摘要:

ABSTRACTTransduction of the murine interferon-(IFN-) gene into various malignant mouse tumor cells has resulted in the loss of tumorigenicity and an acquired capacity to induce long-lasting antitumor immunity following their injection into immunocompetent syngeneic mice. In the present study, we investigated the effectiveness of IFN--producing tumor cells in the therapy of mice with established mouse tumors. In DBA/2 mice bearing subcutaneous (s.c.) Friend erythroleukemia cell (FLC) tumors, we found that to achieve some antitumor response (i) it was necessary to inject high numbers of IFN--producing FLC, which occasionally lead to the formation of slowly growing tumors; and, that (ii) repeated injections of irradiated IFN--FLC did not result in any antitumor effect. The therapeutic potential of IFN--producing FLC rendered sensitive to ganciclovir (GCV), by transfer of the herpes simplex virus thymidine kinase (tk) gene, was investigated. Complete tumor rejection and cure was observed in70% of the animals after injection of high numbers (107) of IFN--producingtk-expressing tumor cells followed 4 days later by repeated GCV treatments, whereas only a slight increase in survival time was obtained after administration of controltk-expressing tumor cells (not producing IFN) and GCV. Tumor rejection was associated with a dramatic destruction of tumor tissue and with the subsequent development of a potent and long-lasting antitumor immunity. No therapeutic effect was observed in immunosuppressed nude mice. These data indicate that this approach may represent an effective and safe therapeutic strategy for antitumor cytokine gene therapy.

ISSN:1043-0342    

DOI:10.1089/hum.1996.7.1-1

出版商:Mary Ann Liebert, Inc.    

年代:1996

数据来源: MAL

摘要:

ABSTRACTProtein delivery from genetically modified skeletal muscle has been reported previously. However, a stable and prolonged secretion was obtained in immunocompromised or newborn animals only. To evaluate the clinical relevance of this approach, we have transduced myoblasts from an adult-glucuronidase-deficient (MPS VII) mouse with retroviral vectors carrying either the human-glucuronidase cDNA or the murine erythropoietin (Epo) cDNA. The cells were then grafted into the tibialis anterior muscle of adult immunocompetent MPS VII recipients. Protein expression was controlled either by ubiquitous or muscle-specific transcriptional regulatory elements. Animals were analyzed over an 8-month period. Thein situdetection of-glucuronidase activity revealed up to 60% of genetically modified myofibers in the recipient muscles. The human desmin promoter and enhancer showed the highestin vivoexpression. Secretion of-glucuronidase induced a disappearance of lysosomal storage lesions in the liver and spleen of recipient animals. Delivery of Epo led to a permanent increase of hematocrit values over 3 months. These results showed that the transplantation of genetically modified myoblasts allowed a sustained secretion of recombinant proteins at therapeutic levels in immunocompetent adult mice. They suggest that the approach may be considered for human applications.

ISSN:1043-0342    

DOI:10.1089/hum.1996.7.1-11

出版商:Mary Ann Liebert, Inc.    

年代:1996

数据来源: MAL

摘要:

ABSTRACTBecause interleukin-10 (IL-10) has potent immunosuppressive and anti-inflammatory properties and is produced by some cancers, we hypothesized that its production might play a role in carcinogenesis by inhibiting adequate antitumoral immune responses. To test this hypothesis, retroviral vectors containing the IL-10 cDNA were generated and used to infect B16F1 melanoma cells that were injected subcutaneously in syngeneic mice. Surprisingly, IL-10 gene transfer resulted in a loss of tumorigenicity that was proportional to the amount of IL-10 secreted. Histological analysis showed massive area of necrosis of these tumor cells, with infiltration of polymorphic inflammatory cells. Parental cells simultaneously implanted had decreased tumorigenicity only when mixed with IL10-producing cells, but not when injected contralaterally, suggesting that their eradication is mediated mostly by a local phenomenon. Host T lymphocytes and natural killer (NK) cells were involved in this eradication because IL-10-producing cells grew in nude mice and in CD8or NK-depleted mice. Finally, mice injected with IL-10-secreting cells developed an antitumoral systemic immune response able to protect them against a subsequent challenge with parental cells. These results demonstrate that, in some settings, IL10 may havein vivoimmunostimulating and proinflammatory properties that need to be considered in its therapeutic development.

