Retroviral Gene Delivery Research Articles

Hematopoietic stem cells expanded by fibroblast growth factor-1 are excellent targets for retrovirus-mediated gene delivery

For the study of the function of genes of interest in hematopoietic stem cells (HSCs) and for successful gene therapy, it is fundamental to develop a method of efficient gene transfer into HSCs. In mice experiments, efforts have been made to raise the transduction efficiency by modifying the vectors, administrating 5-fluorouracil (5-FU) to donor mice, selecting cytokine cocktails to better sustain the long-term repopulating potential of the stem cells, and so on. The objective of this study is to examine whether the use of fibroblast growth factor-1 (FGF-1)-expanded bone marrow cells provide an improved source for retroviral gene delivery to HSCs. Unfractionated bone marrow cells from one mouse were cultured in serum-free medium containing FGF-1. Both floating and attached cells were transferred to retronectin precoated dishes and infected with virus supernatant from MP34 cells stably transduced with pMY/GFP retrovirus. After 3-day infection, the green fluorescence protein-positive fraction was sorted and the cells were transplanted to lethally irradiated mice. The experiments illustrated that the number of bone marrow-derived competitive repopulation units (CRUs) was increased from 600 to 9300 per mouse after a 3-week culture period with FGF-1. Following retroviral transduction of the expanded cells, the absolute number of sorted retrovirus-transduced CRUs was 4200. Using these retrovirus-transduced cells in noncompetitive reconstitution assay, we achieved radiation protection and long-term bone marrow reconstitution in 100% of the recipients with average myeloid and lymphoid chimerisms of 70% and 50%, respectively, even if we transplanted 150 recipients with cells derived from a single donor mouse. In conclusion, FGF-1-expanded bone marrow cells constitute an excellent source of stem cells that could be used in a range of gene delivery protocols.

Retroviral Gene Delivery from Engineering Perspective

Although a few physical and chemical approaches have been documented to enhance the efficiency of retrovirus-mediated gene transfer, systematic analysis of the processes involved in the retroviral transduction system remains to be important for further improvement. With the aid of reasonable mathematical description of an experimental system, we can therefore predict and optimize the retroviral gene delivery on a quantitative basis of understanding.

Gene therapy for severe combined immunodeficiencies

Severe combined immune deficiencies (SCIDs) are a group of monogenic diseases resulting in profound disturbances of lymphocyte development and function. Affected individuals are prone to life-threatening infections and without treatment do not survive beyond the first year of life. Haematopoietic stem cell transplantation from a well-matched donor offers high rates of survival, but in the absence of a suitable matched donor, parental haploidentical transplants are associated with greater complications, lower success rates and in some instances poor long-term immune recovery. Alternative therapeutic options based on correction of the defective gene by retroviral gene delivery have been used to correct X-linked SCID (SCID-X1) and adenosine deaminase-deficient SCID (ADA-SCID). A number of clinical trials have established that ex vivo gene transfer into haematopoietic progenitor cells allows effective recovery of immune defects and that gene therapy can offer a successful alternative to transplantation. The development of leukaemia as a result of insertional mutagenesis in one trial of gene therapy for SCID-X1 has raised concerns regarding the toxicity of retroviral vector-based gene delivery. These side effects are now being studied in detail and measures to prevent such events through alternative vectors delivery systems are in development at present.

FADD and caspase-8 are required for cytokine-induced proliferation of hemopoietic progenitor cells

