Genetic approaches to tissue engineering: breakout session summary.
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Animals↗
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Genetic manipulation of insect vectors as a strategy for the control of vector-borne disease.
A variety of very effective methods have been employed for suppressing insect vector populations, including the application of biological control agents and the elimination of breeding sites, with a continuing and heavy reliance on the use of chemical insecticides. However, the development of insecticide resistance by vector insects, the cost of developing and registering new insecticidal compounds, and the increase in legislation to combat the detrimental effects of insecticidal residues on the environment, have emphasized the need to assess alternative strategies for vector control. What is required is a completely novel approach to either suppress vector populations, or to alter their ability to transmit disease-causing organisms in such a way as to have a profound and long-lasting effect on disease transmission. Genetic manipulation of insect vectors may provide just such an approach. The major requirements for genome manipulation in insects and the progress which has been made to create transgenic vector insects are reviewed. The potential applications of this methodology are then explored in the context of its future use for the control of vector-borne diseases.
Animals↗
Sindbis virus: an efficient, broad host range vector for gene expression in animal cells.
Sindbis virus, an enveloped virus with a single-stranded RNA genome, was engineered to express a bacterial protein, chloramphenicol acetyltransferase (CAT), in cultured insect, avian, and mammalian cells. The vectors were self-replicating and gene expression was efficient and rapid; up to 10(8) CAT polypeptides were produced per infected cell in 16 to 20 hours. CAT expression could be made temperature-sensitive by means of a derivative that incorporated a temperature-sensitive mutation in viral RNA synthesis. Vector genomic RNAs were packaged into infectious particles when Sindbis helper virus was used to supply virion structural proteins. The vector RNAs were stable to at least seven cycles of infection. The expression of CAT increased about 10(3)-fold, despite a 10(15)-fold dilution during the passaging. Sindbis virus vectors should prove useful for expressing large quantities of gene products in a variety of animal cells.
Aedes↗
Dominant lethality and insect population control.
Recent advances in insect genetic engineering have opened up new possibilities in the genetic control of insect vectors of human diseases. We outline the current state of the sterile insect technique and show how the use of engineered dominant lethals can greatly increase the effectiveness of this approach. We consider alternative strategies based on the use of conditional dominant lethals that are not always active in the environment.
Animals↗
Superpolylinkers in cloning and expression vectors.
Versatile DNA polylinkers of more than 300 bp were constructed. They contain the recognition sequences of all restriction enzymes--whether known or still to be discovered--that recognize palindromic hexamers. In addition to these 64 uninterrupted hexameric recognition sites, a number of sites containing interrupted palindromes and nonpalindromic sequences and two recognition sequences with 8 bp are present. Polylinkers (in several variants) were inserted into frequently utilized Escherichia coli cloning vectors such as pBluescript (yielding pSLJ10, pSL250, pSL260, pSL270, and pSL300), pUC18/pUC19 (yielding pSL180 and pSL190, respectively), or pUC118/pUC119 (yielding pSL1180 and pSL1190, respectively). A subtle color discrimination between presence and absence of insert in pSL300 (mid-blue to light-blue or white) was seen in a number of test ligations. The mid-blue color that is generated by pSL300 is presumably due to translational restarts. A different intergenic region for translational restarts was used in plasmids pSL251, pSL261, pSL271, and pSL301. The polylinker was also inserted into expression vector pUC120, yielding pSE1200, and into expression vector pKK233-2, yielding pSE220 and a shortened version thereof, pSE280. Finally, the polylinker was inserted into pTrc99A, resulting in pSE380, which carries a lac repressor gene. This expands the use of the expression system beyond lacIq strains to other bacterial hosts. These versatile vectors have broad applications in genetic engineering.
Amino Acid Sequence↗
Suppression of tumorigenicity and metastasis in murine UV-2237 fibrosarcoma cells by infection with a retroviral vector harboring the interferon-beta gene.
