Clonal gene therapy: transplanted mouse fibroblast clones express human alpha 1-antitrypsin gene in vivo.

A retroviral vector was used to insert human alpha 1-antitrypsin (alpha 1AT) complementary DNA into the genome of mouse fibroblasts to create a clonal population of mouse fibroblasts secreting human alpha 1AT. After demonstrating that this clone of fibroblasts produced alpha 1AT after more than 100 population doublings in the absence of selection pressure, the clone was transplanted into the peritoneal cavities of nude mice. When the animals were evaluated 4 weeks later, human alpha 1AT was detected in both sera and the epithelial surface of the lungs. The transplanted clone of fibroblasts could be recovered from the peritoneal cavities of those mice and demonstrated to still be producing human alpha 1AT. Thus, even after removal of selective pressure, a single clone of retroviral vector-infected cells that expressed an exogenous gene in vitro, continued to do so in vivo, and when recovered, continued to produce the product of the exogenous gene.

Evaluation of "at risk" alpha 1-antitrypsin genotype SZ with synthetic oligonucleotide gene probes.

Alpha 1-antitrypsin (alpha 1AT), a 52,000-mol-wt serum glycoprotein produced by hepatocytes and mononuclear phagocytes, functions as the major inhibitor of neutrophil elastase. The alpha 1AT haplotype S is associated with childhood liver disease and/or adult emphysema when inherited with the Z haplotype to give the phenotype SZ. To accurately identify the SZ phenotype at the level of genomic DNA, four 32P-labeled 19-mer synthetic oligonucleotide probes were prepared; two to identify the M and S difference in exon III, and two to identify the M and Z difference in exon V. These probes were hybridized with various cloned DNAs and genomic DNAs cut with the restriction endonucleases BgII and EcoRI; the genomic DNAs represented all six possible phenotype combinations of the M, S, and Z haplotypes (MM, MS, MZ, SS, ZZ, and SZ). Using the four probes to evaluate 42 samples of genomic DNA, the "at risk" SZ and ZZ phenotypes were correctly identified in all cases, as were the "not at risk" phenotypes SS, MS, MM, and MZ, demonstrating that both exon III and exon V directed probes are necessary to properly identify all of the major "at risk" alpha 1AT genes. However, when used to evaluate a very rare family carrying a null allele, these four oligonucleotide probes misidentified the "at risk" null-null and S null phenotypes as "not at risk" MM and SM combinations. These observations indicate that oligonucleotide gene probes yielded reliable and accurate assessment of "at risk" alpha 1AT genotypes in almost all situations, but in the context of prenatal diagnosis and genetic counseling this approach must be used with caution and in combination with family studies so as not to misidentify rare genotypes that may be associated with a risk for disease.

Bystander killing of melanoma cells using the human tyrosinase promoter to express the Escherichia coli purine nucleoside phosphorylase gene.

We used a gene transfer-based system to generate highly toxic purine bases in tumor cells transfected with the Escherichia coli purine nucleoside phosphorylase (PNP) gene. Because these toxic purines are membrane permeant, they mediate effective killing of neighboring cells that do not express E. coli PNP ("bystander" toxicity). In mixed cultures containing increasing percentages of cells with gene expression, 100% cancer cell growth arrest and total population killing was demonstrated when as few as 1-2% of cells expressed E. coli PNP. We used E. coli PNP to test bystander killing of human melanoma cells. A 529-bp region upstream of the human tyrosinase gene start site was shown to direct melanoma-specific expression in human cell lines. When this human tyrosinase regulatory region was used to control E. coli PNP expression, profound toxicity was observed in melanoma cells after treatment with the relatively nontoxic substrate 6-methylpurine-deoxyriboside, which is converted by E. coli PNP into the highly toxic purine base 6-methylpurine. Bystander toxicity was estimated as at least 100 cells killed for each cell expressing E. coli PNP, a level substantially higher than that of other tumor sensitization genes currently being used in clinical trails. These results suggest that the high bystander activity of the system could lead to significant antimelanoma responses in vivo.

Enhanced efficacy of a novel controlled release paclitaxel formulation (PACLIMER delivery system) for local-regional therapy of lung cancer tumor nodules in mice.

