Transformation of mammalian cells with an amplifiable dominant-acting gene.

We have transferred a mutant hamster gene coding for an altered dihydrofolate reductase to wild-type cultured mouse cells by using total genomic DNA from methotrexate-resistant Chinese hamster ovary A29 cells as donor. By demonstrating the presence of hamster gene sequences in transformants we have provided direct evidence for gene transfer. Transformants selected for increased resistance to methotrexate contain increased amounts of the newly transferred gene. We have used this mutant dhfr gene to introduce the Escherichia coli antibiotic resistance plasmid pBR322 into animal cells. Amplification of the dhfr sequences results in amplification of the pBR322 sequences as well. The use of this gene may allow the introduction and amplification of virtually any genetic element in various new cellular environments.     

A dominant positive and negative selectable gene for use in mammalian cells.

We have constructed three different fusion genes containing the herpes simplex virus thymidine kinase (HSV tk) and the bacterial neomycin phosphotransferase (neo) genes. All three fusion genes utilize the HSV tk promoter but differ at the junction of their components. We have determined if the fusion genes are bifunctional by introducing them into mammalian cells and testing for function of the individual components. One of the fusion genes, TNFUS 69, produced a bicistronic message and a fusion protein that has TK and NEO protein functions. This and other fusion genes of a similar nature could serve as dominant positive and negative selectable markers in mammalian cells.     

Baculovirus-mediated gene transfer into mammalian cells.

This paper describes the use of the baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) as a vector for gene delivery into mammalian cells. A modified AcMNPV virus was prepared that carried the Escherichia coli lacZ reporter gene under control of the Rous sarcoma virus promoter and mammalian RNA processing signals. This modified baculovirus was then used to infect a variety of mammalian cell lines. After infection of the human liver cell lines HepG2, >25% of the cells showed high-level expression of the transduced gene. Over 70% of the cells in primary cultures of rat hepatocytes showed expression of beta-galactosidase after exposure to the virus. Cell lines from other tissues showed less or no expression of lacZ after exposure to the virus. The block to expression in less susceptible cells does not appear to result from the ability to be internalized by the target cell but rather by events subsequent to viral entry. The onset of lacZ expression occurred within 6 hr of infection in HepG2 cells and peaked 12-24 hr postinfection. Because AcMNPV is able to replicate only in insect hosts, is able to carry large (>15 kb) inserts, and is a highly effective gene delivery vehicle for primary cultures of hepatocytes, AcMNPV may be a useful vector for genetic manipulation of liver cells.     

Efficient gene transfer into mammalian primary endocrine cells with lipopolyamine-coated DNA.

A general and efficient transfection procedure, based on compacted lipopolyamine-coated plasmids, has been developed. The active species is obtained by simple addition of excess synthetic lipospermine solution to the DNA and binds within minutes to the cell membrane. This technique has been developed on endocrine cells of the intermediate lobe of the pituitary as a general tool for physiological work on primary cells; it is not toxic and does not interfere with physiological regulations in melanotrope cells. A variety of eukaryotic cell cultures also have been transfected with success for transient and stable expression.     

In vivo and in vitro gene transfer to mammalian somatic cells by particle bombardment.

Chimeric chloramphenicol acetyltransferase and beta-galactosidase marker genes were coated onto fine gold particles and used to bombard a variety of mammalian tissues and cells. Transient expression of the genes was obtained in liver, skin, and muscle tissues of rat and mouse bombarded in vivo. Similar results were obtained with freshly isolated ductal segments of rat and human mammary glands and primary cultures derived from these explants. Gene transfer and transient expression were also observed in eight human cell culture lines, including cells of epithelial, endothelial, fibroblast, and lymphocyte origin. Using CHO and MCF-7 cell cultures as models, we obtained stable gene transfer at frequencies of 1.7 x 10(-3) and 6 x 10(-4), respectively. The particle bombardment technology thus provides a useful means to transfer foreign genes into a variety of mammalian somatic cell systems. The method is applicable to tissues in vivo as well as to isolated cells in culture and has proven effective with all cell or tissue types tested thus far. This technology may therefore prove to be applicable in various aspects of gene therapy.     

