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Dive into the research topics where Joseph M. Le Doux is active.

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Featured researches published by Joseph M. Le Doux.


Proceedings of the National Academy of Sciences of the United States of America | 2008

Engineering graded tissue interfaces

Jennifer E. Phillips; Kellie L. Burns; Joseph M. Le Doux; Robert E. Guldberg; Andrés J. García

Interfacial zones between tissues provide specialized, transitional junctions central to normal tissue function. Regenerative medicine strategies focused on multiple cell types and/or bi/tri-layered scaffolds do not provide continuously graded interfaces, severely limiting the integration and biological performance of engineered tissue substitutes. Inspired by the bone-soft tissue interface, we describe a biomaterial-mediated gene transfer strategy for spatially regulated genetic modification and differentiation of primary dermal fibroblasts within tissue-engineered constructs. We demonstrate that zonal organization of osteoblastic and fibroblastic cellular phenotypes can be engineered by a simple, one-step seeding of fibroblasts onto scaffolds containing a spatial distribution of retrovirus encoding the osteogenic transcription factor Runx2/Cbfa1. Gradients of immobilized retrovirus, achieved via deposition of controlled poly(l-lysine) densities, resulted in spatial patterns of transcription factor expression, osteoblastic differentiation, and mineralized matrix deposition. Notably, this graded distribution of mineral deposition and mechanical properties was maintained when implanted in vivo in an ectopic site. Development of this facile and robust strategy is significant toward the regeneration of continuous interfacial zones that mimic the cellular and microstructural characteristics of native tissue.


Journal of Virology | 2005

Rab9 GTPase Is Required for Replication of Human Immunodeficiency Virus Type 1, Filoviruses, and Measles Virus

James L. Murray; Manos Mavrakis; Natalie Mcdonald; Mamadi Yilla; Jinsong Sheng; William J. Bellini; Lijun Zhao; Joseph M. Le Doux; Michael W. Shaw; Chi-Cheng Luo; Jennifer Lippincott-Schwartz; Anthony Sanchez; Donald H. Rubin; Thomas W. Hodge

ABSTRACT Rab proteins and their effectors facilitate vesicular transport by tethering donor vesicles to their respective target membranes. By using gene trap insertional mutagenesis, we identified Rab9, which mediates late-endosome-to-trans-Golgi-network trafficking, among several candidate host genes whose disruption allowed the survival of Marburg virus-infected cells, suggesting that Rab9 is utilized in Marburg replication. Although Rab9 has not been implicated in human immunodeficiency virus (HIV) replication, previous reports suggested that the late endosome is an initiation site for HIV assembly and that TIP47-dependent trafficking out of the late endosome to the trans-Golgi network facilitates the sorting of HIV Env into virions budding at the plasma membrane. We examined the role of Rab9 in the life cycles of HIV and several unrelated viruses, using small interfering RNA (siRNA) to silence Rab9 expression before viral infection. Silencing Rab9 expression dramatically inhibited HIV replication, as did silencing the host genes encoding TIP47, p40, and PIKfyve, which also facilitate late-endosome-to-trans-Golgi vesicular transport. In addition, silencing studies revealed that HIV replication was dependent on the expression of Rab11A, which mediates trans-Golgi-to-plasma-membrane transport, and that increased HIV Gag was sequestered in a CD63+ endocytic compartment in a cell line stably expressing Rab9 siRNA. Replication of the enveloped Ebola, Marburg, and measles viruses was inhibited with Rab9 siRNA, although the nonenveloped reovirus was insensitive to Rab9 silencing. These results suggest that Rab9 is an important cellular target for inhibiting diverse viruses and help to define a late-endosome-to-plasma-membrane vesicular transport pathway important in viral assembly.


Biotechnology and Bioengineering | 1999

Kinetics of retrovirus production and decay

Joseph M. Le Doux; Howard E. Davis; Jeffrey R. Morgan; Martin L. Yarmush

There has been only limited success in using recombinant retroviruses to transfer genes for the purposes of human gene therapy, in part because the average number of genes delivered to the target cells (transduction efficiency) is often too low to achieve the desired therapeutic effect [Miller, AD. 1990. Blood 76:271-278; Mulligan RC. 1993. Science 260:926-932; Orkin SH, Motulsky AG. 1995. Report and recommendations of the panel to assess the NIH investment in research on gene therapy. Bethesda, MD: National Institutes of Health.]. One strategy to improve transduction efficiency is to focus on understanding and improving the processes used to produce recombinant retroviruses. In this report, we characterized the dynamics of retrovirus production and decay in batch cultures of virus producer cells using a simple mathematical model, a recombinant retrovirus encoding the Escherichia coli lacZ gene, and quantitative assays for virus activity and number. We found that the rate at which recombinant retroviruses spontaneously lose their activity (decay) is a strong function of temperature, decreasing roughly 2-fold for every 5 degrees C reduction in temperature, whereas the rate at which retroviruses are produced is only weakly affected by temperature, decreasing about 10% for every 5 degrees C reduction in temperature. In addition, we developed a simple mathematical model of virus production and decay that predicted that the virus titer in batch cultures of virus producer cells would reach a maximum steady-state at a rate that is inversely proportional to the virus decay rate and to a level that is proportional to the ratio of the virus production rate to the virus decay rate. Consistent with the model, we observed that the steady-state levels of virus titer increased more than 3-fold when the cell culture temperature was reduced from 37 to 28 degrees C. Despite their higher titers, virus stocks produced at 28 degrees C, when used in undiluted form so as to mimic human gene transfer protocols, did not transduce substantially more cells than virus stocks produced at 37 degrees C. The implications of our findings on the production of retroviruses for use in human gene therapy protocols are discussed.