The efficacy of systemic chemotherapy for non-small cell lung cancer (NSCLC) has improved with newer agents. However, the response rates and prolonged survival times achieved by chemotherapy remain modest, and these small gains are obtained at the cost of significant toxicity. In this study, the efficacy of a controlled release formulation of paclitaxel was compared with conventional paclitaxel in animals with human lung cancer xenografts. Paclitaxel (10%) was encapsulated in a proprietary polymer in the form of microspheres (PACLIMER Delivery System). Tumor nodules comprised of two different cell lines (A549 and H1299) were treated by a single i.p. or intratumoral administration of conventionally formulated paclitaxel or a single intratumoral injection of the PACLIMER Delivery System. In vitro testing demonstrated that paclitaxel was released slowly from the microspheres with >80% released after 90 days. Direct comparison of the highest dose for all formulations (24 mg/kg) showed that for nodules comprised of either NSCLC cell line, growth of the PACLIMER Delivery System-treated nodules were inhibited significantly more than the groups treated with conventional paclitaxel or the vehicle controls. Tumor volume doubling times for A549 and H1299 nodules treated with PACLIMER Delivery System were 60 and 35 days, respectively, compared with 10 and 11 days, respectively, in the nodules treated with the conventional paclitaxel by intratumoral administration. We conclude that intratumoral administration of the PACLIMER Delivery System may substantially increase the efficacy of paclitaxel for the therapy of local-regional NSCLC.

Characterization of the anti-stem cell activity of anti-mouse brain serum.

The anti-stem cell activity of a high-titer rabbit anti-mouse brain serum preparation has been further characterized. Following absorption with bone marrow and erythrocytes the antiserum had dose-dependent cytotoxicity against pluripotent stem cells. Rigorous absorptions with bone marrow, spleen, liver, erythrocytes, and thymus failed to remove the anti-stem cell activity of the serum. Adult brain, the immunogen, but not neonatal brain, removed a substantial amount of the activity against stem cells. Maximal cytotoxicity occurred both with and without complement and was maximal following only a 4 degrees C incubation of cells with serum. The anti-stem cell activity was present in the serum globulin fraction. No increase in the frequency of microcolonies or, with longer growth periods, in splenic macrocolonies was observed, suggesting that CFU-s were completely inactivated by exposure to the antiserum. Injected antiserum also reduced CFU-s in vivo.

Quantitative evaluation of retroviral gene transduction efficiency in human lung cancer cells.

Gene therapy may serve as a valuable therapeutic modality for malignancies, such as lung cancer, that are poorly responsive to conventional therapies. Although many methods for transducing new genes into cells have been described, little is known about gene transduction into lung cancer, especially under conditions that might be encountered in clinical use. As a first step in addressing this important issue, the study presented here examined the ability of a recombinant retrovirus to add a selectable marker gene to the A549 non-small cell lung cancer (NSCLC) cell line under a variety of conditions. Examination of viral exposure times ranging from 30 sec to 4 hr revealed that the number of infected cells increased with every increment in time. By increasing the multiplicity of infection to 1.0 and including a polycation, Polybrene, as an infection facilitator, 0.8% of the NSCLC cells were infected with only a 30-sec viral exposure. Nebulization, a potentially attractive route of administration for pulmonary malignancies, had no significant effect on viral titer, proviral structure, or proviral transcripts. A single lyophilization did reduce viral titer by 58 +/- 6%, but did not affect the proviral structure or transcripts produced by the surviving viruses. These results suggest that recombinant retroviruses have the potential to add new genes to malignancies accessible by the airways under conditions likely required for clinical use.

Trans complementation of an E1A-deleted adenovirus with codelivered E1A sequences to make recombinant adenoviral producer cells.

Replication-incompetent adenovirus is conventionally produced by cells that supply replication-enabling proteins from viral sequences present in trans. As an alternative means of recombinant adenovirus production, replication-enabling E1A sequences were cotransduced into human prostate carcinoma cells infected with an E1A-deleted adenovirus containing a luciferase expression cassette. The replication-enabling plasmid was cotransduced by ionic linkage to the recombinant adenovirus exterior. Cells cotransduced with the replication-enabling plasmid made new adenovirus with titers up to 8 x 10(6) in the supernatants 72-120 hr after transduction. Like the parent virus, the virus present in the cotransduced supernatants and lysates was capable of transferring luciferase activity to new cells. The virus present in the cotransduced cell supernatants was amplified and shown to be identical to the parent virus by genomic analysis. It was concluded that simultaneous addition of a replication-defective adenovirus and a replication-enabling gene sequence in a trans configuration converts some of the cotransduced cells into recombinant adenovirus-producing cells.

Surfactant protein A-directed toxin gene kills lung cancer cells in vitro.