High-efficiency gene transfer into mammalian cells: generation of helper-free recombinant retrovirus with broad mammalian host range.

We have constructed a chimeric retrovirus genome containing ecotropic gag-pol sequences from Moloney murine leukemia virus and envelope sequences derived from the amphotropic virus 4070A. This reconstructed genome, termed pMAV-psi-, lacks the psi site required for encapsidation of the viral genome. NIH 3T3 cells transfected with pMAV-psi-, called psi-AM lines, are capable of producing high titer stocks of helper-free recombinant retrovirus with amphotropic host range after transfection with recombinant retroviral vectors carrying the neomycin phosphotransferase gene. Most transfected psi-AM cells remain helper-free, even after months in culture. psi-AM virus stocks infect nearly all human and murine cell lines tested thus far, as assayed by resistance to the neomycin analogue G418. Southern and RNA blot analyses of psi-AM-infected human cells show that recombinant murine retroviruses integrate randomly into genomic DNA as normal proviruses and express high levels of the subgenomic and genomic viral messages in the expected stoichiometry of 1:1.     

Robust in vivo gene transfer into adult mammalian neural stem cells by lentiviral vectors

Stable genetic modification of adult stem cells is fundamental for both developmental studies and therapeutic purposes. Using in vivo marking studies, we showed that injection of lentiviral vectors (LVs) into the subventricular zone of the adult mouse brain enables efficient gene transfer into long-term self-renewing neural precursors and steady, robust vector expression in their neuronal progeny throughout the subventricular zone and its rostral extension, up to the olfactory bulb. By clonal and population analysis in culture, we proved that in vivo-marked neural precursors display self-renewal and multipotency, two essential characteristics of neural stem cells (NSCs). Thus, LVs efficiently target long-term repopulating adult NSCs, and the effect of the initial transduction is amplified by the continuous generation of NSC-derived, transduced progeny. LVs may thus allow novel studies on NSCs' physiology in vivo, and introduction of therapeutic genes into NSCs may allow the development of novel approaches for untreatable CNS diseases.     

Clinical gene transfer studies for hemophilia B

Hemophilia B, a deficiency of functional factor IX (FIX), has been extensively explored as a model for gene transfer. Two U.S. Food and Drug Administration-approved clinical studies for hemophilia B have been undertaken, both using adeno-associated viral vectors (AAV). AAV vectors have tropism for liver, muscle, central nervous system, and the respiratory tract; both skeletal muscle and liver have been used as target tissues in the hemophilia B studies. In both studies, proof of principle was first established in the hemophilia B dog model, with long-term expression of canine FIX at therapeutic levels achieved before clinical studies were initiated. In the AAV-FIX muscle trial, vector was introduced into skeletal muscle of the upper and lower extremities of eight human patients by direct intramuscular injection. Muscle biopsies taken 2 to 10 months postinjection demonstrated gene transfer and expression (by Southern blot and immunofluorescence, respectively) in all patients, but circulating FIX levels were generally not >1%, and escalation of dose to levels that proved therapeutic in animals was thwarted by feasibility issues regarding the number of injections required. Nevertheless, the study demonstrated that parenteral injection of AAV-FIX was safe at the doses tested, and could result in long-term expression of the transgene. Moreover, the general characteristics of transduction of human muscle were similar to those observed in other animal models. The safety and efficacy data established in the first trial formed the basis for a second trial in which AAV-FIX is administered systemically to target the liver. The liver study is currently ongoing, with six patients enrolled to date.     