AbstractThe role of caspase-8 and its adaptor Fas-associated death domain (FADD) in lymphocyte apoptosis is well defined, but their functions in other hemopoietic lineages are not clear. We were unable to generate transgenic mice expressing dominant inhibitors of FADD or caspase-8 in hemopoietic cells, possibly because their expression may have precluded production of vital hemopoietic cells. When using a retroviral gene delivery system, fetal liver stem cells expressing a dominant-negative mutant of FADD (FADD-DN) were unable to generate myeloid or lymphoid cells upon transplantation into lethally irradiated mice. However, fetal liver stem cells expressing very low levels of the caspase-8 inhibitor cytokine response modifier A (CrmA) could reconstitute the hemopoietic system. This level of CrmA expression provided some protection against Fas ligand (FasL)–induced apoptosis and promoted accumulation of myeloid cells in the bone marrow, but it did not inhibit mitogen-induced proliferation of B or T lymphocytes. Using an in vitro colony formation assay, we found that fetal liver stem cells expressing FADD-DN, CrmA, or a dominant-negative mutant of caspase-8 could not proliferate in response to cytokine stimulation. These data demonstrate that the enzymatic activity of caspase-8 and its adaptor FADD are required for cytokine-induced proliferation of hemopoietic progenitor cells.

484. Ultrasound-Mediated Microbubble Destruction and Retroviral Gene Delivery

Although precise and stable transgene expression can be achieved by direct injection of naked DNA into skeletal muscle; therapeutic, non-invasive gene therapy for a range of less accessible tissues ultimately requires systemic introduction of a vector with subsequent targeted delivery. Current approaches tend to focus on vector modification to ease entry and intracellular passage. Liposomal or lipid-based delivery of naked DNA increases uptake by reducing electrostatic repulsion and encouraging endocytosis at the cell membrane; however, uptake in the liver and spleen causes vector wastage and undesirable non-target transgene expression. Adenoviral vectors are more efficient, but they share with liposomal methods a transient transgene expression, high non-specific delivery and a tendency to accumulate in the liver, whilst retroviral vectors mediate stable gene delivery but are not easily produced in sufficient quantity; in neither case are efficient techniques for targeted delivery established.

Metabolism and toxicity of arsenic in human urothelial cells expressing rat arsenic (+3 oxidation state)-methyltransferase

The enzymatic methylation of inorganic As (iAs) is catalyzed by As(+3 oxidation state)-methyltransferase (AS3MT). AS3MT is expressed in rat liver and in human hepatocytes. However, AS3MT is not expressed in UROtsa, human urothelial cells that do not methylate iAs. Thus, UROtsa cells are an ideal null background in which the role of iAs methylation in modulation of toxic and cancer-promoting effects of this metalloid can be examined. A retroviral gene delivery system was used in this study to create a clonal UROtsa cell line (UROtsa/F35) that expresses rat AS3MT. Here, we characterize the metabolism and cytotoxicity of arsenite (iAs III) and methylated trivalent arsenicals in parental cells and clonal cells expressing AS3MT. In contrast to parental cells, UROtsa/F35 cells effectively methylated iAs III, yielding methylarsenic (MAs) and dimethylarsenic (DMAs) containing either As III or As V. When exposed to MAs III, UROtsa/F35 cells produced DMAs III and DMAs V. MAs III and DMAs III were more cytotoxic than iAs III in UROtsa and UROtsa/F35 cells. The greater cytotoxicity of MAs III or DMAs III than of iAs III was associated with greater cellular uptake and retention of each methylated trivalent arsenical. Notably, UROtsa/F35 cells were more sensitive than parental cells to the cytotoxic effects of iAs III but were more resistant to cytotoxicity of MAs III. The increased sensitivity of UROtsa/F35 cells to iAs III was associated with inhibition of DMAs production and intracellular accumulation of MAs. The resistance of UROtsa/F35 cells to moderate concentrations of MAs III was linked to its rapid conversion to DMAs and efflux of DMAs. However, concentrations of MAs III that inhibited DMAs production by UROtsa/F35 cells were equally toxic for parental and clonal cell lines. Thus, the production and accumulation of MAs III is a key factor contributing to the toxicity of acute iAs exposures in methylating cells.