Human surfactant protein A (SPA) expression is considered a marker of respiratory epithelial differentiation. Non-small cell lung cancers (NSCLC) are respiratory epithelial derivatives, and it was previously shown that a minority of these cancers expressed SPA, presumably a consequence of their respiratory epithelial origin. In the studies reported here, SPA-I gene transcriptional regulatory sequences were localized to a 2.75-kb genomic 5'-flanking region fragment obtained by screening a human genomic library. The 2.75-kb fragment was used to direct a luciferase coding sequence transcriptionally within a plasmid construct. In plasmid transduction experiments, the SPA-directed luciferase plasmid produced significant luciferase activity in the SPA-expressing NSCLC cell line, H441, but only background levels in the non-SPA-expressing A549 cells. Because Northern blot analysis of resected NSCLC showed that the majority expressed SPA, an SPA-transcriptional targeting strategy was investigated using chimeric toxin genes comprising the coding sequence for herpes simplex virus thymidine kinase (HSV-TK) under transcriptional control of SPA or SV40 regulatory sequences. As expected, transduction of the constitutive, SV40-directed plasmid followed by ganciclovir treatment reduced numbers of both the A549 and H441 cells. In contrast, the SPA-directed plasmid reduced only the SPA-expressing H441 cells and had no significant effect on the A549 cells. The results of these in vivo experiments suggest the concept of transcriptionally directing toxin genes with SPA can produce targeted toxicity in NSCLC.

Overexpression of adenovirus-encoded transgenes from the cytomegalovirus immediate early promoter in irradiated tumor cells.

Efficient expression of therapeutic genes in irradiated tumor cells would facilitate the conversion of a malignant tumor nodule into a cancer vaccine in situ. We reported previously that transgene expression from an adenoviral vector could be markedly enhanced by treating transduced tumor cells with butyrate. In this study, we demonstrated that a similar butyrate effect could be achieved in irradiated tumor cells. In addition, irradiating cells at doses of 2-40 Gy prior to transduction could also amplify recombinant adenoviral transgene products in a cell-type-specific manner. This suggests that adenovirus-mediated gene therapy, radiation therapy, and butyrate-mediated cancer therapy may potentially be formulated into one synergistic protocol for cancer treatment.

Quantitative and in vivo activity of adenoviral-producing cells made by cotransduction of a replication-defective adenovirus and a replication-enabling plasmid.

Achieving limited recombinant viral replication may provide a means of amplifying viral-mediated gene transfer in vivo. We have previously shown that cotransduction of an E1-defective adenovirus with a plasmid containing the deleted E1 functions into prostate carcinoma cells resulted in E1-defective virus production by those cells. The studies described here have extended these findings to more firmly establish the capacity of the trans complementation approach to achieve amplification of recombinant viral transgene expression. The recombinant virus used for all the studies was AdCMV-luc which contained a luciferase expression cassette; the replication-enabling plasmid, pE1, encoded the E1 functions deleted from AdCMV-luc. Quantitative in vitro studies with the HeLa cell line showed that for each plaque forming unit of AdCMV-luc originally exposed to the cells, 0.54 x 10(3) new replication-defective viruses were detected in supernatants and lysates over the following 4 days. Multiple cell lines were shown to support new virus production following cotransduction of AdCMV-luc and pE1. Small numbers of replication-competent viruses were detected in the lysates and supernatants from the cotransduced cells such that for every 10(5) replication-defective viruses approximately two replication-competent viruses were produced. Tumor nodules produced by engrafting a mixture of AdCMV-luc/pE1-cotransduced HeLa cells with uninfected HeLa cells yielded much higher levels of luciferase expression than control tumors containing mixtures of cells infected with AdCMV-luc alone. In total, these results demonstrate new virus production by cells receiving a replication-defective adenovirus and a replication-enabling plasmid are capable of amplifying recombinant viral transgene expression in vivo.

Coacervate microspheres as carriers of recombinant adenoviruses.

The therapeutic utility of recombinant adenoviruses (rAds) is limited in part by difficulties in directing the viruses to specific sites and by the requirement for bolus administration, both of which limit the efficiency of target tissue infection. As a first step toward overcoming these limitations, rAds were encapsulated in coacervate microspheres comprised of gelatin and alginate followed by stabilization with calcium ions. Ultrastructural evaluation showed that the microspheres formed in this manner were 0.8-10 microM in diameter, with viruses evenly distributed. The microspheres achieved a sustained release of adenovirus with a nominal loss of bioactivity. The pattern of release and the total amount of virus released was modified by changes in microsphere formulation. Administration of the adenovirus-containing microspheres to human tumor nodules engrafted in mice showed that the viral transgene was transferred to the tumor cells. It is concluded that coacervate microspheres can be used to encapsulate bioactive rAd and release it in a time-dependent manner.

Use of a tissue-specific promoter for targeted expression of the herpes simplex virus thymidine kinase gene in cervical carcinoma cells.