Clinical gene transfer studies for hemophilia A

The recent advances in gene transfer technology have expedited the development of gene therapy for the treatment of hemophilia A. Three different U.S. Food and Drug Administration-approved phase I clinical trials had been initiated using different gene therapy approaches each with their own advantages and limitations. In the first gene therapy trial for hemophilia A, a non-viral approach was being explored for patients with severe hemophilia A using ex vivo transfected dermal fibroblast expressing B-domain-deleted factor VIII ( BDD-FVIII). There were no serious adverse events and some patients appeared to have experienced fewer bleeding episodes with very low levels of FVIII near baseline. In the second trial, onco-retroviral vectors expressing BDD-FVIII were injected by peripheral intravenous infusion in adult patients suffering from severe hemophilia A. The procedure was safe and in some patients FVIII-transduced cells were detectable in the peripheral blood for more than a year. Although no sustained FVIII expression was detectable, occasional modest changes in FVIII levels were apparent, and in some cases a reduced bleeding frequency occurred compared with historical rates. In another trial, one patient suffering from severe hemophilia A has been treated with a high-capacity (or gutless) adenoviral vector expressing full-length FVIII, which appeared to have resulted in 1% of normal FVIII levels for several months. However, a transient inflammatory response with hematologic and liver abnormalities was observed. In conclusion, although modest improvements in clinical end points have been detected in some patients in these early phase I trials, further improvements in gene delivery technologies are warranted to bring hemophilia A gene therapy one step closer to reality.     

Positive genetic selection for gene disruption in mammalian cells by homologous recombination.

Efficient modification of genes in mammalian cells by homologous recombination has not been possible because of the high frequency of nonhomologous recombination. An efficient method for targeted gene disruption has been developed. Cells with substitution of exogenous sequences into a chromosomal locus were enriched, by a factor of 100, using a positive genetic selection that specifically selects for homologous recombination at the targeted site. The selection is based on the conditional expression of a dominant selectable marker by virtue of in-frame gene fusion with the target gene. The dominant selectable marker was derived by modification of the Escherichia coli neo gene so that it retains significant activity in mammalian cells after in-frame fusion with heterologous coding sequences. In the example presented here, homologous recombinants were efficiently recovered from a pool in which the targeted gene was disrupted in 1 per 10,000 cells incorporating exogenous DNA.     

Ecdysone-inducible gene expression in mammalian cells and transgenic mice.

During metamorphosis of Drosophila melanogaster, a cascade of morphological changes is triggered by the steroid hormone 20-OH ecdysone via the ecdysone receptor, a member of the nuclear receptor superfamily. In this report, we have transferred insect hormone responsiveness to mammalian cells by the stable expression of a modified ecdysone receptor that regulates an optimized ecdysone responsive promoter. Inductions reaching 4 orders of magnitude have been achieved upon treatment with hormone. Transgenic mice expressing the modified ecdysone receptor can activate an integrated ecdysone responsive promoter upon administration of hormone. A comparison of tetracycline-based and ecdysone-based inducible systems reveals the ecdysone regulatory system exhibits lower basal activity and higher inducibility. Since ecdysone administration has no apparent effect on mammals, its use for regulating genes should be excellent for transient inducible expression of any gene in transgenic mice and for gene therapy.     

Expression of a preproinsulin-beta-galactosidase gene fusion in mammalian cells.

As an approach to the study of mammalian gene expression, the promoters and translation initiation regions of the rat preproinsulin II and the simian virus 40 early genes were fused to the structural gene of Escherichia coli beta-galactosidase, a sensitive probe for gene expression. These fusions were introduced into COS-7 cells, a simian virus 40 large tumor-antigen-producing monkey kidney cell line, where they directed the synthesis of enzymatically active hybrid beta-galactosidase proteins. Conditions for transfection were varied to optimize the expression of beta-galactosidase activity in the transfected cells. The pH optimum of this activity was found to be 7.0, the same as that of native E. coli beta-galactosidase and distinct from the major lysosomal "acid" beta-galactosidase. The fused preproinsulin-beta-galactosidase was further characterized by gel electrophoresis of nondenatured cell extracts stained by a fluorogenic substrate and by immunoprecipitation and gel electrophoresis of 3H-labeled cell proteins. These results all indicate that fully active tetrameric beta-galactosidase hybrids can be produced in mammalian cells. The expression of preproinsulin-beta-galactosidase activity was measured in the presence of high glucose, insulin, dexamethasone, or epidermal growth factor but no regulatory changes were observed.     

