ISSN:1043-0342    

DOI:10.1089/hum.1991.2.1-1

出版商:Mary Ann Liebert, Inc.    

年代:1991

数据来源: MAL

ISSN:1043-0342    

DOI:10.1089/hum.1991.2.1-3

出版商:Mary Ann Liebert, Inc.    

年代:1991

数据来源: MAL

摘要:

ABSTRACTThe first three approved human clinical trials utilizing retroviral-mediated gene transfer are now underway. While this technology holds great promise for the study and treatment of human disease, it also poses a number of safety concerns. In evaluating clinical protocols, potential complications and the likelihood of their occurrence are estimated by review committees so that a risk/benefit assessment can be made. Current knowledge, reviewed in this article, suggests that no acute complications secondary to retroviral-mediated gene transfer are likely, but the possibility of long-term or unforeseen sequelae in patients suggests the need for post-treatment monitoring.

ISSN:1043-0342    

DOI:10.1089/hum.1991.2.1-5

出版商:Mary Ann Liebert, Inc.    

年代:1991

数据来源: MAL

摘要:

ABSTRACTThe ability of skeletal muscle to regenerate provides an excellent therapeutic entry,viagenetic engineering, for correcting diseases of skeletal muscle and other tissues. We have used a retrovirus to transfer the cDNA for the human multidrug transporter, encoded by theMDR1 gene, into the genomes of the rat muscle cell line L6 and into primary rat myocytes. TheMDR1 gene confers drug resistance to cells, and thus serves as a selectable markerin vitro. In cultured cells, the retroviral promoter-driven humanMDR1 cDNA was shown to be stable in the presence or absence of drug selection or muscle cell fusion.MDR1 mRNA was synthesized, as shown by RNA blot analysis andin situhybridization. The protein product was localized to the plasma membrane of transduced myocytes and myotubes by immunofluorescence. As a model for skeletal muscle gene therapy, transduced L6 myocytes were implanted into the tibialis anterior muscle of Wistar rats. The retroviral sequences of the humanMDR1 gene and its mRNA were present in the muscles of Wistar rats 5 days, but not 12 days, after implantation, possibly because of immunorejection. On the other hand, the humanMDR1 cDNA was stable in the tibialis anterior muscle of nude mice, which are incapable of immunorejection, at least 4 weeks after implantation of myocytes. Immunosuppression of Wistar rats with cyclosporine A delayed immunorejection of recombinant myocytes, andMDR1 cDNA and mRNA was detected 34 weeks after implantation.In situhybridization revealed that injected recombinant myocytes remain in discrete foci in adult rodent skeletal muscle and expressMDR1 mRNA for at least 30 days in nude mice and cyclosporine-treated rats.

ISSN:1043-0342    

DOI:10.1089/hum.1991.2.1-15

出版商:Mary Ann Liebert, Inc.    

年代:1991

数据来源: MAL

摘要:

ABSTRACTThe development of liver-directed gene therapy protocols depends upon the ability to transfer genes into a large number of liver cells such that the genes are expressed persistently. We used a retroviral vector to transfer the gene for neomycin phosphotransferase(neo)into mouse liver cellsin vivo. Direct injection of the retrovirus preparation into mitotically active (regenerating) liver parenchyma resulted in efficient gene transfer, withneosequences detectable in the livers of every animal tested 10 weeks to 6 months later. Theneogene was expressed for at least 3 months. This methodology may eventually be applicable to the treatment of human disease.

ISSN:1043-0342    

DOI:10.1089/hum.1991.2.1-27

出版商:Mary Ann Liebert, Inc.    

年代:1991

数据来源: MAL

摘要:

ABSTRACTRetroviral vectors carrying the neomycin phosphotransferase(neo)gene have been shown to confer G418 resistance to canine keratinocytes at relatively high frequency. To investigate the usefulness of keratinocytes as potential target cells for gene therapy, we used a retroviral vector (LASN) that contains both human adenosine deaminase (hADA) andneogenes. We show here that LASN-transduced canine keratinocytes expressed high levels of hADA, a human protein of therapeutic relevance. Selection of LASN-transduced keratinocytes in medium containing G418 resulted in a population of cells that expressed even higher levels of hADA, about 80-fold higher than the endogenous canine ADA level. However, the G418-selected cells had a reduced proliferative potential and altered morphology indicative of terminal differentiation. To test whetherl-histidinol is more beneficial for selection of keratinocytes than G418, we constructed two retroviral vectors that contain both theneoand the histidinol dehydrogenase(hisD)genes. Cocultivation of primary keratinocytes with lethally irradiated PA317 retrovirus packaging cells that produce these vectors gave rise to 1253% drug-resistant colonies in either G418 orl-histidinol. In contrast to G418, selection of transduced keratinocytes inl-histidinol had no apparent effect on the proliferative potential or morphology of drug-resistant cells containing the vectors. Given the utility of this selection system, twohisD-based generic constructs containing cloning sites for cDNA expression from either the retroviral promoter or from an internal human cytomegalovirus immediate early promoter were constructed. Our results suggest thathisDwill be a useful selectable marker for use in studies of keratinocyte differentiation and for transfer of genes into keratinocytes for the purposes of gene therapy.