Enhanced retroviral gene delivery in ultrasonic standing wave fields

Enhancement of retroviral transduction efficiency has been achieved by several physical and chemical approaches. However, the application of those methods is hampered by not easily scalable configurations. In this study, instead of looking into the effect of sonoporation, the potential of ultrasonic standing wave fields (USWF) to facilitate retroviral transduction rate was explored. We reasoned that, driven by the primary acoustic radiation force, suspended cells moved to the pressure nodal planes first and formed cell bands. Nanometer-sized retroviruses, circulated between nodal planes by acoustic microstreaming, then used the preformed cell bands as the nucleating sites to attach on. As a result, the encounter opportunity between retroviruses and cells was increased and further facilitated the gene delivery efficiency. Our results showed that mega-Hertz USWF brought K562 erythroleukemia cells (10(6) cells/ml) and vesicular stomatitis virus G-protein (VSV-G) pseudotyped retroviruses (titer of 5 x 10(6) CFU/ml) into close contact at the pressure nodal planes, yielding a four-fold increment of enhanced green fluorescent protein transgene expression after 5-min USWF exposure in the presence of Polybrene. Furthermore, with a fixed titer of retrovirus, the transduction rate was augmented with the increase of cell concentration. In summary, USWF offer a feasible means to enhance retroviral transduction efficiency in large-scale settings.

Synergy between Genetic and Tissue Engineering: Runx2 Overexpression and in Vitro Construct Development Enhance in Vivo Mineralization

Tissue engineering has emerged as a promising strategy to generate bone-grafting substrates. These approaches, however, are limited by an insufficient supply of committed osteoprogenitor cells and dedifferentiation of osteogenic cells during in vitro culture. To address these limitations, we engineered bone marrow stromal cells to constitutively express the osteoblastic transcription factor Runx2/Cbfa1, using retroviral gene delivery. These Runx2-modified cells were integrated into three-dimensional polymeric scaffolds to create tissue-engineered constructs. Compared with control stromal cells, Runx2 overexpression significantly upregulated osteoblastic differentiation and mineralization in vitro and in vivo in an ectopic, nonosseous subcutaneous site. More importantly, in vitro construct development to create a mineralized template before implantation dramatically enhanced subsequent in vivo mineralized tissue formation, providing a novel templating tissue-engineering strategy to improve in vivo mineralization. Finally, Runx2 overexpression and in vitro construct development synergistically enhanced in vivo mineralization compared with in vitro construct development or genetic engineering alone. This work provides a novel integrated genetic and tissue-engineering strategy to create mineralized templates for generating robust bone-grafting material.

Exogenous Runx2 Expression Enhances in Vitro Osteoblastic Differentiation and Mineralization in Primary Bone Marrow Stromal Cells

Bone marrow stromal cells represent a promising cell source for cell-based therapeutic and bone tissue-engineering applications, but are restricted by a low frequency in healthy marrow, an age-related decrease in osteogenic capacity, and a propensity for dedifferentiation during in vitro expansion. To address these limitations, retroviral gene delivery was used to examine the effects of sustained and elevated expression of the Runx2 osteoblastic transcription factor on osteoblastic gene and protein expression and mineralization in primary rat bone marrow stromal cells. Runx2 overexpression upregulated several osteoblast-specific genes, including collagen type I and osteocalcin, and enhanced alkaline phosphatase activity and biological mineral deposition. Forced Runx2 expression in combination with dexamethasone increased matrix mineralization compared with exogenous Runx2 expression or dexamethasone treatment alone, whereas dexamethasone-free control cultures displayed minimal mineralization. These additive effects suggest complementary interactions between Runx2 and dexamethasone-responsive regulatory factors. Finally, Runx2 overexpression in stromal cell cultures undergoing considerable in vitro expansion resulted in higher matrix mineralization capacity compared with controls, which completely lost the ability to produce mineralized matrix even in the presence of dexamethasone. These findings provide a novel strategy for cell-based therapeutic applications requiring significant numbers of osteogenic cells to synthesize mineralized constructs for the treatment of large bone defects.