Biotechnology Progress | 1999

Large‐Scale Processing of Recombinant Retroviruses for Gene Therapy

Stylianos T. Andreadis; Charles M. Roth; Joseph M. Le Doux; Jeffrey R. Morgan; Martin L. Yarmush

Gene therapy is a new therapeutic modality with the potential of treating inherited and acquired diseases. Several viral and physicochemical vehicles have been used for the transfer of genes to mammalian cells, but recombinant retroviruses are used in the majority of gene therapy clinical trials today. In this communication, we review the major concerns associated with the large‐scale production and processing of retroviral particles. While some of the current processes for manufacturing recombinant proteins will be applicable to recombinant retroviruses, the instability, sensitivity to inhibitors, complexity, and size of retroviral particles require that new technologies be designed and evaluated. Here, we examine those issues critical to the design of strategies for production, concentration, and purification as well as formulation and storage of recombinant retroviruses. Processes for large‐scale manufacturing of recombinant retroviruses that can produce high gene transfer efficiencies will have significant impact on the clinical implementation of gene therapy.


Human Gene Therapy | 2001

Complexation of Retrovirus with Cationic and Anionic Polymers Increases the Efficiency of Gene Transfer

Joseph M. Le Doux; Natalia Landázuri; Martin L. Yarmush; Jeffrey R. Morgan

Previously, we have demonstrated that chondroitin sulfate proteoglycans and glycosaminoglycans inhibit retrovirus transduction. While studying the mechanism of inhibition, we found that the combined addition of equal-weight concentrations (80 microg/ml) of Polybrene and chondroitin sulfate C to retrovirus stocks resulted in the formation of a high-molecular-weight retrovirus-polymer complex that could be pelleted by low-speed centrifugation. The pelleted complex contained more than 80% of the virus particles, but less than 0.3% of the proteins that were originally present in the virus stock. Surprisingly, the virus in the complex remained active and could be used to transduce cells. The titer of the pelleted virus, when resuspended in cell culture medium to the starting volume, was three-fold greater than the original virus stock. The selectivity (CFU/mg protein) of the process with respect to virus activity was more than 1000-fold. When the pelleted virus-polymer complex was resuspended in one-eighth of the original volume and used to transduce NIH 3T3 murine fibroblasts and primary human fibroblasts, gene transfer was increased 10- to 20-fold over the original unconcentrated retrovirus stock. The implications of our findings for the production, processing, and use of retrovirus stocks for human gene therapy protocols are discussed.


Journal of Virology | 2000

Toward a More Accurate Quantitation of the Activity of Recombinant Retroviruses: Alternatives to Titer and Multiplicity of Infection

Stylianos T. Andreadis; Thomas Lavery; Howard E. Davis; Joseph M. Le Doux; Martin L. Yarmush; Jeffrey R. Morgan

ABSTRACT In this paper, we present a mathematical model with experimental support of how several key parameters govern the adsorption of active retrovirus particles onto the surface of adherent cells. These parameters, including time of adsorption, volume of virus, and the number, size, and type of target cells, as well as the intrinsic properties of the virus, diffusion coefficient, and half-life (t 1/2), have been incorporated into a mathematical expression that describes the rate at which active virus particles adsorb to the cell surface. From this expression, we have obtained estimates of C vo, the starting concentration of active retrovirus particles. In contrast to titer,C vo is independent of the specific conditions of the assay. The relatively slow diffusion (D = 2 × 10−8 cm2/s) and rapid decay (t 1/2 = 6 to 7 h) of retrovirus particles explain why C vo values are significantly higher than titer values. Values ofC vo also indicate that the number of defective particles in a retrovirus stock is much lower than previously thought, which has implications especially for the use of retroviruses for in vivo gene therapy. With this expression, we have also computed AVC (active viruses/cell), the number of active retrovirus particles that would adsorb per cell during a given adsorption time. In contrast to multiplicity of infection, which is based on titer and is subject to the same inaccuracies, AVC is based on the physicochemical parameters of the transduction assay and so is a more reliable alternative.


