Therapeutic efficacy in a hemophilia B model using a biosynthetic mRNA liver depot system.

DNA-based gene therapy has considerable therapeutic potential, but the challenges associated with delivery continue to limit progress. Messenger RNA (mRNA) has the potential to provide for transient production of therapeutic proteins, without the need for nuclear delivery and without the risk of insertional mutagenesis. Here we describe the sustained delivery of therapeutic proteins in vivo in both rodents and non-human primates via nanoparticle-formulated mRNA. Nanoparticles formulated with lipids and lipid-like materials were developed for delivery of two separate mRNA transcripts encoding either human erythropoietin (hEPO) or factor IX (hFIX) protein. Dose-dependent protein production was observed for each mRNA construct. Upon delivery of hEPO mRNA in mice, serum EPO protein levels reached several orders of magnitude (>125 000-fold) over normal physiological values. Further, an increase in hematocrit (Hct) was established, demonstrating that the exogenous mRNA-derived protein maintained normal activity. The capacity of producing EPO in non-human primates via delivery of formulated mRNA was also demonstrated as elevated EPO protein levels were observed over a 72-h time course. Exemplifying the possible broad utility of mRNA drugs, therapeutically relevant amounts of human FIX (hFIX) protein were achieved upon a single intravenous dose of hFIX mRNA-loaded lipid nanoparticles in mice. In addition, therapeutic value was established within a hemophilia B (FIX knockout (KO)) mouse model by demonstrating a marked reduction in Hct loss following injury (incision) to FIX KO mice.

Long-term production and delivery of human growth hormone in vivo.

The application of somatic cell gene therapy to large patient populations will require the development of safe and practical approaches to the generation and characterization of genetically manipulated cells. Transkaryotic implantation is a gene therapy system based on the production of clonal strains of engineered primary and secondary cells, using non-viral methods. We demonstrate here that, on implantation, these clonal cell strains stably and reproducibly deliver pharmacologic quantities of protein for the lifetime of the experimental animals.

Breakage of the human Y-chromosome short arm between two blocks of tandemly repeated DNA sequences.

Y-chromosomal rearrangements, a common cause of sex reversal in man, frequently occur between two blocks of repeated DNA. Both blocks are composed of 20-kb tandemly repeated Y-chromosome-specific DNA sequences. They are located in the proximal portion of the Y short arm on a NotI restriction fragment of approximately 5.3 Mb and on an MluI fragment of approximately 5.5 Mb. Chromosome breaks positioned between the two blocks were detected in two of three 46,XY females with deletions of Yp and in five of six 46,XX males positive for the repeat sequences. The rearranged NotI fragments in the 46,XX males were 4.4 Mb and the MluI fragments were 2.0 Mb in length. This indicates that breaks occur within a small region of Yp defined by the two blocks of specific repeated DNA sequences. The region between the two blocks thus appears to be a focus of structural lability in the human Y chromosome.

Amplified inverted duplications within and adjacent to heterologous selectable DNA.

Plasmids containing a dihydrofolate reductase (DHFR) expression unit were transfected into DHFR-deficient Chinese hamster ovary (CHO) cells. Methotrexate exposure was used to select cells with amplified DHFR sequences. Three cell lines were isolated containing amplified copies of transfected DNA that had integrated into the Chinese hamster genome. Plasmid DNA was found to co-amplify with flanking hamster sequences that were repetitive (2 cell lines) and unique (1 cell line). Fragments comprising the junctions of amplified plasmid and CHO DNA were found to exist as inverted duplications in all three cell lines. These observations provide evidence that inverted duplication occurred prior to DNA amplification, thus underscoring the importance of inverted duplication in the DNA amplification process.

Chromosome instability associated with human alphoid DNA transfected into the Chinese hamster genome.