Coordinated expression of desmoglein 1 and desmocollin 1 regulates intercellular adhesion

Desmoglein 1 (Dsg1) is a component of desmosomes present in the upper epidermis and can be targeted by autoimmune antibodies or bacterial toxins, resulting in skin blistering diseases. These defects in tissue integrity are believed to result from compromised desmosomal adhesion; yet, previous attempts to directly test the adhesive roles of desmosomal cadherins using normally non-adherent L cells have yielded mixed results. Here, two complementary approaches were used to better resolve the molecular determinants for Dsg1-mediated adhesion: (1) a tetracycline-inducible system was used to modulate the levels of Dsg1 expressed in L cell lines containing desmocollin 1 (Dsc1) and plakoglobin (PG) and (2) a retroviral gene delivery system was used to introduce Dsg1 into normal human epidermal keratinocytes (NHEK). By increasing Dsg1 expression relative to Dsc1 and PG, we were able to demonstrate that the ratio of Dsg1:Dsc1 is a critical determinant of desmosomal adhesion in fibroblasts. The distribution of Dsg1 was organized at areas of cell–cell contact in the multicellular aggregates that formed in these suspension cultures. Similarly, the introduction of Dsg1 into NHEKs was capable of increasing the aggregation of single cell suspensions and further enhanced the adhesive strength of intact epithelial sheets. Endogenous Dsc1 levels were also increased in NHEKs containing Dsg1, providing further support for the coordination of these two desmosomal cadherins in regulating adhesive structures. These Dsg1-mediated effects on intercellular adhesion were directly related to the presence of an intact extracellular domain as ETA, a toxin that specifically cleaves this desmosomal cadherin, inhibited adhesion in both fibroblasts and keratinocytes. Collectively, these observations demonstrate that Dsg1 promotes the formation of intercellular adhesion complexes and suggest that the relative level of Dsg and Dsc expressed at the cell surface regulates this adhesive process.

Runx2/Cbfa1 stimulates transdifferentiation of primary skeletal myoblasts into a mineralizing osteoblastic phenotype

Runx2, a transcriptional activator downstream of bone morphogenetic protein (BMP) signaling, is essential to osteoblastic differentiation and bone formation and maintenance. BMPs activate complex signaling networks, utilizing numerous signaling molecules and transcription factors to induce expression of osteoblastic markers in mesenchymal cell types. However, the role of Runx2 in this process, particularly in an environment independent of the other regulatory elements modulated by BMPs, remains poorly understood. In the present study, we used retroviral gene delivery to examine the effects of sustained Runx2 expression in primary myoblasts. Runx2 inhibited myogenesis, as demonstrated by suppression of MyoD and myogenin mRNA levels and reduced myotube formation. Additionally, Runx2-stimulated osteogenesis including osteoblastic gene expression, alkaline phosphatase activity, and biological mineral deposition. Notably, these osteogenic markers were induced to significantly greater levels than those observed in BMP-2-treated controls. These results demonstrate that direct exogenous expression of the Runx2 transcription factor, only one of numerous downstream targets of BMP signaling, is sufficient to induce transdifferentiation of myogenic cells into a mineralizing osteogenic lineage. This work underscores the potency of Runx2 as a regulator of osteogenesis and cell differentiation and provides new insights into the plasticity of committed mesenchymal cells.

