Joseph Le Doux

This paper describes the problem-solving studio (PSS) learning environment. PSS was designed to teach students how to solve difficult analytical engineering problems without resorting to rote memorization of algorithms, while at the same time developing their deep conceptual understanding of the course topics. There are several key features of PSS. First, students work in teams of two to solve problems, working at the same table with another team of two. The student teams and tables are stable, remaining together for most of the semester. The teams work in a public, shared problem-solving space that allows in-class mentors (near peers of the students) and the instructor to observe and critique their work. The public nature of the work enables the instructor to provide students with real-time, situated feedback. In addition, it enables the instructor to tailor the challenge level to the needs of each team, such that the problem is too difficult for any one student to solve on their own, but reasonable enough that the team can solve it together, given the support that the PSS environment provides. We call this targeted adjustment of the problem's difficulty dynamic scaffolding. PSS provides the support students need through a specific set of participant structures that govern how the instructors, in-class mentors, and students interact during class. This paper describes the PSS approach and how it has been implemented in an entry-level course at Georgia Tech called " Conservation Principles of Biomedical Engineering (BMED 2210) ". Our results show that even though PSS emphasizes engineering problem-solving skills, the students' conceptual understanding of the material significantly improves, as measured by Shallcross' material and energy balances concept inventory. The implications of our work, particularly with respect to the use of PSS in flipped classrooms, are discussed.

Abstract: When most teachers hear the phrase" professional development," skepticism clouds their faces. But research-based professional development can provide interesting experiences and valuable content knowledge. The Research Experience for Teachers ( ...

We have previously shown that the envelope glycoproteins of human parainfluenza type 3 (HPIV3), F and HN, are able to pseudotype lentiviruses, but the titers of these viruses are too low for use in clinical gene transfer. In this study we investigated the cause of these low titers. We compared the mRNA and protein expression levels of HN and F in transfected cells and in cells infected with wild-type HPIV3. Transfected cells contained similar levels of HN and F cytosolic mRNA, but fewer cell-surface HN and F proteins (3.8- and 1.3-fold less, respectively), than cells infected with wild-type HPIV3. To increase expression of HN in transfected cells, we codon-optimized HN and used it to transfect lentivirus producer cells. Cell surface expression of HN, as well as the amount of HN incorporated into virus particles, increased two- to threefold. Virus titers increased 1.2- to 6.4-fold, and the transduction efficiency of polarized MDCK cells via their apical surfaces increased 1.4-fold. Interestingly, even though codon optimization improved the expression levels of HN and virus titers, we found that HPIV3 pseudotyped viruses contained about 14-fold fewer envelope proteins than lentiviruses pseudotyped with the amphotropic envelope protein. Taken together, our findings suggest that titers are low, not because virus producer cells express levels of HPIV3 envelope proteins that are too low, but because too few of these proteins are incorporated by the lentiviruses for them to be able to efficiently transduce cells.

Recombinant retroviruses have frequently been used to transfer genes to cells in ex vivo human gene therapy protocols. Retroviruses have rarely been used for in vivo gene transfer protocols, however, because of the likelihood that innocent bystander cells, adjacent to the diseased cells that are the target of the therapy, would be inadvertently modified. This has limited the use of

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.

A difficulty in the field of gene therapy is the need to increase the susceptibility of hematopoietic stem cells (HSCs) to ex vivo genetic manipulation. To overcome this obstacle a high-throughput screen was performed to identify compounds that could enhance the transduction of target cells by lentiviral vectors. Of the 1280 compounds initially screened using the myeloid-erythroid-leukemic K562 cell line, 30 were identified as possible enhancers of viral transduction. Among the positive hits were known enhancers of transduction (camptothecin, etoposide and taxol), as well as the previously unidentified phorbol 12-myristate 13-acetate (PMA). The percentage of green fluorescent protein (GFP)-positive-expressing K562 cells was increased more than fourfold in the presence of PMA. In addition, the transduction of K562 cells with a lentiviral vector encoding fVIII was four times greater in the presence of PMA as determined by an increase in the levels of provirus in genetically modified cells. PMA did not enhance viral transduction of all cell types (for example, sca-1(+) mouse hematopoietic cells) but did enhance viral transduction of human bone marrow-derived CD34(+) cells. Notably, the percentage of GFP-positive CD34(+) cells was increased from 7% in the absence of PMA to greater than 22% in the presence of 1 nM PMA. PMA did not affect colony formation of CD34(+) cells or the expression of the hematopoietic markers CD34 and CD45. These data demonstrate that high-throughput screening can be used to identify compounds that increase the transduction efficiency of lentiviral vectors, identifying PMA as a potential enhancer of lentiviral HSC transduction.

