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.     

High-efficiency gene transfer and expression in normal human hematopoietic cells with retrovirus vectors

Retroviral vectors containing the selectable bacterial gene for G418 resistance (neo) were used to demonstrate gene transfer into primary human bone-marrow progenitor cells. To obtain populations of cells in which a high proportion of cells were expressing the neo gene, several important modifications were made to earlier procedures. Cells from normal donors were infected in vitro, were exposed to high concentrations of G418 for two days in liquid culture to enrich for cells expressing the neo gene, and were plated in semisolid medium. Gene transfer and expression were detected in colonies arising from progenitors of granulocyte-macrophage and erythroid lineages. Survival curves indicated that a high proportion of progenitor cells, approaching 100%, were G418 resistant. Furthermore, addition of growth factors contained in 5637-conditioned medium to the bone marrow improved the recovery of G418-resistant progenitors twofold to threefold. In addition to these biological measurements of gene expression in progenitor cells, significant levels of neo-specific RNA, similar to the levels of RNA expression in the virus-producing fibroblast cell line, were detected in the bone marrow cells after preselection. These results demonstrate that retrovirus vectors can be used successfully to transfer genes at high efficiency into progenitor cells in the human blood-forming system.     

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.     

Gene transfer into murine hematopoietic stem cells with helper-free foamy virus vectors

Gene transfer into hematopoietic stem cells (HSCs) is an ideal treatment strategy for many genetic and hematologic diseases. However, progress has been limited by the low HSC transduction rates obtained with retroviral vectors based on murine leukemia viruses. This study examined the potential of vectors derived from the nonpathogenic human foamy virus (HFV) to transduce human CD34(+) cells and murine HSCs. More than 80% of human hematopoietic progenitors present in CD34(+) cell preparations derived from cord blood were transduced by a single overnight exposure to HFV vector stocks. Mice that received transduced bone marrow cells expressed the vector-encoded transgene long term in all major hematopoietic cell lineages and in over 50% of cells in some animals. Secondary bone marrow transplants and integration site analysis confirmed that gene transfer occurred at the stem cell level. Transgene silencing was not observed. Thus vectors based on foamy viruses represent a promising approach for HSC gene therapy. (Blood. 2001;98:604-609)     

Location and nucleotide sequence of the transfer origin of the broad host range plasmid RK2.

The origin of plasmid DNA transfer, oriT, has been localized on RK2, a conjugative drug-resistance plasmid of the IncP group with a very broad host range in gram-negative bacteria. The transfer origin is contained in a 760-base-pair Hae II restriction fragment that maps in the same region as the single-strand nick made by the RK2 relaxation complex. The functional oriT was subcloned as a 112-base-pair Hpa II fragment, and the DNA sequence of this region was determined. The dominant structural feature of the oriT sequence is a 19-base-pair inverted repeat, with 15 of the 19 bases able to form pairs in a hairpin structure. This inverted repeat may be the recognition site for the relaxation complex proteins, which nick the plasmid DNA molecule and initiate the transfer process.     

Mutant and recombinant avian retroviruses with extended host range.

Avian retroviruses of subgroups B and D efficiently infect chicken (C/E) but not turkey (T/BD) cells. We describe here three variants of subgroup B and D viruses that infect both cell types equally well. One of these viruses, NTRE-4, was a recombinant between transformation-defective Prague (Pr) strain Rous sarcoma virus (RSV) subgroup B and the endogenous virus RAV-0; the second, SR-DE-1, was a recombinant between Schmidt-Ruppin RSV subgroup D and defective endogenous virus information. T1 oligonucleotide fingerprint analysis of the genomes of these two viruses showed only a small alteration in the portion of the env gene responsible for subgroup specificity, as indicated by the presence of a single subgroup E oligonucleotide in an otherwise purely subgroup B or D gene. The third virus, hrBO1Pr-B, was a variant of Pr-RSV-B that did not appear to be a recombinant and whose altered host range we attribute to mutation. Analysis of the host range of all three viruses by infection of selectively resistant cells and by interference testing indicates that all use the subgroup B receptor on chicken cells and the subgroup E receptor on turkey cells. These viruses may be analogous to the polytropic recombinant viruses recently found to be associated with leukemia in some strains of mice.     