401. Engineering Retroviral Vectors for Stem Cell Therapies - Overcoming the Effect of Gene Silencing

The combined application of gene therapy and stem cell engineering for tissue regeneration have has drawn immense recent interest. In recent clinical trials with several patients who suffered from different forms of severe combined immunodeficiency syndrome (SCID), corrective genes were introduced into the patients' hematopoeitic stem cells via Moloney Murine Leukemia (MoMLV) viral vectors. This marked the first successful reports of gene therapy clinical trials. However, improvements to the vectors are needed to improve gene expression and safety of retroviral gene delivery to stem cells. For example, MoMLV vectors suffer from transcriptional silencing in many stem cell systems in vitro and in vivo, and enhancing gene expression after vector delivery could improve therapeutic efficacy, as well as improve safety by lowering the necessary dosage. The mechanism by which stem cells shutdown expression from an integrated retrovirus is not completely understood, but it is thought to be achieved via a combination of DNA methylation, histone modification, and chromatin remodeling. So far, five different regions within the retroviral genome have been identified as recognized targets that promote silencing, most of which lie within the U3 region of the viral Long Terminal Repeat (LTR). The effect of silencing can be reduced by modification and/or deletions of these regions; however, complete relief has never been achieved. Furthemore, improvements in long-term expression by these LTR modifications usually come at the cost of significant reductions in viral titer.

The use of retroviral vectors for gene therapy-what are the risks? A review of retroviral pathogenesis and its relevance to retroviral vector-mediated gene delivery

Retroviral vector-mediated gene transfer has been central to the development of gene therapy. Retroviruses have several distinct advantages over other vectors, especially when permanent gene transfer is the preferred outcome. The most important advantage that retroviral vectors offer is their ability to transform their single stranded RNA genome into a double stranded DNA molecule that stably integrates into the target cell genome. This means that retroviral vectors can be used to permanently modify the host cell nuclear genome. Recently, retroviral vector-mediated gene transfer, as well as the broader gene therapy field, has been re-invigorated with the development of a new class of retroviral vectors which are derived from lentiviruses. These have the unique ability amongst retroviruses of being able to infect non-cycling cells. Vectors derived from lentiviruses have provided a quantum leap in technology and seemingly offer the means to achieve significant levels of gene transfer in vivo.The ability of retroviruses to integrate into the host cell chromosome also raises the possibility of insertional mutagenesis and oncogene activation. Both these phenomena are well known in the interactions of certain types of wild-type retroviruses with their hosts. However, until recently they had not been observed in replication defective retroviral vector-mediated gene transfer, either in animal models or in clinical trials. This has meant the potential disadvantages of retroviral mediated gene therapy have, until recently, been seen as largely, if not entirely, hypothetical. The recent clinical trial of γc mediated gene therapy for X-linked severe combined immunodeficiency (X-SCID) has proven the potential of retroviral mediated gene transfer for the treatment of inherited metabolic disease. However, it has also illustrated the potential dangers involved, with 2 out of 10 patients developing T cell leukemia as a consequence of the treatment. A considered review of retroviral induced pathogenesis suggests these events were qualitatively, if not quantitatively, predictable. In addition, it is clear that the probability of such events can be greatly reduced by relatively simple vector modifications, such as the use of self-inactivating vectors and vectors derived from non-oncogenic retroviruses. However, these approaches remain to be fully developed and validated. This review also suggests that, in all likelihood, there are no other major retroviral pathogenetic mechanisms that are of general relevance to replication defective retroviral vectors. These are important conclusions as they suggest that, by careful design and engineering of retroviral vectors, we can continue to use this gene transfer technology with confidence.

Early osteoblastic differentiation induced by dexamethasone enhances adenoviral gene delivery to marrow stromal cells

We investigated the implications of induced osteogenic differentiation on gene delivery in multipotent rat marrow stromal cells (MSCs). Prior to genetic manipulation cells were cultured with or without osteogenic supplements (5 × 10 −8 M dexamethasone, 160 μM l-ascorbic acid 2-phosphate, and 10 mM β-glycerophosphate). Comparison of liposome, retroviral, and adenoviral vectors demonstrated that all three vectors could mediate gene delivery to primary rat MSCs. When these vectors were applied in the absence or presence of osteogenic supplements, we found that MSCs differentiated prior to transduction with adenovirus type 5 vectors produced a 300% increase in transgene expression compared to MSCs that were not exposed to osteogenic supplements. This differentiation effect appeared specific to adenoviral mediated gene delivery, since there was minimal increase in retroviral gene delivery and no increase in liposome gene delivery when MSCs were treated with osteogenic supplements. In addition, we also determined this increase in transgene production to occur at a higher concentration of dexamethasone (5 × 10 −8 M) in the culture medium of MSCs prior to adenoviral transduction. We found that this increased transgene production could be extended to the osteogenic protein, human bone morphogenetic protein 2 (hBMP-2). When delivered by an adenoviral vector, hBMP-2 transgene production could be increased from 1.4 ng/10 5 cells/3 days to 4.3 ng/10 5 cells/3 days by culture of MSCs with osteogenic supplements prior to transduction. These results indicate that the utility of MSCs as a therapeutic protein delivery mechanism through genetic manipulation can be enhanced by pre-culture of these cells with dexamethasone.