Repetitive DNA sequences have been implicated in the mediation of DNA rearrangement in mammalian cells. We have tested this hypothesis by using a dihydrofolate reductase (DHFR) expression vector into which candidate sequences were inserted. DHFR- Chinese hamster ovary (CHO) cells were transfected with this vector, the amplification of which was then selected for by methotrexate (MTX) exposure. Cells transfected with the vector alone (and resistant to 0.02 or 1.0 microM MTX) or with a poly(dG-dT) insert (and resistant to 0.05 or 1.0 microM MTX) showed little change in chromosome aberrations or sister chromatid exchange frequencies. In contrast, transfection of DHFR- CHO cells with a vector containing either of two distinct 0.34-kilobase human alphoid DNA segments (and selection to 0.05 to 10.0 microM MTX) showed an approximately 50% increase in chromosome number and marked changes in chromosome structure, including one or two dicentric or ring forms per cell. The sister chromatid exchange frequency also increased, to more than double the frequency of that in cells transfected without insert or those containing poly(dG-dT). In situ hybridization of one 0.34-kilobase insert in some cells suggested clustering of homologous sequences in structurally abnormal recipient CHO cell chromosomes. The approach described provides an introduction to a unique means for a coordinate molecular and cytological study of dynamic changes in chromosome structure.

Disparate effects of 5-bromodeoxyuridine on sister-chromatid exchanges and chromosomal aberrations in Bloom syndrome fibroblasts.

The existence of a high frequency of spontaneous sister-chromatid exchanges (SCEs) in Bloom syndrome (BS) has thus far been supported by data on a small number of BS cell lines. To examine the cause of baseline SCEs more broadly, the frequencies of SCEs, as well as chromosomal aberrations (CAs) in 4 additional BS fibroblast strains were compared, under different assay and cell culture conditions, with those of normal cells in the range of approximately 0.9-90% 5-bromodeoxyuridine (BrdUrd) substitution into template DNA. SCEs at low levels of BrdUrd substitution were detected by an extremely sensitive immunofluorescent technique. From approximately 0.9% to 4.5% BrdUrd substitution, the SCE frequency in BS cells remained constant, at a level (40/cell) 8 times higher than that of normal cells. As BrdUrd substitution increased further, the SCE frequency in BS cells increased almost linearly, reaching 70-100 per cell at approximately 90% substitution, while the SCE increment in control fibroblasts was less than 5 per cell. Analysis of SCEs in 3 successive replication cycles similarly revealed that the SCE increment in BS cells depended on BrdUrd only at a high BrdUrd substitution level. In contrast to data on SCEs, CA induction by incorporated BrdUrd in BS cells was only slightly higher than that in normal cells. Thus, BS cells are extremely sensitive to BrdUrd for SCE induction, but much less so for CA induction.

5-Bromodeoxyuridine-dependent increase in sister chromatid exchange formation in Bloom's syndrome is associated with reduction in topoisomerase II activity.

Bloom's syndrome is characterized by a high sister chromatid exchange (SCE) frequency, the basis for which is not yet understood. Immunofluorescent detection of SCE formation in dermal fibroblasts was employed over a wide range of 5-bromodeoxyuridine (BrdU) substitution into template DNA to show that this SCE elevation reflects both an increased baseline SCE frequency and an exaggerated increment in SCE formation as BrdU substitution increases. The impact of BrdU on SCE formation in Bloom's syndrome is paralleled by its ability to reduce the activity in nuclear extracts of topoisomerase II, an enzyme important for DNA replication and interchange. The extractable topoisomerase II activity of Bloom's syndrome fibroblasts, as measured by unknotting of page P4 DNA, is much more strongly inhibited by cell growth in medium containing BrdU than is that of normal fibroblasts. The results are consistent with the hypothesis that much of the BrdU-dependent component of SCE formation in Bloom's syndrome may be mediated by an effect of BrdU substitution of template DNA on topoisomerase II activity.

An explanation of interspecific differences in sensitivity to X-ray-induced chromosome aberrations and a consideration of dose-response curves.