ISSN:1043-0342    

DOI:10.1089/hum.1996.7.1-23

出版商:Mary Ann Liebert, Inc.    

年代:1996

数据来源: MAL

摘要:

ABSTRACTThe use of defined or serum-free culture conditions during retroviral transduction of hematopoietic cells would be desirable for standardization and safety reasons, as well as potentially allowing greater expansion of progenitor cells. Retroviral vector supernatants were concentrated and purifiedviatangential flow filtration polyethylene glycol (PEG)-precipitation, and ultracentrifugation, allowing serum-free transductions at standard multiplicities of infection (moi). Protein content of transductions using these concentrated vectors was 56 logs lower than in standard transductions. Transduction efficiencies of these concentrated vector preparations added back to serum-free or serum-containing media were equivalent to standard retroviral supernatant transductions of CD34-enriched progenitors. Absolute progenitor (CFU-C) numbers at the end of transduction were higher in serum-freeconcentrated virus transductions, as opposed to transductions in standard vector supernatants containing fetal calf serum.

ISSN:1043-0342    

DOI:10.1089/hum.1996.7.1-33

出版商:Mary Ann Liebert, Inc.    

年代:1996

数据来源: MAL

摘要:

ABSTRACTApolipoprotein (apo) B100 is an essential component of low-density lipoproteins (LDL) and lipoprotein(a)Lp(a). In mammals, apoB can be edited post-transcriptionally to encode a truncated form of apoB (apoB48) that is unable to form either of these atherogenic lipoproteins. To study the effect of increasing hepatic apoB editing activity on formation of Lp(a), a recombinant adenovirus encoding rat apoBEC-1, the cytidine deaminase component of the apoB mRNA editing complex, was administered to human apoB/apo(a) transgenic mice. This resulted in expression of apoBEC-1 in hepatocytes of these mice, increased hepatic editing of human apoB mRNA, and decreased plasma levels of human apoB100 and Lp(a). The apoBEC-1 recombinant adenovirus was also administered to rabbits, an animal which, like humans, naturally lacks hepatic apoB editing. Expression of the exogenous apoBEC-1 in rabbit liver resulted in editing of up to 10% of apoB mRNA. Hepatic apoB editing was associated with lower LDL levels in these rabbits relative to those treated with a control adenovirus. However, LDL levels were elevated significantly in both animals as a result of adenovirus injection. These studies demonstrate that introduction of the cytidine deaminase apoBEC-1 is sufficient to induce hepatic apoB editing in an animal lacking this activity, and that induction of editing could serve as a novel approach for lowering plasma concentrations of the atherogenic lipoproteins Lp(a) and LDL.

ISSN:1043-0342    

DOI:10.1089/hum.1996.7.1-39

出版商:Mary Ann Liebert, Inc.    

年代:1996

数据来源: MAL

摘要:

ABSTRACTPrimary cultures of airway epithelia were used to evaluate variables pertinent to adenovirus (Ad)-mediated gene transfer efficiency and efficacy including: (i) Ad-vectors with different promoters, (ii) the duration of vector incubation with cells, (iii) the concentration and depth of vector-containing medium at constant multiplicity of infection (moi) (103), and (iv) the relative sensitivity of reverse transcription polymerase chain reaction (RT-PCR)versusfunctional analysis for the detection of transduced cystic fibrosis transmembrane conductance regulator (CFTR). An Ad5-lacZvector with a cytomegalovirus (CMV) enhancer/promoter transduced the greatest amount of-galactosidase (-Gal) activity, while an Ad2-lacZvector with an E1a enhancer/promoter transduced the least. Ad5-lacZvectors with the Rous sarcoma virus (RSV), E1a/RSV, or CMV enhancer/-actin (CB) promoters transduced intermediate levels of-Gal. Optimal gene transfer efficiency was detected with a 48 hr incubation of Ad5-CMVlacZwith cells, although optimal CFTR Cltransport function was detectable after only a 30 min incubation of Ad5-CBCFTRwith cells, consistent with correction of610% of cells in the epithelial sheet. Ad5-CBCFTRtransduction of CF airway epithelial cells (moi103) was optimal when higher concentrations, lower volumes, or smaller depths of vector-containing medium were utilized. RT-PCR was at least 100-fold more sensitive for the detection of transduced CFTR than functional analysis, and could detect as few as 0.001 % Ad5-CBCFTR-infected CF cells admixed with uninfected CF cells. In summary, the variables studied clearly affect the efficiency of Ad-mediated gene transferin vitroand potentiallyin vivo. They also suggest that RT-PCR is a poor marker of gene transfer efficiency and efficacy.