Introduction Cell-based approaches to bone regeneration often use gene therapy to induce osteogenesis in non-osteoblastic cells. However, unregulated overexpression of osteogenic proteins risks aberrant effects of uncontrolled signaling, including tumorigenesis and abnormal bone formation. Additionally, the incorporation of regulation into these systems allows for studying unique patterns of gene expression, including varying duration and magnitude, cycling of expression over time, and the sequential presence of multiple exogenous genes. We utilized a retroviral tetracycline-inducible system to modulate Runx2 expression and demonstrate regulation of mineralization in vitro and in vivo. This work is significant to developing controlled and effective gene therapy methods for tissue engineering, as well as novel strategies for investigating the effects of temporal modulation and magnitude of expression levels on osteoblastic differentiation.Methods Primary myoblasts were transduced with Runx2 and tTA retroviral stocks. Regulation of osteogenesis was evaluated by Western blotting, qRT-PCR, alkaline phosphatase activity, and matrix mineralization. Cells seeded on collagen scaffolds and implanted intramuscularly into hind limbs of syngeneic mice were analyzed for DNA content and mineralization by micro-CT, histology, and FT-IR.Results Cells expressed anhydrotetracycline (aTc)-dependent levels of Runx2 protein, as well as osteocalcin, bone sialoprotein, and osterix mRNA. Additionally, aTc levels regulated mineralization by these cells, as shown by alkaline phosphatase activity, von Kossa staining, and calcium content (Fig. 1A). FT-IR spectroscopy confirmed this mineral to be biological hydroxyapatite. Osteocalcin expression and mineralization were upregulated in response to removal of aTc, and were repressed when aTc was replaced, demonstrating temporal regulation of osteogenesis (Fig. 1B). These cells also demonstrated aTc-dependent mineralization in vivo (Fig. 2).Conclusions We have stimulated osteogenesis in a Runx2- dependent manner. This conversion to a osteoblastic phenotype is inducible, repressible, and recoverable after suppression. Regulated mineralization was evident in both 2D and 3D settings in vitro, as well as ectopically in vivo. These results present a novel combination of Runx2 and tetracycline-regulated gene expression as a promising, controllable, and effective approach to bone tissue engineering and addressing limitations of constitutive gene therapy. Additionally, this system is useful for elucidating time- and dose-dependent biological mechanisms of Runx2-stimulated osteogenesis.

Using a model recombinant retrovirus encoding theEscherichia coli lacZgene, we have found that medium conditioned with NIH 3T3 cells and packaging cell lines derived from NIH 3T3 cells inhibits infection. Most of the inhibitory activity was greater than 100 kDa and was sensitive to chondroitinase ABC digestion, which is consistent with the inhibitor being a chondroitin sulfate proteoglycan. Proteoglycans secreted

Introduction Inefficient and uncontrolled gene delivery has hampered the widespread efficacy of gene therapy. Biomaterial- mediated gene transfer represents a promising strategy to address these limitations by immobilizing the gene carrier onto a biocompatible substrate. This approach permits control of gene transfer by co- localizing cell adhesion and the gene delivery vehicle. These interactions must be carefully balanced to adequately

Methods are needed to manipulate natural nanoparticles. Viruses are particularly interesting because they can act as therapeutic cellular delivery agents. Here we examine a new method for rapidly modifying retroviruses that uses lipid conjugates composed of a lipid anchor (1,2-distearoyl-sn-glycero-3-phosphoethanolamine), a polyethylene glycol chain, and biotin. The conjugates rapidly and stably modified retroviruses and enabled them to bind streptavidin. The implication of this work for modifying viruses for gene therapy and vaccination protocols is discussed.