High-efficiency retroviral-mediated gene transfer into human and nonhuman primate peripheral blood lymphocytes.

Peripheral blood lymphocytes (PBLs) are primary targets for gene therapy of inherited and acquired disorders of the immune system. We describe the development of an optimized transduction system that provides for high-efficiency retrovirus-mediated gene transfer into primary PBLs. This optimized transduction protocol combines centrifugation of the lymphocytes (1000 x g) at the inception of transduction with phosphate depletion, low-temperature incubation (32 degrees C), and the use of the packaging cell line PG13. Gene marking studies of human and primate PBLs using these optimized transduction conditions demonstrated that the transduction efficiency exceeded 50% of the total lymphocyte population. The optimized transduction efficiency of PBLs with amphotropic retroviral vectors was in excess of 25%. The transduction procedure does not alter phenotype, viability, or expansion of the transduced cells. Our data indicate that this optimized transduction system leads to high-efficiency gene transfer into primary human lymphocytes, which obviates the requirement for selection of transduced cells prior to gene-therapy procedures. Thus, large quantities of healthy retrovirally transduced lymphocytes containing a broad immunological repertoire can be generated for use in clinical protocols. Our results represent a significant improvement in the methodology for the transduction of lymphocytes for gene therapy.     

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.     

Pathogenic and host range determinants of the feline aplastic anemia retrovirus.

Feline leukemia virus (FeLV) C-Sarma (or FSC) is a prototype of subgroup C FeLVs, which induce fatal aplastic anemia in outbred specific-pathogen-free (SPF) cats. FeLV C isolates also possess an extended host range in vitro, including an ability, unique among FeLVs, to replicate in guinea pig cells. To identify the viral determinants responsible for the pathogenicity and host range of FSC we constructed a series of proviral DNAs by exchanging gene fragments between FSC and FeLV-61E (or F6A), the latter of which is minimally pathogenic and whose host range in vitro is restricted to feline cells. Transfer of an 886-base-pair (bp) fragment of FSC, encompassing the codons for 73 amino acids at the 3' end of pol (the integrase/endonuclease gene) and the codons for 241 amino acids of the N-terminal portion of env [the extracellular glycoprotein (gp70) gene], into the F6A genome was sufficient to confer onto chimeric viruses the ability to induce fatal aplastic anemia in SPF cats. In contrast, no chimera lacking this sequence induced disease. When assayed in vitro, all chimeric viruses containing the 886-bp fragment of FSC acquired the ability to replicate in heterologous cells, including dog and guinea pig cells. Thus, the pathogenic and the host range determinants of the feline aplastic anemia retrovirus colocalize to a 3' pol-5' env region of the FSC genome and likely reside within a region encoding 241 amino acid residues of the N terminus of the extracellular glycoprotein.     

Introduction of a selectable gene into different animal tissue by a retrovirus recombinant vector.

The potential use of retrovirus vectors to transduce foreign genetic information into cells of different tissues of an animal was explored by introducing a recombinant genome carrying the Eco gpt gene into postimplantation mouse embryos. To obviate the need for preparing concentrated virus stocks, psi 2-2-5 cells producing the replication-defective murine sarcoma virus (MSV)-gpt virus were microinjected directly into embryos. The psi 2-2-5 cells were mixed with cells producing replication-competent Moloney murine leukemia virus (Mo-MuLV) to facilitate spread of the vector. A high percentage of the manipulated embryos continued to develop without disturbance and were analyzed either prior to birth or as adults for expression of both helper and Eco gpt virus. Microinjection of as few as 10 Mo-MuLV-producing cells resulted in viremia of greater than 50% of the embryos or adults, 25%-30% of which produced MSV-gpt recombinant virus in a variety of organs including thymus, spleen, lung, kidney, and brain. The fraction of vector-producing cells, however, was 3 to 5 orders of magnitude lower than that of helper-virus-producing cells. Our results demonstrate that a selectable gene can be introduced by retroviral vectors into animals and can be expressed in a wide variety of different somatic tissues.     