Transduction of a bacterial gene into mammalian cells.

The transduction of an Escherichia coli gene into mammalian cells is described. A supressor tRNA gene was linked to a simian virus 40 (SV40) vector in vitro and the recombinant was used to transfect rat embryo cells and monkey kidney cells. The hybrid SV40 genome, SV40-su+ III, retained genetic information required for autonomous replication and cellular transformation and had a 1300-base-pair DNA segment in the late gene region (between the restriction endonuclease sits Hpa II at 0.735 and EcoRI at 0/1.0 on the SV40 genetic map) replaced by an 870-base-pair bacterial DNA segment containing the suppressor tRNA gene, su+ III (tRNATyrsu+III). The structure and fate of the SV40-su+III chimera were determined by DNA reassociation kinetic analysis and restriction enzyme cleavage of the total cellular DNA from transformed rat embryo cells and persistently infected monkey cells. Hybridization with radiolabeled probes specific for vector (SV40) or su+III DNA sequences revealed primarily nonintegrated or free hybrid genomes. In cloned lines of both cell types, the bacterial DNA segment was recovered intact, as judged by the length of the segment excised by restriction endonucleases and its ability to hybridize to the radiolabeled bacterial DNA probe and not to the SV40 probe.     

Direct transfer of cloned genes from bacteria to mammalian cells.

Induction of a virus infection by cloned simian virus 40 DNA was chosen as a test system to detect transfer of genes from bacteria to cultured mammalian cells. Escherichia coli cells containing a recombinant plasmid with three tandem inserts of simian virus 40 DNA were able to infect CV-1 monkey cells under various conditions. The gene transfer was resistant to DNase I and therefore seems not to occur via free DNA but most likely via uptake of whole bacteria, followed by release of plasmid DNA and generation of infectious circular simian virus 40 DNA in a recombination-excision process. Spontaneous transfer was found to be infrequent, 4 x 10(9) bacteria yielding one infection per 10(7) monkey cells. The frequency was greatly increased by adding bacteria as a calcium phosphate coprecipitate or by fusion of lysozyme-treated bacteria (protoplasts) with monkey cells in the presence of polyethylene glycol. With the latter technique, 10(4) protoplasts gave rise to one infection per 15 monkey cells. Experiments with other cell lines of human, monkey, and mouse origin, and also with bacteria harboring another recombinant plasmid, indicate that DNA transfer from bacteria to mammalian cells is a general phenomenon.     

Amplification of expression of hepatitis B surface antigen in 3T3 cells cotransfected with a dominant-acting gene and cloned viral DNA.

3T3 cells containing hepatitis B virus DNA sequences can be efficiently selected by exposure to methotrexate after cotransfection with cloned viral DNA and DNA coding for a methotrexate-resistant dihydrofolate reductase. More than 75% of methotrexate-resistant cells isolated after cotransfection with a head-to-tail tandem of the hepatitis B virus genome synthesized viral surface antigen. The antigen was released into the culture medium in the form of 22-nm particles with buoyant density of 1.20 g/ml. No other virally coded proteins were detected in the cells or the culture medium. Application of selective pressure by increasing the concentration of methotrexate resulted in an amplification of viral DNA sequences and a concomitant increase in the rate of synthesis and release of hepatitis B surface antigen. The ability to produce large amounts of surface antigen appears to be a stable trait and has been maintained in these cultures through more than 30 passages.