ISSN:1043-0342    

DOI:10.1089/hum.1996.7.1-51

出版商:Mary Ann Liebert, Inc.    

年代:1996

数据来源: MAL

摘要:

ABSTRACTRecently, we have succeeded in using nonautologous myoblasts engineered to secrete mouse growth hormone (GH) to correct partially the growth retardation of the Snell dwarf mice, which suffer from pituitary GH deficiency. The allogeneic myoblasts were protected from immune rejection by enclosure in permselective microcapsules fabricated from alginate, thus validating the clinical efficacy of using universal nonautologous cells for somatic gene therapy. Because GH therapy is considered also for treating patients with normal pituitary function, we now apply this protocol to treat normal mice to evaluate the potential consequences of using GH gene therapy in subjects with no demonstrated GH deficiency. When microencapsulated allogeneic myoblasts engineered to secrete mouse GH were implanted into normal male and female mice, contrary to expectation, the treated animals became significantly shorter and lost weight; their internal organs became smaller and their tibial growth plates were less differentiated, indicating reduced skeletal growth. Females were more severely affected than males and 2 animals died by day 13 of unknown cause. By day 70, most of the abnormalities were restored to normal except for body weights, which remained below normal. In conclusion, although somatic gene therapy for GH delivery is beneficial for pituitary dwarfism, it may have adverse metabolic consequences in those with normal hypothalamicpituitary functions, and the female mice were more severely affected than males.

ISSN:1043-0342    

DOI:10.1089/hum.1996.7.1-61

出版商:Mary Ann Liebert, Inc.    

年代:1996

数据来源: MAL

摘要:

ABSTRACTRat myoblast primary cultures were tested as a model for proinsulin synthesis and processing and unregulated insulin delivery for insulin-dependent diabetes mellitus (IDDM) gene therapy. Three human proinsulin cDNA constructs containing genetically engineered furin endoprotease cleavage sites between the B-chain and C-peptide (IFur) and between the C-peptide and A-chain (IIFur) and/or containing a histidine B10 to aspartic acid point mutation were subcloned into a mammalian expression vector (pCMV) containing the cytomegalovirus (CMV) promoter. The altered cleavage sites enable the insulin to be processed by the ubiquitous endoprotease furin. The histidine B10 to aspartic acid mutation creates a more stable form of insulin leading to an increase in insulin accumulation. Myoblasts transfected with a proinsulin cDNA construct mutated at all three sites (pCMV.IFur.IIFur.B10), a construct with only the furin sites (pCMV.IFur.IIFur), and a construct containing only the mutation at the B10 position (pCMV.B10) accumulated 85216, 15013, and 88339U (pro)insulin/ml, respectively, in the culture medium during a 48-hr incubation. (Pro)insulin was detected in the culture medium within 2 hr post-transfection. Significant (pro)insulin release continued for 1 week and gradually diminished over a month. Approximately 50% of the proinsulin released from rat myoblasts transfected with pCMV.IFur.IIFur.B10 was completely processed into mature insulin based on densitometric analysis of autoradiographs of gels containing immunoprecipitated35S-Cys-labeled (pro)insulin. However, only a trace of the proinsulin encoded by pCMV.B10 was processed. In an isolated rat adipocyte14Cglucose oxidation assay, insulin released from myoblasts transfected with pCMV.IFur.IIFur.B10 was active biologically, displaying more biological activity than normal human insulin. Plasmid expression was studied by transfecting myoblasts with the-galactosidase (-Gal) gene in pCMV, allowing them to divide and fuse into multinucleated myotubes, followed by staining for-Gal. Approximately 80% of myotubes expressed-Gal. The results indicate that proinsulin encoded by genetically modified proinsulin cDNA is processed into mature insulin, which is secreted at high levels, making myoblasts a viable target cell for gene therapy of IDDM.