Development of a high-titer retrovirus producer cell line capable of gene transfer into rhesus monkey hematopoietic stem cells.

Retroviral-mediated gene transfer into primitive hematopoietic cells has been difficult to achieve in large-animal models. We have developed an amphotropic producer clone that generates greater than 10(10) recombinant retroviral particles (colony-forming units) per ml of culture medium. Autologous rhesus monkey bone-marrow cells were cocultured with either high (2 x 10(10) colony-forming units/ml) or low (5 x 10(6) colony-forming units/ml) titer producer clones for 4-6 days and reinfused into sublethally irradiated animals. The proviral genome was detected in blood and bone-marrow cells from all three animals reconstituted with cells cocultured with the high-titer producer cells. In contrast, three animals reconstituted with bone marrow cocultured with the low-titer producer clone exhibited no evidence of gene transfer.     

DNA-mediated transfer of the adenine phosphoribosyltransferase locus into mammalian cells.

In this report, we demonstrate the feasibility of transforming mouse cells deficient in adenine phosphoribosyltransferase (aprt; AMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.7) to the aprt+ phenotype by means of DNA-mediated gene transfer. Transformation was effected by using unfractionated high molecular weight genomic DNA from Chinese hamster, human, and mouse cells and restriction endonuclease-digested DNA from rabbit liver. The transformation frequency observed was between 1 and 10 colonies per 10(6) cells per 20 microgram of donor DNA. Transformants displayed enzymatic activity that was donor derived as demonstrated by isoelectric focusing of cytoplasmic extracts. These transformants fall into two classes: those that are phenotypically stable when grown in the absence of selective pressure and those that are phenotypically unstable under the same conditions.     

Restoration of superoxide generation to a chronic granulomatous disease-derived B-cell line by retrovirus mediated gene transfer.

Failure of a superoxide generating system, the NADPH oxidase, present in neutrophils and other phagocytes gives rise to chronic granulomatous disease (CGD), a group of single-gene inherited disorders all characterized by an extreme susceptibility to pyogenic infection, with potentially fatal consequences. About 30% of CGD cases are caused by an autosomally inherited deficiency of a 47-Kd cytoplasmic component of the oxidase (p47-phox). Epstein-Barr virus (EBV) immortalized B-lymphocyte lines established from these CGD patients also express this NADPH oxidase defect and consequently are rendered incapable of generating superoxide on stimulation. We have used a p47-phox-deficient EBV-transformed B-cell line as a recipient for retroviral transfer of a functional p47-phox cDNA. The presence and activity of the retrovirally encoded p47-phox in the transduced cells is demonstrated and we show that this restores their capacity to generate superoxide.     

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.     

Replication of the broad host range plasmid RSF1010: requirement for three plasmid-encoded proteins.

Cloning of specific regions of plasmid RSF1010, in conjunction with in vitro replication studies, has revealed three novel genes: repA, repB, and repC. They are clustered in one region of the plasmid, separated from the origin of replication by regions that are not essential for plasmid viability in an Escherichia coli host. In vivo, a 2.1-kilobase segment of the plasmid, bearing the replication origin, can establish itself as an autonomous replicon if the DNA region carrying the three rep genes is present in the same cell on an independent plasmid. In vitro, RSF1010 DNA is efficiently replicated by an ammonium sulfate fraction from the E. coli extract, provided the extracts are prepared from cells that can supply the required rep gene products. Using cells containing the cloned rep gene region as a source of elevated levels of the rep proteins, we have partially purified these proteins in functional form. When added to an enzyme fraction derived from plasmid-free cells, they specifically promote the replication of plasmid DNA bearing the RSF1010 origin.     