In this study, we endeavored to determine the effectiveness of interferon beta (IFNbeta) gene therapy against highly metastatic murine UV-2237m fibrosarcoma cells. UV-2237m cells were engineered to produce murine IFNbeta constitutively following infection by a retroviral vector harboring the murine IFNbeta gene. Parental (UV-2237m-P), control-vector-transduced (UV-2237m-Neo), and IFNbeta-transduced (UV-2237m-IFNbeta) cells were injected subcutaneously (s.c.) or intravenously (i.v.) into syngeneic mice. Parental and control-transduced cells produced rapidly growing tumors, whereas IFNbeta-transduced cells did not. The tumorigenicity of IFNbeta-sensitive or -resistant parental cells was significantly suppressed when they were injected s.c. together with IFNbeta-transduced cells. The IFNbeta-transduced cells did not inhibit growth of parental cells injected s.c. at a distant site. UV-2237m-IFNbeta cells produced s.c. tumors in nude, SCID/Beige, and natural killer(NK)-cell-compromised syngeneic mice. The IFNbeta-transduced cells were more sensitive to in vitro splenic cell-mediated lysis than were the parental or control-transduced cells. Pretreatment of C3H/HeN mice with the NK-cell-selective antiserum (anti-asialoGM1) partially abrogated the cytotoxic activity of the cells. Cytotoxic activity was not observed in mixed culture of UV-2237m-IFNbeta cells and splenic cells from SCID/Beige mice. Significant cytotoxicity against UV-2237m-IFNbeta cells was mediated by macrophages activated by either IFNgamma, lipopolysaccharide, or a combination of both. Our data led us to conclude that the constitutive expression of IFNbeta can suppress tumorigenicity and metastasis of UV-2237m cells, which is due, in part, to activation of host effector cells.
Animals↗
Towards efficient cell targeting by recombinant retroviruses.
Efficient human gene therapy will rely on efficient gene delivery systems. To date, recombinant, engineered retroviruses are the most widely used vectors for stable clinical gene transfer. However, this technology still suffers from a number of drawbacks that limit its application. The ability to target cells of therapeutic interest, in particular, would improve both the efficiency and the safety of retroviral vectors. It might also allow the development of new animal models of human diseases and probably extend the scope of gene therapy itself.
Gene Transfer Techniques↗
Tissue-specific gene expression from Mo-MLV retroviral vectors with hybrid LTRs containing the murine tyrosinase enhancer/promoter.
Transcriptional tissue specificity was engineered directly into Moloney Murine Leukaemia Virus (Mo-MLV)-derived retroviral vectors by replacing the viral enhancer in the 3' long terminal repeat (LTR) with two different lengths (2.5 kbp or 769 bp) of the murine tyrosinase promoter/enhancer. The hybrid tyrosinase-LTR was transferred to the proviral 5' LTR following viral packaging and infection of target cell lines. Hybrid tyrosinase-LTR-driven IL-2 production was barely above background levels in infected nonmelanoma cell lines containing intact provirus, whereas infected melanoma cell lines expressed high levels of IL-2 and the larger tyrosinase promoter/enhancer fragment directed higher levels of transgene expression. By replacing the viral enhancer with the tyrosine promoter/enhancer sequences, promoter interference effects which we have previously observed when the tyrosinase promoter was included as an internal promoter within a similar retroviral vector were effectively abolished. Our data show that the hybrid tyrosinase-LTR behaves as a tightly regulated melanocytic-specific regulatory element when embedded in an enhancer-deleted Mo-MLV LTR. The use of other heterologous cellular promoter/enhancer elements in similar vectors should allow the development of simpler, targeted retroviral vectors for the expression of genes in selected cell types and may eventually provide for the development of safer, more efficient vectors for use in human gene therapy.
3T3 Cells↗
Avidin-based targeting and purification of a protein IX-modified, metabolically biotinylated adenoviral vector.