Using 1-beta-D-arabinofuranosylcytosine (AraC) which is an inhibitor of DNA-repair resynthesis, previous studies have shown that the frequency of chromosome-type aberrations is influenced by the rate of repair of araC-inhibitable DNA damage. The experiments described here are a further test of this hypothesis and also an attempt to determine if the different sensitivities of lymphocytes of different species to X-ray-induced aberrations are related to the rate of endonucleolytic incision during repair of DNA damage. Unstimulated lymphocytes from 4 species were exposed to an X-ray dose of 200 rad, and then incubated with araC for 0, 1, 2, 3 or 4 h. The aberration frequencies increased in all species up to 3-4 h. It was also clear that the rate of increase was different between species and was approximately proportional to the ratios of X-ray-induced aberrations observed in the absence of araC. For example, human lymphocytes are approximately twice as sensitive as rabbit lymphocytes to the induction of aberrations by X-rays and the rate of increase of aberrations in the presence of araC was about twice as great in human as rabbit lymphocytes. In addition, using 50, 100, 200 or 300 rad of X-rays and treating human lymphocytes for 0, 1, 2 or 3 h in araC post-irradiation, we have shown that the rate of increase in aberrations is proportional to the amount of araC-inhibitable DNA damage; with a limiting dose at about 50 rad. These results appear to provide a basis for interpreting differences in sensitivities to aberration induction among mammalian species.

The effect of 3-aminobenzamide on the frequency of X-ray- or neutron-induced chromosome aberrations in cycling or non-cycling human lymphocytes.

3-Aminobenzamide (3-AB) is a potent inhibitor of poly(ADP-ribose) synthesis in mammalian cells. It was found to cause a 2-fold increase in dicentric frequency following X-irradiation of 9-18 h PHA-stimulated human lymphocytes, while 3-AB by itself had no effect on aberration frequency. In contrast to previously reported data, however, our results indicate that 3-AB does not increase the frequency of aberrations following either neutron or X-ray exposure of unstimulated (G0) human lymphocytes. Although 3-AB incubation after X-ray exposure in G1 caused a large increase in dicentrics, there was no effect of 3-AB incubation following neutron exposure in G1. The implications of these experiments are presently uncertain, but they do, however, suggest the importance of cycling cells for 3-AB to exert its enhancement effect, presumably on some step of DNA repair. Furthermore, these data support the hypothesis that there are different mechanisms of chromosome aberration induction with fission neutrons and X-rays (at X-ray doses above 50 rad).

The replication of unsubstituted and 5-bromodeoxyuridine- or 5-chlorodeoxyuridine-substituted DNA regulates the rate of induction of sister chromatid exchanges.

The thymidine (dThd) analog 5-bromodeoxyuridine (BrdUrd) is widely used in studies of the induction of sister chromatid exchanges (SCEs), since growth in the presence of BrdUrd allows the subsequent differential staining of the chromosomes through the fluorescence-plus-Giemsa (FPG) method. However, the analog itself induces SCEs, an aspect of its use which is often not considered. We have studied the induction of SCE by BrdUrd and a second dThd analog 5-chlorodeoxyuridine (CldUrd). Growth of Chinese hamster ovary (CHO) cells for 2 rounds of replication in the presence of different concentrations of either analog results in increasing results in increasing SCE frequencies which are linearly proportional to the degree of analog substitution for dThd in the DNA. However, CldUrd causes 3 to 5 times the number of SCEs found with BrdUrd, at equivalent substitution for dThd. With both analogs the increase in SCE frequency is due to the replication of the analog-substituted DNA and not to the incorporation of analog into nascent DNA. This induction of SCE can be considered at the level of a single strand of DNA since the replication of bifilarly substituted DNA results in twice the number of SCEs that are induced by the replication of unifilarly substituted DNA.

Sister-chromatid exchanges and gene mutations are induced by the replication of 5-bromo- and 5-chloro-deoxyuridine substituted DNA.