ISSN:1043-0342    

DOI:10.1089/hum.1991.2.1-33

出版商:Mary Ann Liebert, Inc.    

年代:1991

数据来源: MAL

摘要:

ABSTRACTFor most genetic deficiencies manifested in the liver, maximization of gene expression in hepatocytes will be an important factor in achieving successful gene therapy. A rapid, highly efficient, and nontoxic method for transfecting DNA into hepatocytes was used to compare directly promoter strengths of various cellular and viral promoters. Conditions are described here for transfecting 510% of primary hepatocytes using the positively charged liposomes, Lipofectin. Cells are not damaged by this method as they continue to transcribe genes controlled by liver specific promoters and can survive for over 2 weeks in culture. We find that the cytomegalovirus, SR, and-actin promoters are more active than the SV40, RSV, RNA polymerase II, albumin,1-antitrypsin, or phosphoenolpvriivatc carboxykinase promoters. A simple TK promoter and a TK promoter with the polyoma enhancer (MCI) were almost completely inactive. This information will be useful in the construction of vectors designed to express genes efficiently in primary hepatocytes for purposes of gene therapy, although the stability of expression from these promoters will need to be demonstrated in hepatocytesin vivo.

ISSN:1043-0342    

DOI:10.1089/hum.1991.2.1-41

出版商:Mary Ann Liebert, Inc.    

年代:1991

数据来源: MAL

摘要:

ABSTRACTExpression of a gene encoding the diphtheria toxin A (DT-A) fragment, controlled by tissue specific regulatory elements, has previously been used to kill selected cell populations. Here, we have examined the feasibility of controlling DT-A expression using regulatory systems from the human immunodeficiency virus (HIV-1) genome. Plasmids were constructed which express either DT-A or, as a model system, the luciferase(luc)reporter gene, under control of HIV-1 long terminal repeat (LTR) sequences (167 to80). Whiletrans-activation by expression of the viral protein Tat was demonstrated, significant basal expression was observed. To reduce basal expression,cis-acting negative regulatory elements from theenvregion of the HIV-1 genome were inserted in the 3untranslated region of both thelucand DT-A constructs. This dramatically reduced basal expression from the HIV LTR, and now both viral regulatory proteins Tat and Rev were required for maximaltrans-activation. Such regulation of DT-A expression might be therapeutically applied to selectively kill HIV-infected cells in acquired immunodeficiency syndrome (AIDS) and AIDS-related complex (ARC).

ISSN:1043-0342    

DOI:10.1089/hum.1991.2.1-53

出版商:Mary Ann Liebert, Inc.    

年代:1991

数据来源: MAL

摘要:

ABSTRACTRetroviral vectors are considered to be the most suited vehicles for somatic gene therapy with hematopoietic stem cells as targets. Retrovirus-mediated gene transfer into differentiation-restricted hematopoietic precursor (FDC-P1, FDC-P2) and multipotent progenitor (stem) cell lines (FDC-Pmix) is inefficient. Two cellular restrictions are involved. One is specific for stem but not precursor cells and is at the level of transcription. Due to a unique property of the transcriptional control region of the myeloproliferative sarcoma virus (MPSV), vectors derived from MPSV are not affected by this block. The second restriction occurs before proviral DNA synthesis and integration. This inhibition of effective viral infection depends on the state of differentiation, being more pronounced in multipotent clonogenic blast cells. This block to retroviral infection affects all retroviral vectors tested.

ISSN:1043-0342    

DOI:10.1089/hum.1991.2.1-61

出版商:Mary Ann Liebert, Inc.    

年代:1991

数据来源: MAL

摘要:

ABSTRACTManipulationper se is not bad. The crucial question in the moral debate about genetic engineering is: When and how are we allowed to manipulate? Unfortunately, the moral discussion surrounding this question is itself being manipulated. There are moral manipulations (by those who wish to either reassure or to alarm) and there are ethical manipulations (the failed utilitarian calculus and the centering of the discussion only around rules, rights, and duties). A different ethical approach is needed: one based on virtues. The duty of ethics is to help us understand the moral possibilities in each situation,i.e., to develop our moral sensibility. In the area of genetic engineering research we are motivated by a will to know, but at the same time we fear total self knowledge. We want to control, to improve our world and ourselves, but we recoil at obtaining ultimate perfection. Therefore, we must value the unknowable, the uncontrollable. Our everincreasing capacity to mould the world and ourselves is making it more difficult to develop a sensitivity for what is given and cannot be made. It is dangerous for our ethics to assume the activistic traits of our technology. We risk losing a fundamental element of what we are, or ought to be. We should train ourselves in moral passivity.

ISSN:1043-0342    

DOI:10.1089/hum.1991.2.1-71

出版商:Mary Ann Liebert, Inc.    

年代:1991

数据来源: MAL