High-throughput screening identifies compounds that enhance lentiviral transduction.

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.

Inducible regulation of Runx2-stimulated osteogenesis.

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.

[The role of structural elements in dimerization of RNA fragments in avian retroviruses]. / Rol' strukturnykh élementov v dimerizatsii fragmentov RNK retrovirusov ptits.

The slipped loop structure, earlier identified as an unusual DNA structure, was found to be a possible element of the RNA folding. In order to experimentally test this suggestion, model oligoribonucleotides capable of forming the SLS were synthesized. Treatment of the oligoribonucleotides with nuclease S1 and RNases specific for single- and double-stranded RNA demonstrated the steric possibility of SLS formation. To determine the possible functional role of SLS-RNA, various naturally occurring RNAs were screened in silico. Among the most interesting findings were dimerization initiation sites of avian retroviral genomic RNAs. Analysis of RNA from 31 viruses showed that formation of the intermolecular SLS during RNA dimerization is theoretically possible, competing with the formation of an alternative hairpin structure. Identification of the secondary structure of selected RNA dimers employing nuclease digestion techniques as well as covariance analysis of the retroviral RNA dimerization initiation site sequences were used to show that the alternative conformation (loop-loop interaction of two hairpins) is the most preferred. Alternative structures and conformational transitions in RNA dimerization mechanisms in avian retroviruses are discussed.

Complexation of retrovirus with cationic and anionic polymers increases the efficiency of gene transfer.

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.

Fear memories require protein synthesis in the amygdala for reconsolidation after retrieval.

'New' memories are initially labile and sensitive to disruption before being consolidated into stable long-term memories. Much evidence indicates that this consolidation involves the synthesis of new proteins in neurons. The lateral and basal nuclei of the amygdala (LBA) are believed to be a site of memory storage in fear learning. Infusion of the protein synthesis inhibitor anisomycin into the LBA shortly after training prevents consolidation of fear memories. Here we show that consolidated fear memories, when reactivated during retrieval, return to a labile state in which infusion of anisomycin shortly after memory reactivation produces amnesia on later tests, regardless of whether reactivation was performed 1 or 14 days after conditioning. The same treatment with anisomycin, in the absence of memory reactivation, left memory intact. Consistent with a time-limited role for protein synthesis production in consolidation, delay of the infusion until six hours after memory reactivation produced no amnesia. Our data show that consolidated fear memories, when reactivated, return to a labile state that requires de novo protein synthesis for reconsolidation. These findings are not predicted by traditional theories of memory consolidation.

Toward a more accurate quantitation of the activity of recombinant retroviruses: alternatives to titer and multiplicity of infection.

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.

Toward a more accurate quantitation of the activity of recombinant retroviruses: alternatives to titer and multiplicity of infection.

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 (t1/2), 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 Cvo, the starting concentration of active retrovirus particles. In contrast to titer, Cvo is independent of the specific conditions of the assay. The relatively slow diffusion (D = 2 x 10(-8) cm2/s) and rapid decay (t1/2 = 6 to 7 h) of retrovirus particles explain why Cvo values are significantly higher than titer values. Values of Cvo 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.

Kinetics of retrovirus production and decay.

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.

Differential inhibition of retrovirus transduction by proteoglycans and free glycosaminoglycans.

We have previously shown that the efficiency of retrovirus-mediated gene transfer is limited in part due to the presence of chondroitin sulfate proteoglycans in virus stocks. In this study, we have used a model recombinant retrovirus encoding the Escherichia coli lacZ gene, bovine aorta chondroitin sulfate proteoglycan (CSPG), various free glycosaminoglycan chains (GAGs), and quantitative assays for retrovirus transduction to explore the mechanism by which proteoglycans and glycosaminoglycans inhibit retroviruses. We found that CSPG and GAGs block an early step in virus-cell interactions but do not act by inactivating viruses or by reducing the growth rate of the target cells. CSPG and most of the GAGs tested (chondroitin sulfate A, chondroitin sulfate B, heparin, heparan sulfate, and hyaluronic acid) inhibited transduction, but with widely varying degrees of activity. The chemical structure of GAGs was found to be an important determinant of their inhibitory activity, which suggests that GAGs do not inhibit transduction simply because they are highly negatively charged polymers. When GAGs were used in combination with a cationic polymer (Polybrene), however, their inhibitory activity was neutralized, and interestingly, at optimal doses of GAG and Polybrene, transduction efficiency was actually enhanced by as much as 72%. In contrast, the inhibitory activity of CSPG, due to the influence of its core protein, was not substantially reduced by Polybrene. The importance of these findings to our understanding of retrovirus-cell interactions and to the development of more efficient retrovirus gene transfer protocols is discussed.

Large-scale processing of recombinant retroviruses for gene therapy.

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.

Removal of proteoglycans increases efficiency of retroviral gene transfer.

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.

Targeted antisense modulation of inflammatory cytokine receptors.

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.

Proteoglycans secreted by packaging cell lines inhibit retrovirus infection.

Using a model recombinant retrovirus encoding the Escherichia coli lacZ gene, 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 by NIH 3T3 cells and purified by anion-exchange chromatography inhibited amphotropic retrovirus infection. Pretreatment of amphotropic retrovirus stocks with chondroitinase ABC boosted the level of transduction efficiency by more than twofold. The implications of these findings with respect to retrovirus-cell interactions and the production of high-titer retroviral stocks are discussed.

Sympathetic nervous system and control of blood pressure during natural behaviour.

The role of the sympathetic nervous system in generating behaviourally linked blood pressure (BP) responses was investigated in awake rats instrumented for chronic intra-arterial BP recording. All data from the recording sessions were digitalized and processed by a computer. Frequency interval histograms of behaviourally associated changes of BP and heart rate (HR) were generated. 6-Hydroxydopamine (100 and 200 mg/kg intravenously) was used for chemosympathectomy. In the intact rats (n = 8) three different frequency histograms of mean BP values for three behavioural groupings were generated (from lowest to highest average values): (1) resting, (2) exploring-grooming and (3) eating-drinking. Chemosympathectomy (n = 5) abolished this order of BP changes; mean BP did not rise from rest to other behaviours, nor did it vary during different behaviours. It is concluded that the sympathetic nervous system is responsible for producing the changes in BP that are appropriate and characteristic of each behaviour in awake, unrestrained rats.