem cells thereby limiting gene transfer rates in vivo. This can be a critical obstacle in candidate applications with exhausted autologous stem cell pools, such as Fanconi Anemia. Among the advantages of HIV-derived lentivirus vectors is their ability to transduce non dividing cells, permitting shortened ex vivo culture durations while maintaining gene transfer to long-term repopulating cells. We have previously reported long-term gene transfer rates of 12–40% after VSV-G/ lentivirus vector transduction of murine stem cells by targeting unseparated marrow cells after reduced prestimulation and a single 12 hour vector exposure (Kurre et al., Mol. Ther. 2004 Jun;9(6):914–22). We herein report studies showing maintenance of gene transfer efficiency in this model at drastically reduced ex vivo vector exposure times. In initial in vitro experiments we studied cytokine support, vector particle density, and minimum exposure duration requirements for efficient gene transfer to unseparated marrow cells. We determined that fibronectin fragment support was critical in maintaining minimum gene transfer efficiencies, even during brief 1, or 3-hour exposures. In an effort to extend these in vitro findings targeting a mixed leukocyte population and explore the feasibility in vivo, we next performed repopulation experiments in myeloablated murine recipients. Unseparated marrow cells harvested from donor animals were depleted of red blood cells, washed and immediately transduced on fibronectin fragment in the presence of murine stem cell factor. Following a 1 hour exposure to lentivector (VSV-G/RRLsin-cPPThPGK-EGFPwpre), cells were washed repeatedly, resuspended and injected into myeloablated recipients (n=10). Animals showed ready hematopoietic reconstitution and demonstrated average GFP marking of 31% (range: 17–41.2%) in peripheral blood 20 weeks after transplantation. Gene marking in secondary recipients 9 weeks after reconstitution (n=15, 3 recipient animals per donor) persisted at 29% on average (range 14.9–66%). Results also demonstrate transduction of granulocytes, B- and T-lymphocytes, as well as stable long-term GFP expression in primary and secondary animals. Copy number determination by real-time PCR in marrow cells from primary recipients shows an average of 4 proviral copies (range 2.1–8.1) per GFP-expressing cell. Our studies confirm that HIV-derived lentivirus vectors are ideally suited for the transduction of murine long-term repopulating cells. We hypothesize that ultra-short transduction actively preserves stem cell content in the inoculum. Moreover, this protocol represents an ideal platform for subsequent in vivo selection to achieve complete phenotype correction and high-level therapeutic chimerism required for some applications. We anticipate that our strategy may prove particularly useful in situations where the target stem cell quantity is greatly limited and cells are of poor ex vivo viability.

scholarly journals Dynamic tracking and identification of tissue-specific secretory proteins in the circulation of live mice

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Kwang-eun Kim ◽  
Isaac Park ◽  
Jeesoo Kim ◽  
Myeong-Gyun Kang ◽  
Won Gun Choi ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Ex Vivo ◽  
Secretory Protein ◽  
Spatiotemporal Dynamics ◽  
Whole Body ◽  
Accurate Information ◽  
Secretory Proteins ◽  
Specific Cell

AbstractSecretory proteins are an essential component of interorgan communication networks that regulate animal physiology. Current approaches for identifying secretory proteins from specific cell and tissue types are largely limited to in vitro or ex vivo models which often fail to recapitulate in vivo biology. As such, there is mounting interest in developing in vivo analytical tools that can provide accurate information on the origin, identity, and spatiotemporal dynamics of secretory proteins. Here, we describe iSLET (in situ Secretory protein Labeling via ER-anchored TurboID) which selectively labels proteins that transit through the classical secretory pathway via catalytic actions of Sec61b-TurboID, a proximity labeling enzyme anchored in the ER lumen. To validate iSLET in a whole-body system, we express iSLET in the mouse liver and demonstrate efficient labeling of liver secretory proteins which could be tracked and identified within circulating blood plasma. Furthermore, proteomic analysis of the labeled liver secretome enriched from liver iSLET mouse plasma is highly consistent with previous reports of liver secretory protein profiles. Taken together, iSLET is a versatile and powerful tool for studying spatiotemporal dynamics of secretory proteins, a valuable class of biomarkers and therapeutic targets.

Limiting Carryover Events Resulting from Cell Surface Retention of VSV-G Pseudotyped HIV-Lentivector Particles on Hematopoietic Targets

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3735-3735
Author(s):  
Lee O’Neill ◽  
Yung-Wei Pan ◽  
Amy M. Skinner ◽  
Peter Kurre
Keyword(s):  
Gene Transfer ◽  
Cell Surface ◽  
Ex Vivo ◽  
Target Cells ◽  
Vector Particle ◽  
Dependent Manner ◽  
Dose Dependent ◽  
Vector Particles

Abstract Preclinical evidence and clinical trials speak to the therapeutic potential of retrovirus vectors for the heritable genetic modification of cells. Careful evaluation of the antecedent risks is critical to move these applications forward. Others previously demonstrated the persistence of intact vector particles on the surface of target cells. Inadvertent particle transfer after in vivo applications could lead to the transduction of bystander tissues, or provoke immunological responses. We recently demonstrated prolonged adherence of VSV-G pseudotyped, HIV-1 derived lentivirus particles after ex vivo transduction culture of murine hematopoietic target cells (1°) with subsequent transduction of secondary (2°) targets in vitro and in vivo. Extended particle adherence is independent of Env pseudotype and routine wash procedures (Pan et al., J Virol. Jan 2007). We hypothesized that unwanted carryover could be minimized by disrupting the vector particle attachment to 2° cells while maintaining uptake to 1° targets. Initial studies indicated that the transduction of 1° targets at 4°C (to prevent uptake) for up to 6 hours followed by serial PBS washes and subsequent direct co-culture with fibroblasts resulted in undiminished 2° gene transfer compared to transduction at 37°C. Conversely, post-transduction exposure to escalating concentrations of citric acid resulted in a systematic decrease in both 1° and 2° gene transfer rates. This is consistent with separable mechanisms for pH sensitive VSV-G mediated uptake of particles in 1° targets and the receptor independent attachment responsible for carryover and 2° transduction, respectively. Glycosaminoglycans, including heparin, quantitatively bind to pseudotyped vector particles. We found that exposure of particles to heparin effectively abrogated subsequent transduction of cells by disrupting attachment. Remarkably, serial heparin washes at the conclusion of transduction had only minimal effects on gene transfer to 1° targets, but resulted in a two-log reduction in 2° gene transfer. Increases in the concentration of protamine sulfate (a polycation) during transduction partly reversed the effect of heparin (a polyanion), demonstrating the residual impact of electrostatic interactions on attachment of retrovirus particles from the 1° cell. In further studies we showed that trypsin washes following vector exposure incompletely cleaved 1° cell surface bound particles while pronase effectively degraded cell surface bound particles in a dose dependent manner, abrogating carryover. Because pronase at high concentrations also compromised cell surface epitope integrity we studied the expression of chemokine receptor (CXCR) 4, both a critical mediator of progenitor cell homing to the bone marrow and a representative protease-sensitive surface molecule. These experiments revealed a dose dependent degradation of CXCR4 on the cell surface of 1° target cells and rapid regeneration within three hours, critical for applications involving the injection of ex vivo modified hematopoietic cells. In conclusion, our results demonstrate that select wash procedures can disrupt the ability of virus particles to bind secondary targets, degrade residual surface bound particles and reduce gene transfer to inadvertent 2° targets in vitro by up to 99%. These studies are important first steps in understanding and limiting inadvertent carryover in the context of gene therapy while maximizing target cell transduction.

Sign in / Sign up

Export Citation Format

Share Document