An improved understanding of retrovirus-cell interactions is needed to achieve more predictable outcomes in gene transfer protocols. Transduction efficiency is determined largely by the interaction between the virus envelope proteins and their receptors. Nevertheless, little quantitative information is available about the relationship between gene transfer and envelope protein number. This motivated us to examine the relationship between the number of envelope proteins per retrovirus particle and the number of genes transferred when undiluted virus stocks are used, as is done in most human gene therapy clinical trials. We constructed a panel of several clonal amphotropic murine leukemia virus packaging cell lines, each of which were derived from the same parental cell line (TELCeB6 cells). These packaging cell lines (TELCeB6-A) differed only in the amount of envelope protein that they expressed. In agreement with the findings of others, we found that virus titer increased as the number of envelope proteins that were associated with the virus particles increased. In addition, we found that the virus stocks contained high levels of free envelope protein that strongly inhibited infection when undiluted virus stocks were applied directly to cells.These free envelope proteins complicated our efforts to examine the relationship between gene transfer and the number of envelope proteins per virus, so we purified the viruses from the free envelope proteins using a previously described polymer complexation method. Remarkably, purified virus transferred an average of 80-fold more genes than unpurified virus. Next, to examine the relationship between gene transfer and the number of envelope proteins per virus, we used purified virus to transduce NIH 3T3, Hela, and TE671 cells. Interestingly, we found that gene transfer increased as the number of envelope proteins per virus particle decreased, exactly the opposite trend that we and others have previously observed between virus titer and the number of envelope proteins per virus. In addition, we found that when highly purified virus stocks were used to transduce cells, the level of gene transfer increased linearly with virus concentration at low doses of virus, but reached a 'plateau' (i.e., a level maximum) when high doses of virus were used. When cells were transduced with high doses of purified amphotropic virus, further transduction by amphotropic retroviruses, but not ecotropic retroviruses, was blocked. These results suggest that envelope proteins that are associated with a virus particle block or otherwise prevent cell surface receptors from interacting with subsequent virus particles that are pseudotyped with the same envelope protein. Our results demonstrate the importance of using purified virus stocks towards achievable more predictable outcomes. In addition, we speculate that it may be possible to adjust the maximum number of genes that can be transferred to a particular cell line by 'tuning' the number of envelope proteins associated per virus particle to the cell line that is being transduced.

The effectiveness of retrovirus or lentivirus transduction of embryonic stem (ES) cells is often limited because transgene expression is silenced or variegated. We wondered if other steps of transduction, in addition to gene expression, were restricted in ES cells. We quantitatively compared (1) the amount of virus binding, (2) the number of integrated transgenes, and (3) the resulting level of gene expression. We found that three- to fourfold fewer retroviruses and lentiviruses bound to R1 mES cells than to NIH 3T3 cells, suggesting that both types of viruses bind less efficiently to mES cells. Retroviruses and lentiviruses differed in the efficiency with which they completed post-binding steps of transduction. In R1 mES cells, we detected 3-fold fewer integrated retrovirus transgenes and 11-fold lower expression levels than in NIH 3T3 cells, which suggests that the primary limitation to retrovirus transduction may be low levels of transgene expression. In contrast, we detected 10-fold fewer integrated lentivirus transgenes and 8-fold lower expression levels in R1 mES cells than in NIH 3T3 cells, which suggests that lentivirus transduction may be limited by inefficient intracellular post-binding steps of transduction. The implications of our findings for developing improved viral vectors for transducing mES cells are discussed.

Lentiviral vectors pseudotyped with the envelope glycoproteins of amphotropic murine leukemia virus and the G protein of vesicular stomatitis virus have been previously developed for use in human gene therapy trials. We report here the generation of infectious replication-defective HIV-1 particles pseudotyped with hemagglutinin-neuraminidase (HN) and fusion (F) proteins derived from human parainfluenza virus type 3 (HPIV3). The HIV-1(HPIV3) pseudotype

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.