ISSN:1043-0342    

DOI:10.1089/hum.1996.7.1-71

出版商:Mary Ann Liebert, Inc.    

年代:1996

数据来源: MAL

摘要:

ABSTRACTRecombinant, replication-deficient adenovirus (Ad) vectors have been successfully used to transfer and express the normal human cystic fibrosis transmembrane conductance regulator (CFTR) cDNAin vivoin the respiratory epithelium of experimental animals and humans with cystic fibrosis (CF). Since Ad-directed gene expression wanes over time, repeat administration is necessary to achieve an effective treatment for CF. A major hurdle to such a strategy is the possibility that anti-Ad humoral immunity may prevent gene expression in individuals with pre-existing anti-Ad immunity or following repeat administration. One strategy to circumvent such a problem would be alternating the use of Ad vectors belonging to different subgroups. Neutralizing antibodies developed with the administration of one Ad serotype do not cross-react with an Ad belonging to a second serotype in a manner that blocks infection and gene expression. To test this hypothesis, an immunizing dose of wild-type Ad5 (subgroup C), Ad4 (subgroup E), or Ad30 (subgroup D) was administered intratracheally to experimental animals, followed by an intratracheal administration of a replication-deficient subgroup C-derived vector coding for marker genes (chloramphenicol acetyl transferase or-galactosidase) or for the normal human CFTR cDNA. As expected, studies with vectors coding for marker genes or for CFTR cDNA demonstrated that airway administration of a vector does not yield efficient gene transfer, if there has been prior recent airway administration of the same Ad subgroup. In contrast, effective expression from the second administration can be achieved with an adenovirus vector belonging to a subgroup different from the first adenovirus administered. These data support the paradigm of alternating Ad vectors derived from different subgroups as strategy to circumvent anti-Ad humoral immunity, thus permitting the use of Ad vectors as a means to treat the respiratory manifestations of CF.

ISSN:1043-0342    

DOI:10.1089/hum.1996.7.1-79

出版商:Mary Ann Liebert, Inc.    

年代:1996

数据来源: MAL

摘要:

ABSTRACTIn 1991 we reported gene transduction into autologous long-term repopulating marrow cells in dogs using amphotropic helper-free retrovirus vectors containing the bacterial neomycin phosphotransferase gene (neo) and the human adenosine deaminase gene (ADA). Two of the dogs are still alive and healthy now more than 5 years after transplantation of transduced autologous marrow cells. In one of the surviving dogs, polymerase chain reaction (PCR) analysis showed theneoand ADA genes to be present in peripheral blood granulocytes and lymphocytes up to the present time. The estimated percentage ofneo-positive cells ranged from<0.001% to 0.1%. ADA mRNA expression was detected by reverse transcriptase PCR (RT-PCR) in granulocytes 63 months after transplantation. The other surviving dog failed to show either persistence or expression of the transduced genes after 50 months. Three additional dogs have been transplanted according to the same transduction protocols and with the same retrovirus vectors, and persistence of the transducedneogene has been documented in peripheral blood myeloid and lymphoid cells along with G418-resistant colony-forming unit-granulocyte/macrophage (CFU-GM) for now more than 2 years. These findings represent the longest follow-up of retrovirus-mediated gene transduction in any animal species. Long-term transduction efficiency, though, has remained low and will need to be improved for therapeutic application to be possible.

ISSN:1043-0342    

DOI:10.1089/hum.1996.7.1-89

出版商:Mary Ann Liebert, Inc.    

年代:1996

数据来源: MAL