While genetic modification of adenoviral vectors can produce vectors with modified tropism, incorporation of targeting peptides/proteins into the structural context of the virion can also result in destruction of ligand targeting or virion integrity. To combat this problem, we have developed a versatile targeting system using metabolically biotinylated adenoviral vectors bearing biotinylated fiber proteins. These vectors have been demonstrated to be useful as a platform for avidin-based ligand screening and vector targeting by conjugating biotinylated ligands to the virus using high-affinity tetrameric avidin (K(d) = 10(-15) M). The biotinylated vector could also be purified by biotin-reversible binding on monomeric avidin (K(d) = 10(-7) M). In this report, a second metabolically biotinylated adenovirus vector, Ad-IX-BAP, has been engineered by fusing a biotin acceptor peptide (BAP) to the C-terminus of the adenovirus pIX protein. This biotinylated vector displays twice as many biotins and was markedly superior for single-step affinity purification on monomeric avidin resin. However, unlike the fiber-biotinylated vector, Ad-IX-BAP failed to retarget to cells with biotinylated antibodies including anti-CD71 against the transferrin receptor. In contrast, Ad-IX-BAP was retargeted if transferrin, the cognate ligand for CD71, was used as a ligand rather than the anti-CD71. This work demonstrates the utility of metabolic biotinylation as a molecular screening tool to assess the utility of different viral capsid proteins for ligand display and the biology and compatibility of different ligands and receptors for vector targeting applications. These results also demonstrate the utility of the pIX-biotinylated vector as a platform for gentle single-step affinity purification of adenoviral vectors.
Adenoviridae↗
Controlled release of bioactive agents in gene therapy and tissue engineering.
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Genetic Therapy↗
Strategies for achieving multiple layers of selectivity in gene therapy.
Here we review the progress towards the development of targeted vectors for direct in vivo delivery in gene therapy. Currently, there are many separate approaches. These include: simple physical/anatomical localization of administration of the vector at the site where gene transfer is required; exploitation of natural tropisms of plasmid, viral and cellular vectors; and the use of molecular engineering to change the specificity of proteins and nucleic acids so that they specifically recognize target ligands expressed on/in the target cells. Unfortunately, each of these approaches is usually imperfect by itself. However, combinations of these strategies might produce vectors in which several layers of imperfect targeting give an overall level of specificity that can justify systemic delivery of vectors to treat human disease.
Biotechnology↗
Characterization of five evolutionary conserved regions of the human tyrosine hydroxylase (TH) promoter: implications for the engineering of a human TH minimal promoter assembled in a self-inactivating lentiviral vector system.
A DNA fragment of about 13 kb containing the human tyrosine hydroxylase (TH) promoter was previously isolated from a genomic DNA library and sequenced. The 11 kb from the transcription start of the human TH promoter was successively joined to the green fluorescent protein (GFP) to generate a transgenic mouse model. High levels of GFP expression could be observed in TH-positive cells of the Substantia nigra of embryonic and adult mice. Intriguingly, the sequence of the human TH promoter showed a low degree of homology with the mouse and rat TH promoters. In fact, comparative analysis of the sequences of human, rat, and mouse TH promoters revealed only five small regions of high homology. These five evolutionarily conserved regions were numbered in numeric progression from the 5' end of human TH promoter. In the present study, a panel of minimal human TH promoters was generated to analyze the transcriptional activity and specificity of gene expression conferred by the five conserved regions (CRs). The series of constructs was termed 250 bp and contained the first -194 bp of the human TH promoter immediately upstream of the transcription start, the first 35 bp the human TH messenger RNA leader, plus one or more of the five CRs. All the constructs were assembled in a self-inactivating form of the latest series of lentiviral vector system based on the human immunodeficiency virus type 1 (HIV-1). Lentiviral-mediated gene transfer was highly efficient for the in vitro transduction of human neuronal progenitor cells (hNPCs). Since a subset of hNPCs express TH following in vitro treatment with a mixture of differentiating agents, it was possible to assess specificity of expression for all the minimal human TH promoters. Overall, the successive addition of the five conserved regions produced a greater degree of specificity in induced TH-positive hNPCs, in particular after the addition of CRI (-8,917, -8,876). However, the human TH minimal promoters did not show any specificity for TH-positive differentiated mouse primary striatal and S. nigra cells, indicating a difference of TH gene regulation between the human and mouse systems. The human TH minimal promoters may provide the opportunity for the selection of TH-positive human embryonic and adult stem cells for brain transplantation experiments in animal models for Parkinson's disease.