Addressing cell-sourcing limitations with gene therapy.

Genetic engineering with Run/spl times/2/Cbfa 1 for an alternative to biological grafts. Forced Runx2 expression via retroviral gene delivery promotes osteoblastic differentiation and mineralization in bone marrow stromal cells and stimulates conversion of myoblasts and dermal fibroblasts into a mineralizing osteoblastic phenotype. This genetic engineering strategy provides a promising approach to overcome cell-sourcing limitations in bone tissue engineering.

Lentiviruses in gene therapy clinical research.

Gene therapy vectors derived from lentiviruses offer many potentially unique advantages over more conventional retroviral gene delivery systems. Principal amongst these is their ability to provide long-term and stable gene expression and to infect non-dividing cells, such as neurons. However, the use of lentiviral-based vectors in the clinic also raises specific safety and ethical issues. Concerns include the possible generation of replication competent lentiviruses during vector production, mobilisation of the vector by endogenous retroviruses in the genomes of patients, insertional mutagenesis leading to cancer, germline alteration resulting in trans-generational effects and dissemination of new viruses from gene therapy patients. Investigators proposing to conduct this type of research should take due account of the potential risks for interaction of lentiviral gene therapy vectors with other retroviral elements in human subjects, such as Human Immunodeficiency Virus. In addition, strict quality control for replication competent lentiviruses and suitable measurements of lentiviral infectious particle number will be required before these types of viral vector can proceed to the clinic.

Cell-type-dependent up-regulation of in vitro mineralization after overexpression of the osteoblast-specific transcription factor Runx2/Cbfal.

Functional expression of the transcriptional activator Runx2/Cbfal is essential for osteoblastic differentiation and bone formation and maintenance. Forced expression of Runx2 in nonosteoblastic cells induces expression of osteoblast-specific genes, but the effects of Runx2 overexpression on in vitro matrix mineralization have not been determined. To examine whether exogenous Runx2 expression is sufficient to direct in vitro mineralization, we investigated sustained expression of Runx2 in nonosteoblastic and osteoblast-like cell lines using retroviral gene delivery. As expected, forced expression of Runx2 induced several osteoblast-specific genes in NIH3T3 and C3H10T1/2 fibroblasts and up-regulated expression in MC3T3-E1 immature osteoblast-like cells. However, Runx2 expression enhanced matrix mineralization in a cell-type-dependent manner. NIH3T3 and IMR-90 fibroblasts overexpressing Runx2 did not produce a mineralized matrix, indicating that forced expression of Runx2 in these nonosteogenic cell lines is not sufficient to direct in vitro mineralization. Consistent with the pluripotent nature of the cell line, a fraction (25%) of Runx2-expressing C3H10T1/2 fibroblast cultures produced mineralized nodules in a viral supernatant-dependent manner. Notably, bone sialoprotein (BSP) gene expression was detected at significantly higher levels in mineralizing Runx2-infected C3H10T1/2 cells compared with Runx2-expressing cultures which did not mineralize. Treatment of Runx2-infected C3H10T1/2 cultures with dexamethasone enhanced osteoblastic phenotype expression, inducing low levels of mineralization independent of viral supernatant. Finally, Runx2 overexpression in immature osteoblast-like MC3T3-E1 cells resulted in acceleration and robust up-regulation of matrix mineralization compared with controls. These results suggest that, although functional Runx2 is essential to multiple osteoblast-specific activities, in vitro matrix mineralization requires additional tissue-specific cofactors, which supplement Runx2 activity.