The thymidine (dT) analogue 5-chlorodeoxyuridine (CldU) induces 7-8-fold more sister-chromatid exchanges (SCE) than does 5-bromodeoxyuridine (BrdU) at equal substitution for dT in Chinese hamster ovary cells in culture. This difference facilitates study of the mechanism of induction of SCE by these analogues. Cultures were incubated with either BrdU or CldU for one cell cycle, followed by incubation in the presence of dT alone or BrdU or CldU for the second cell cycle and the SCE frequency determined in M2 cells. The results suggest that the induction of SCE is dependent only on the replication of the analogue-substituted DNA during the second cell cycle. Additional studies employed cultures grown in the presence of BrdU or CldU for 7 days to obtain mainly bifilarly substituted DNA, followed by 2 rounds of replication in the presence of dT alone. The SCE frequencies were approximately twice those found in cultures which had undergone the usual 2 rounds in the presence of the analogue; this is consistent with the replication of twice the amount of analogue-substituted DNA. Furthermore, such long-term growth in the presence of BrdU or CldU also results in concentration-dependent increases in the frequency of 6-thioguanine-resistant mutants, suggesting that gene mutations also result from the replication of analogue-substituted DNA.

SCE induction is proportional to substitution in DNA for thymidine by CldU and BrdU.

The incorporation of 5-bromo- and 5-chlorodeoxyuridine into cellular DNA and the induction of sister-chromatid exchanges (SCE) was examined with Chinese hamster ovary cells in culture. Although CldU caused 3-5 times more SCE than BrdU at equal extracellular concentrations, the 2 analogs were incorporated into DNA in place of thymidine similarly over a concentration range of 0.5-20 microM. The results show that SCE induction is linearly proportional to substitution for thymidine by both BrdU and CldU. Finally, based on linear extrapolation, a spontaneous level of approximately 6 SCEs per cell is estimated.

The induction of specific-locus mutations and sister-chromatid exchanges by 5-bromo- and 5-chloro-deoxyuridine.

The induction of 6-thioguanine-resistant (TGr) mutants and sister-chromatid exchanges (SCE) by 5-bromo- and 5-chloro-deoxyuridine (BrdU and CldU) was determined with Chinese hamster ovary cells in culture. Over a concentration range of 0.5-200 microM, CldU treatment caused 3-5 times the number of SCEs found with BrdU; for example, with 10 microM CldU, 60.0 +/- 14.2 SCEs/metaphase (mean +/- S.D.) were induced, while with 10 microM BrdU, only 14.6 +/- 4.1 SCEs/metaphase were found. Despite this difference in SCE induction, both compounds were mutagenic only at concentrations greater than 50 microM, and BrdU was more mutagenic than CldU. These results indicate that SCEs and gene mutations are induced by these thymidine analogs through 2 different mechanisms.

Mutagenicity of municipal water obtained from an agricultural area.

We have studied the mutagenicity of the water from Lake Bloomington and of the tap water that is made from the lake water. The lake, which is the source of drinking water for Bloomington, Illinois (pop. 44,000), is surrounded by land that is farmed intensively--being mainly in maize and soybeans. Samples were collected monthly from May through October 1979 and concentrated 3,000X with XAD-2 resin. Nearly all of the lake and tap water concentrates were mutagenic in Salmonella typhimurium TA98 and TA100 in the presence of S-9 mix, and the May tap water concentrate was highly mutagenic. In addition, many of the concentrates were toxic to the bacteria in the absence of S-9 mix. Chemical analysis of the highly mutagenic tap water concentrate from May revealed the presence of a number of organic contaminants that were absent from control concentrates prepared from deionized and distilled treated-well water. In addition, unconcentrated lake and tap water were tested in a reverse-mutation test in maize (Zea mays); no mutagenicity was detected. This study indicates that the contamination of drinking water with agricultural and/or industrial chemicals may result in a potential health hazard.