Animals↗
Picolinic acid- or desferrioxamine-inducible autocrine activation of macrophages engineered to produce IFNgamma: an approach for gene therapy.
Macrophage (Mphi)-based vectors are highly mobile cellular shuttles designed to deliver therapeutic genes within the tissues. We engineered a mouse Mphi cell line to express the murine interferon-gamma (IFNgamma) under the control of an inducible promoter containing the hypoxia-responsive element, which can be triggered by hypoxia and other stimuli. We show that this Mphi vector can be induced to produce IFNgamma under normoxic conditions by stimulation with picolinic acid (PA), a catabolite of tryptophan, or desferrioxamine (DFX), an iron-chelating drug. The Mphi vector responds to IFNgamma with the induction of IRF-1 and of other IFNgamma-inducible genes, the expression of Ia antigens and induction of phagocytic activity. Inducible nitric oxygen synthase gene expression, nitric oxide production, as well as TNFalpha secretion were enhanced by PA or DFX as the sole stimuli. None of the above responses could be triggered individually by PA or DFX in control, normal Mphi, indicating that the Mphi vector overcame the need for costimulatory molecules derived from the immune system for its full activation. Furthermore, we demonstrate that extracellular iron can downregulate such response, thereby identifying an additional tool for the fine tuning of the Mphi vector response to stimulation.
Animals↗
Characterization of novel safe lentiviral vectors derived from simian immunodeficiency virus (SIVmac251) that efficiently transduce mature human dendritic cells.
We describe the generation and the characterization of new lentiviral vectors derived from SIVmac251, a simian immunodeficiency virus (SIV). A methodical approach was used to engineer both efficient and safe packaging constructs allowing the production of SIV viral core proteins. SIV-vectors encoding GFP (green fluorescent protein) were generated as VSV-G-pseudotyped particles upon transient expression of the vector construct and helper functions in 293 cells. The SIV vectors were able to transduce efficiently various target cell types at low multiplicity of infection, including monocyte-differentiated human dendritic cells (DCs) which retained their capacity to differentiate into mature DCs after gene transfer. Transduction of the DCs by the SIV vectors was prevented when infections were performed in the presence of AZT, a reverse-transcriptase inhibitor. After gene transfer, expression of the GFP in the target cells remained constant after several weeks, indicating that the vectors had been stably integrated into the genome of the host cells. Preparations of SIV vectors were systematically checked for the absence of replication-competent and recombinant retroviruses but remained negative, suggesting the innocuousness of these novel gene delivery vectors. Side-to-side comparisons with vectors derived from HIV-1 (human immunodeficiency virus) indicated that the SIV vectors were equally potent in transducing proliferating target cells. Finally, we have determined the infectivity of SIV vectors pseudotyped with surface glycoproteins of several membrane-enveloped viruses.
Animals↗
Effect of genetic background and culture conditions on the production of herpesvirus-based gene therapy vectors.
Herpes simplex virus type-1 (HSV-1) represents a unique vehicle for the introduction of foreign DNA to cells of a variety of tissues. The nature of the vector, the cell line used for propagation of the vector, and the culture conditions will significantly impact vector yield. An ideal vector should be deficient in genes essential for replication as well as those that contribute to its cytotoxicity. Advances in the engineering of replication-defective HSV-1 vectors to reduce vector-associated cytotoxicity and attain sustained expression of target genes make HSV-1 an attractive gene-delivery vehicle. However, the yield of the less-cytotoxic vectors produced in standard tissue-culture systems is at least three order of magnitudes lower than that achieved with wild-type virus. The low overall yield and the complexity involved in the preparation of HSV vectors at high concentrations represent major obstacles in use of replication-defective HSV-derived vectors in gene therapy applications. In this work, the dependence of the vector yield on the genetic background of the virus is examined. In addition, we investigated the production of the least toxic, lowest-yield vector in a CellCube bioreactor system. After initial optimization of the operational parameters of the cellcube system, we were able to attain virus yields similar to or better than those values attained using the tissue culture flask system for vector production with significant savings with respect to time, labor, and cost.