Using a panel of amphotropic murine leukemia virus packaging cell lines that differed only in their levels of envelope protein (gp70) expression, we examined the relationship between transduction and the number of envelope proteins per virus. We generated virus stocks that contained different levels of virus-associated envelope proteins, purified them from gp70 that was not associated with the viruses, quantified their titers, and measured the efficiency with which they transduced NIH 3T3, TE671, and HeLa cells. As expected, titers increased monotonically with viral envelope protein number. Titers are measured using highly dilute virus, however, and are often not predictive of gene transfer when high doses of virus are used, as is done in gene therapy protocols. Interestingly, when we used high doses of virus, we observed significantly different trends: gene transfer increased, reached a maximum, and then declined sharply as the number of envelope proteins per virus increased. The highest levels of gene transfer occurred when cells were transduced with a moderate dose of virus that contained low levels of envelope protein. Our results indicate that transduction is inhibited when viruses that contain large numbers of envelope proteins are used. This is most likely because each virus, when it binds to a cell, delivers a large payload of envelope proteins that occupy or inactivate multiple virus receptors, reducing or eliminating the susceptibility of the cell to being transduced by additional viruses. The implications of our findings for the design of improved retroviral vectors for human gene therapy are discussed.

We have previously shown that the envelope glycoproteins of human parainfluenza type 3 (HPIV3), F and HN, are able to pseudotype lentiviruses, but the titers of these viruses are too low for use in clinical gene transfer. In this study we investigated the cause of these low titers. We compared the mRNA and protein expression levels of HN and F in transfected cells and in cells infected with wild-type HPIV3. Transfected cells contained similar levels of HN and F cytosolic mRNA, but fewer cell-surface HN and F proteins (3.8- and 1.3-fold less, respectively), than cells infected with wild-type HPIV3. To increase expression of HN in transfected cells, we codon-optimized HN and used it to transfect lentivirus producer cells. Cell surface expression of HN, as well as the amount of HN incorporated into virus particles, increased two- to threefold. Virus titers increased 1.2- to 6.4-fold, and the transduction efficiency of polarized MDCK cells via their apical surfaces increased 1.4-fold. Interestingly, even though codon optimization improved the expression levels of HN and virus titers, we found that HPIV3 pseudotyped viruses contained about 14-fold fewer envelope proteins than lentiviruses pseudotyped with the amphotropic envelope protein. Taken together, our findings suggest that titers are low, not because virus producer cells express levels of HPIV3 envelope proteins that are too low, but because too few of these proteins are incorporated by the lentiviruses for them to be able to efficiently transduce cells.

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.

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.

Antisense technology is potentially a powerful means by which to selectively control gene expression. We have used antisense oligonucleotides to modulate the response of the hepatoma cell line, HepG2, to the inflammatory cytokine, IL-6, by inhibiting the expression of its multifunctional signal transducer, gp130. HepG2 cells respond to IL-6 by upregulating acute phase proteins, such as haptoglobin, by five- to tenfold. Gp130 is central to this response, as the upregulation of haptoglobin is almost completely blocked by the addition of high concentrations ( approximately 100 microg/ml) of a monoclonal antibody to gp 130. Antisense oligodeoxynucleotides complementary to the mRNA encoding gp 130 inhibited the upregulation of haptoglobin by IL-6-stimulated HepG2 cells by about 50%. However, a nonsense sequence also inhibited haptoglobin secretion by about 20%. To improve the specificity and efficiency of action, we targeted the antisense oligonucleotides to HepG2 cells using a conjugate of asialoglycoprotein-poly-L-lysine. The targeted antisense reduced the binding of IL-6 to HepG2 cells, virtually eliminating high affinity binding. In addition, it inhibited haptoglobin upregulation by over 70%. Furthermore, the dose of targeted antisense required for biological effect was reduced by about an order of magnitude as compared with unconjugated antisense. These results demonstrate the potential of antisense oligonucleotides as a means to control the acute phase response as well as the need for a greater understanding of the mechanism and dynamics of antisense molecules as they are developed toward therapeutic application.