Analysis of gene transfer efficiency of retrovirus producer cell transplantation for in situ gene transfer to hematopoietic cells

OBJECTIVE: The aim of this study was to assess the gene transfer efficiency of an in situ administration protocol for hematopoietic stem/progenitor cells in the rhesus macaque (Macaca mulatta) animal model. MATERIALS AND METHODS: Moloney murine leukemia virus amphotropic vector producer cells (1--2 x 10(8) cells/animal) were transplanted into the femoral bone marrow cavities of six macaques. To determine if the levels of gene transfer could be increased, a second injection at the same dose of producer cells was performed into the iliac crest in three of the six macaques. RESULTS: We demonstrated that 0.02-0.1% of peripheral blood mononuclear cells contained the vector transgene for up to 12 months following the initial administration of producer cells. Hematopoietic progenitor cell assays indicated that the neomycin phosphotransferase gene was detected in 10--30% of progenitor cell colonies. A humoral immune response directed toward viral particles was demonstrated in all animals. Additionally, we demonstrated that an increase in the levels of transduced cells, up to 1% of circulating peripheral blood mononuclear cells and granulocytes, contain the transgene following producer cell readministration. CONCLUSIONS: These data demonstrate the successful in situ gene transfer to hematopoietic stem/progenitor cells and circulating peripheral blood mononuclear cells that persists as long as 12 months postinjection, in the absence of any preconditioning.     

Efficient transfer of genetic material into mammalian cells using Starburst polyamidoamine dendrimers.

Starburst polyamidoamine dendrimers are a new class of synthetic polymers with unique structural and physical characteristics. These polymers were investigated for the ability to bind DNA and enhance DNA transfer and expression in a variety of mammalian cell lines. Twenty different types of polyamidoamine dendrimers were synthesized, and the polymer structure was confirmed using well-defined analytical techniques. The efficiency of plasmid DNA transfection using dendrimers was examined using two reporter gene systems: firefly luciferase and bacterial beta-galactosidase. The transfections were performed using various dendrimers, and levels of expression of the reporter protein were determined. Highly efficient transfection of a broad range of eukaryotic cells and cell lines was achieved with minimal cytotoxicity using the DNA/dendrimer complexes. However, the ability to transfect cells was restricted to certain types of dendrimers and in some situations required the presence of additional compounds, such as DEAE-dextran, that appeared to alter the nature of the complex. A few cell lines demonstrated enhanced transfection with the addition of chloroquine, indicating endosomal localization of the complexes. The capability of a dendrimer to transfect cells appeared to depend on the size, shape, and number of primary amino groups on the surface of the polymer. However, the specific dendrimer most efficient in achieving transfection varied between different types of cells. These studies demonstrate that Starburst dendrimers can transfect a wide variety of cell types in vitro and offer an efficient method for producing permanently transfected cell lines.     

Two dominant-acting selectable markers for gene transfer studies in mammalian cells.

We report the development of two dominant-acting genetic markers useful for monitoring gene transfer in mammalian cells that are based on prokaryotic genes encoding key steps in the synthesis of the essential amino acids, tryptophan and histidine. Under appropriate conditions, expression of these genes obviates the nutritional requirements for their respective amino acid products. Expression of the trpB gene of Escherichia coli, which encodes the beta subunit of tryptophan synthase (EC 4.2.1.20), allows mammalian cell survival and multiplication in medium containing indole in place of tryptophan. The hisD gene of Salmonella typhimurium encodes histidinol dehydrogenase (EC 1.1.1.23), which catalyzes the two-step NAD+-dependent oxidation of L-histidinol to L-histidine. In medium lacking histidine and containing histidinol, only mammalian cells expressing the hisD product survive. The selection is a double one in that the provided precursor histidinol is itself toxic to animal cells through its inhibition of histidyl-tRNA synthetase; thus, the dehydrogenase both removes an inhibitor and forms a required end product. Alternatively, the his selection may be carried out under conditions in which the dehydrogenase serves mainly to detoxify histidinol. For either the trp or his selections the substitute nutrient (indole or histidinol) is readily available, inexpensive, stable, permeable to cells, and convertible to the end product in a step controlled by a single gene. Vectors based upon murine retrovirus and papovavirus backbones have been successfully employed for both genes, allowing selection in a range of cell types, including 3T3, CV-1, and HeLa. These dominant selective schemes should provide generally useful and inexpensive alternatives to others currently in use, such as the gpt, neo, hygro, dhfr, and tk selections.