Gene Therapy | 2006

Inducible regulation of Runx2-stimulated osteogenesis

Charles A. Gersbach; Joseph M. Le Doux; Robert E. Guldberg; Andrés J. García

Ex vivo gene therapy is a promising approach to orthopedic regenerative medicine. These strategies typically focus on the constitutive overexpression of osteogenic factors to induce osteoblastic differentiation and matrix mineralization. However, the unregulated production of osteoinductive molecules has also resulted in abnormal bone formation and tumorigenesis. To address these limitations, this work describes a retroviral system to deliver the Runx2 osteoblastic transcription factor under control of the tetracycline-inducible (tet-off) promoter in primary skeletal myoblasts. Runx2 expression was tightly regulated by anhydrotetracyline (aTc) concentration in cell culture media. Osteoblastic gene expression, alkaline phosphatase activity, and matrix mineralization were also controlled by aTc in a dose-dependent manner. Additionally, osteoblastic differentiation was temporally regulated by adding and removing aTc from the culture media. Engineered cells were seeded onto collagen scaffolds and implanted intramuscularly in the hind limbs of syngeneic mice. In vivo mineralization by these constructs was regulated by supplementing the drinking water with aTc, as demonstrated by micro-computed tomography and histological analyses. Collectively, these results present a novel system for regulating osteoblastic differentiation of a clinically relevant autologous cell source. This system is significant to developing controlled and effective orthopedic gene therapy strategies and studying the regulation of osteoblastic differentiation.


Journal of Virology | 2000

Erratum in print version of "Toward a More Accurate Quantitation of the Activity of Recombinant Retroviruses: Alternatives to Titer and Multiplicity of Infection"

Stylianos T. Andreadis; Thomas Lavery; Howard E. Davis; Joseph M. Le Doux; Martin L. Yarmush; Jeffrey R. Morgan

In this paper, we present a mathematical model with experimental support of how several key parameters govern the adsorption of active retrovirus particles onto the surface of adherent cells. These parameters, including time of adsorption, volume of virus, and the number, size, and type of target cells, as well as the intrinsic properties of the virus, diffusion coefficient, and half-life (t(1/2)), have been incorporated into a mathematical expression that describes the rate at which active virus particles adsorb to the cell surface. From this expression, we have obtained estimates of C(vo), the starting concentration of active retrovirus particles. In contrast to titer, C(vo) is independent of the specific conditions of the assay. The relatively slow diffusion (D = 2 x 10(-8) cm(2)/s) and rapid decay (t(1/2) = 6 to 7 h) of retrovirus particles explain why C(vo) values are significantly higher than titer values. Values of C(vo) also indicate that the number of defective particles in a retrovirus stock is much lower than previously thought, which has implications especially for the use of retroviruses for in vivo gene therapy. With this expression, we have also computed AVC (active viruses/cell), the number of active retrovirus particles that would adsorb per cell during a given adsorption time. In contrast to multiplicity of infection, which is based on titer and is subject to the same inaccuracies, AVC is based on the physicochemical parameters of the transduction assay and so is a more reliable alternative.


Journal of Gene Medicine | 2004

Complexation of retroviruses with charged polymers enhances gene transfer by increasing the rate that viruses are delivered to cells.

Natalia Landázuri; Joseph M. Le Doux

We have previously found that retrovirus transduction is enhanced when an anionic polymer (chondroitin sulfate C) is added to virus stocks that contain an equal weight concentration of a cationic polymer (Polybrene). This observation was unexpected given that previous work has shown that cationic polymers enhance transduction while anionic polymers have the opposite effect.


Biotechnology and Bioengineering | 1998

Removal of proteoglycans increases efficiency of retroviral gene transfer.

Joseph M. Le Doux; Jeffrey R. Morgan; Martin L. Yarmush

We have previously shown that medium conditioned by virus producer cells inhibits retrovirus transduction, and that a portion of the inhibitory activity is sensitive to chondroitinase ABC. In this study, we have quantitatively evaluated the fraction of the inhibitory activity that is due to chondroitinase ABC-sensitive material and partially characterized the inhibitors. The kinetics of chondroitinase ABC digestion of glycosaminoglycans and virus inhibitory activity in cell culture medium were measured, and the results used to estimate the amount of the chondroitinase ABC-sensitive virus inhibitory activity that was initially in the medium. We found that up to 76% of the inhibitory activity of medium conditioned by packaging cells derived from NIH 3T3 cells is sensitive to chondroitinase ABC. The remainder of the inhibitory activity is not sensitive to other glycosaminoglycan lyases (heparitinase I or heparinase I), which suggests that substances other than glycosaminoglycans or proteoglycans are present in virus stocks and inhibit transduction. To further characterize the inhibitors, proteoglycans from conditioned medium were purified by batch anion exchange and size exclusion chromatography. Two major size groups (100 kDa and 950 kDa) of proteoglycans were isolated. Transduction was inhibited 50% by 0.6 microg/mL of the high-molecular-weight proteoglycan or by 1.7 microg/mL of the low-molecular-weight proteoglycan. Significantly, the proteoglycans, because of their large size and poor sieving properties, coconcentrated with virus particles concentrated by ultrafiltration and prevented any significant increases in transduction efficiency. Transduction efficiencies of virus stocks were increased more than tenfold by ultrafiltration, but only when the concentrated virus was treated with chondroitinase ABC.

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Delfi Krishna

Georgia Institute of Technology

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Alisha A. Waller

Georgia Institute of Technology

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Andrés J. García

Georgia Institute of Technology

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Howard E. Davis

Massachusetts Institute of Technology

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Natalia Landazuri

Georgia Institute of Technology

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