Molecular chemotherapy strategies have been developed for a number of epithelial malignancies based on selective delivery and expression of a toxin-encoding gene into the cancer cells. To date, these strategies have not been explored in the context of carcinoma of the cervix, despite the fact that a variety of factors suggest this as an appropriate disease for this gene therapy approach. One limitation in this respect is that appropriate tissue-specific promoters for selective toxin gene expression have not been defined for cervical carcinoma. In this regard, the secretory leukoprotease inhibitor (SLPI) gene has been shown to be constitutively expressed in many epithelial carcinoma cells including the uterine cervix. Thus, we investigated the utility of the SLPI gene as a tissue-specific promoter for regulatory control of the herpes simplex virus thymidine kinase gene for in vitro treatment of cervical carcinoma cells. For this analysis, a gene construct was derived with the herpes simplex virus thymidine kinase gene under regulatory control of the 5' upstream regions of the SLPI gene. Transient transduction of three human cervical carcinoma cell lines with the SLPI-thymidine kinase (TK) construct was followed by treatment with the prodrug ganciclovir. Crystal violet staining was subsequently used to assess cell viability. In this analysis, it was shown that the SLPI-TK construct directed TK-mediated killing in two of three tested cervical cell lines, with the two cell lines being positive for SLPI. In addition, mixing experiments established that cervical carcinoma cells could exhibit a bystander effect which potentially augments the efficacy of molecular chemotherapy approaches. These findings may allow for the development of efficacious, target-specific, toxin gene therapy strategies for cervical carcinoma in human patients.

Transforming growth factor beta within fibrotic scleroderma lungs.

PURPOSE, PATIENTS, AND METHODS: Since transforming growth factor beta (TGF beta) has been implicated as an important mediator of pulmonary fibrosis, we measured TGF beta protein and gene expression in alveolar epithelial lining fluid (ELF) of fibrotic scleroderma lungs sampled by bronchoalveolar lavage (BAL). TGF beta protein was qualitatively examined by Western blot analysis, and quantitatively by radioreceptor assays. Gene expression was evaluated in BAL mononuclear cells by Northern blot analysis with quantification of relative gene expression by densitometric analysis of the autoradiograms. RESULTS: Normal and scleroderma subjects had a 24-kd protein that comigrated with defined human TGF beta 1 and immunoreacted with anti-TGF beta antibody. The normal population had a significantly higher average TGF beta concentration (705 pM) compared with the scleroderma subjects (177 pM). The TGF beta 1 gene was expressed in amounts that did not significantly differ between the scleroderma and normal groups. On an individual subject basis, the TGF beta concentration variability did not correlate with variations in BAL cellularity or TGF beta 1 gene expression within the recovered mononuclear cells. CONCLUSIONS: It is concluded that both normal and fibrotic lungs have TGF beta 1 present at the alveolar epithelial surface. However, in the fibrotic scleroderma lungs, TGF beta protein content and gene expression were not increased at the alveolar epithelial surface. The simultaneous analysis of TGF beta protein content, gene expression, and cellular constituents within individual ELF specimens showed that the cellular components of the ELF do not appear to be major determinants of TGF beta protein concentration at the alveolar epithelial surface.

Supernatant rescue assay vs. polymerase chain reaction for detection of wild type adenovirus-contaminating recombinant adenovirus stocks.

The certification of recombinant adenoviruses prepared for clinical use requires the exclusion of contaminating, replication-competent adenovirus (wild type virus). Polymerase chain reaction (PCR)-based detection assays have been developed that detect the presence of viral sequences present only in wild type adenoviruses. As an alternative, this report describes a novel bioassay, designated the 'supernatant rescue assay', that detected minimal amounts of wild type virus mixed with high numbers of recombinant adenoviruses. This assay is based on the observation that minimal numbers of wild type adenovirus can be rescued from the supernatants of cells exposed to high multiplicities of infection (mois) of recombinant virus mixed with a known, minimal number of wild type virions. This assay was highly reproducible and routinely detected the presence of a single wild type virus mixed within 10(9) recombinant viruses. Under the experimental conditions employed, the supernatant rescue assay was significantly more sensitive than all three different PCR-detection assays.

Reciprocal expression of pleiotrophin and midkine in normal versus malignant lung tissues.

Abundant evidence suggests that growth factors are important mediators of non-small cell lung cancer (NSCLC) growth. Although multiple growth factors have been found to be produced by NSCLC tissues, little is known about possible differences in growth factor expression between malignant and adjacent normal lung tissues. Variation in growth factor expression between normal and malignant lung tissues could be potentially useful diagnostically and therapeutically. In studies reported here, the expression of the angiogenic growth factor pleiotrophin (PTN) and homolog midkine (MK) was assessed in resected normal and malignant lung tissues. Primers specific for the two growth factors were used to amplify reverse transcriptase-produced DNA copies of RNA transcripts harvested from the tissues. This analysis revealed that all normal lung tissues examined (n = 17) expressed PTN but only two expressed MK. Conversely, all of the resected lung cancers (n = 20) expressed MK but only one expressed PTN. These results demonstrated a striking reciprocal expression pattern of MK and PTN in normal versus malignant lung tissue.