Constitutive Expression of Preproendothelin in the Cardiac Neural Crest Selectively Promotes Expansion of the Adventitia of the Great Vessels in Vivo

Cardiac neural crest cells are essential for normal development of the great vessels and the heart, giving rise to a range of cell types, including both neuronal and non-neuronal adventitial cells and smooth muscle. Endothelin (ET) signaling plays an important role in the development of cardiac neural crest cell lineages, yet the underlying mechanisms that act to control their migration, differentiation, and proliferation remain largely unclear. We examined the expression patterns of the receptor, ET A, and the ET-specific converting enzyme, ECE-1, in the pharyngeal arches and great vessels of the developing chick embryo. In situ hybridization analysis revealed that, while ET A is expressed in the pharyngeal arch mesenchyme, populated by cardiac neural crest cells, ECE-1 expression is localized to the outermost ectodermal cells of the arches and then to the innermost endothelial cells of the great vessels. This dynamic pattern of expression suggests that only a subpopulation of neural crest cells in these regions is responsive to ET signaling at particular developmental time points. To test this, retroviral gene delivery was used to constitutively express preproET-1, a precursor of mature ET-1 ligand, in the cardiac neural crest. This resulted in a selective expansion of the outermost, adventitial cell population in the great vessels. In contrast, neither differentiation nor proliferation of neural crest-derived smooth muscle cells was significantly affected. These results suggest that constitutive expression of exogenous preproET-1 in the cardiac neural crest results in expansion restricted to an adventitial cell population of the developing great vessels.

Induction of ovarian cancer by defined multiple genetic changes in a mouse model system.

We have developed a mouse model for ovarian carcinoma by using an avian retroviral gene delivery technique for the introduction of multiple genes into somatic ovarian cells of adult mice. Ovarian cells from transgenic mice engineered to express the gene encoding the avian receptor TVA were efficiently infected in vitro with multiple vectors carrying coding sequences for oncogenes and marker genes. When target cells were derived from TVA transgenic mice deficient for p53, the addition of any two of the oncogenes c-myc, K-ras, and Akt were sufficient to induce ovarian tumor formation when infected cells were injected at subcutaneous, intraperitoneal, or ovarian sites. We demonstrated that the ovarian surface epithelium is the precursor tissue for these ovarian carcinomas, and that introduction of oncogenes causes phenotypic changes in the ovarian surface epithelial cells. The induced ovarian tumors in mice resembled human ovarian carcinomas in their rapid progression and intraperitoneal metastatic spread.

Transduction rate constant as more reliable index quantifying efficiency of retroviral gene delivery

Although the efficiency of retrovirus-mediated gene delivery can be enhanced by several physicochemical approaches reported (e.g., addition of polycations and spinoculation), systematic analysis of retroviral transduction combined with experimental data remains to be challenged. With the aid of a reasonable mathematical description of an experimental system, we can therefore predict and optimize the retroviral gene delivery on a quantitative basis of understanding. In this study, we formulated a mathematical model involved with diffusion, decay and uptake of retroviral vectors onto the target cells resided on a solid culture surface. The model was solved analytically by the Laplace transform method. The analytical solutions were then fitted with experimental data to compute two unknown parameters: concentration of infectious retrovirus and transduction rate constant. Our results showed that the concentration of infectious retrovirus determined by the titration method was approximately hundred-fold lower than the one calculated by fitting experimental data with the mathematical solutions. More importantly, effects of polycation (i.e., Polybrene) on ex vivo retroviral transduction were illustrated in a quantitative way by estimating the transduction rate constant, which represents a more reliable parameter to determine the degree of transduction of a retroviral vector to a given target cell.