Bioreactors↗
Sequential cloned gene integration in the yeast Kluyveromyces lactis.
Two integrating vectors developed for use in Saccharomyces cerevisiae were successfully employed for cloned gene integration in the yeast Kluyveromyces lactis. A delta-integrating vector carrying the dominant selection marker neo allowed tandem integrations of a CUP1p-lacZ cassette into one or two chromosomal sites. A delta/UB-integrating vector, which contains a reusable selection cassette, enabled multiple rounds of integration and the sequential insertion of stable, dispersed copies of CUP1p-lacZ. Subsequent gene expression was closely correlated with integrated copy number illustrating the promise of this method for metabolic engineering in K. lactis. While both vectors contain an S. cerevisiae delta target sequence, the presence of delta-like elements in K. lactis has not been confirmed. Given the degree of illegitimate recombination in this yeast species, the insertions likely occurred at random locations in the chromosomes.
Chromosomes, Fungal↗
Endogenous microRNA regulation suppresses transgene expression in hematopoietic lineages and enables stable gene transfer.
MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression by repressing translation of target cellular transcripts. Increasing evidence indicates that miRNAs have distinct expression profiles and play crucial roles in numerous cellular processes, although the extent of miRNA regulation is not well known. By challenging mice with lentiviral vectors encoding target sequences of endogenous miRNAs, we show the efficiency of miRNAs in sharply segregating gene expression among different tissues. Transgene expression from vectors incorporating target sequences for mir-142-3p was effectively suppressed in intravascular and extravascular hematopoietic lineages, whereas expression was maintained in nonhematopoietic cells. This expression profile, which could not be attained until now, enabled stable gene transfer in immunocompetent mice, thus overcoming a major hurdle to successful gene therapy. Our results provide novel in situ evidence of miRNA regulation and demonstrate a new paradigm in vector design with applications for genetic engineering and therapeutic gene transfer.
Animals↗
Strategies for retargeted gene delivery using vectors derived from lentiviruses.
With the development of the first viral vector systems 20 years ago [Mann et al., 1983; Watanabe and Temin, 1983] gene therapy strategies have come to the forefront of novel therapeutics [Cavazzana-Calvo et al., 2000]. A deeper understanding of vector biology and the molecular mechanisms of disease alongside tremendous advances in vector technology have significantly advanced the field of human gene therapy. Over the last few years several challenges needed to be overcome in order to bring gene therapy strategies closer to the clinic. These hurdles include the preparation of large amounts of stable, high titre vectors, minimising vector-related immunology and last but not least targeting infection and transgene expression to tissue or cells, which in many cases are not or only slowly dividing. Viral vectors are useful vehicles for the delivery of foreign genes into target cells, and retroviral vectors have been popular because of their ability to integrate into the host cell genome and maintain persistent gene expression. Moreover, lentiviruses, members of the retroviral family, have the ability to infect cells at both mitotic and post-mitotic stages of the cell cycle thus opening up the possibility to target non-dividing target cells and tissues. Human immunodeficiency virus (HIV) based vectors have been used in vitro and in vivo in a number of situations, however, safety concerns still exist, and therefore the development of vector systems based on primate as well as non-primate lentiviruses is ongoing. Concomitantly with lentiviral vector design, much has been learned about the incorporation of heterologous env proteins on lentiviral cores in order to combine specific targeting properties of envelope glycoproteins with the biological properties of lentiviral vectors. In this review article we will give an overview over advantages lentiviral vector systems offer. We will then discuss the current state of our understanding of the structure and function of viral envelope glycoproteins and emerging targeting strategies based on retroviral and lentiviral vector systems.
Animals↗