Amphotropic Packaging Cell Line Research Articles

Molecular Biology Techniques Q&A

Molecular Biology Techniques Q&A

Regulation of cAMP Responses by the G12/13 Pathway Converges on Adenylyl Cyclase VII

Regulation of intracellular cAMP by multiple pathways enables differential function of this ubiquitous second messenger in a context-dependent manner. Modulation of G(s)-stimulated intracellular cAMP has long been known to be modulated by the G(i) and G(q)/Ca(2+) pathways. Recently, the G(13) pathway was also shown to facilitate cAMP responses in murine macrophage cells. We report here that this synergistic regulation of cAMP synthesis by the G(s) and G(13) pathways is mediated by a specific isoform of adenylyl cyclase, AC7. Furthermore, this signaling paradigm exists in several hematopoietic lineages and can be recapitulated by exogenous expression of AC7 in HEK 293 cells. Mechanistic characterization of this synergistic interaction indicates that it occurs downstream of receptor activation and it can be mediated by the alpha subunit of either G(12) or G(13). Our results demonstrate that AC7 is a specific downstream effector of the G(12/13) pathway.

Amphotropic retrovirus transduction is inhibited by high doses of particle‐associated envelope proteins

Using a panel of amphotropic murine leukemia virus packaging cell lines that differed only in their levels of envelope protein (gp70) expression, we examined the relationship between transduction and the number of envelope proteins per virus. We generated virus stocks that contained different levels of virus-associated envelope proteins, purified them from gp70 that was not associated with the viruses, quantified their titers, and measured the efficiency with which they transduced NIH 3T3, TE671, and HeLa cells. As expected, titers increased monotonically with viral envelope protein number. Titers are measured using highly dilute virus, however, and are often not predictive of gene transfer when high doses of virus are used, as is done in gene therapy protocols. Interestingly, when we used high doses of virus, we observed significantly different trends: gene transfer increased, reached a maximum, and then declined sharply as the number of envelope proteins per virus increased. The highest levels of gene transfer occurred when cells were transduced with a moderate dose of virus that contained low levels of envelope protein. Our results indicate that transduction is inhibited when viruses that contain large numbers of envelope proteins are used. This is most likely because each virus, when it binds to a cell, delivers a large payload of envelope proteins that occupy or inactivate multiple virus receptors, reducing or eliminating the susceptibility of the cell to being transduced by additional viruses. The implications of our findings for the design of improved retroviral vectors for human gene therapy are discussed.

The Proinflammatory Actions of Angiotensin II Are Dependent on p65 Phosphorylation by the IκB Kinase Complex

The vasoactive hormone angiotensin II (Ang II) probably triggers inflammatory cardiovascular diseases by activating transcription factors such as NF-kappaB. We describe here a novel mode of NF-kappaB activation in cultured vascular smooth muscle cells exposed to Ang II. Ang II treatment resulted in an increase in the phosphotransferase activity of the IKK complex, which was mediated through the AT1 receptor subtype. The typical phosphorylation and proteasome-dependent degradation of the NF-kappaB inhibitor IkappaBalpha were not observed. Rather, Ang II treatment of vascular smooth muscle cells led to the phosphorylation of p65 on serine 536, a signal detected in both the cytoplasm and the nuclear compartments. The use of pharmacological inhibitors that inhibit the activation of MEK by Ang II revealed that phosphorylation of p65 on serine 536 did not require the MEK-ERK-RSK signaling pathway. On the other hand, specifically targeting the IKKbeta subunit of the IKK complex by overexpression of a dominant negative version of IKKbeta (IKKbeta K44A) or silencing RNA technology demonstrated that the IKKbeta subunit of the IKK complex was responsible for the detected phosphoserine 536 signal in Ang II-treated cells. Characterization of the signaling pathway leading to activation of the IKK complex by Ang II revealed that neither epidermal growth factor receptor transactivation nor the phosphatidylinositol 3-kinase-AKT signaling cascade were involved. Collectively, our data demonstrate that the proinflammatory activity of Ang II is independent of the classical pathway leading to IkappaBalpha phosphorylation and degradation but clearly depends on the recruitment of an IKK complex signaling cascade leading to phosphorylation of p65 on serine 536.

344. Application of a suicide Gene to X-SCID Gene Therapy

X-linked severe combined immunodeficiency (X-SCID) is a fatal disease characterized by the absence of humoral and cellular immunity due to mutations in the gene encoding the common gamma (|[gamma]|c) chain. X-SCID is a good candidate for the somatic gene therapy because T and NK cells will develop once the |[gamma]|c chain is introduced into patients' CD34 positive cells in vitro. In addition, recent reports of retrovirus-based X-SCID gene therapy clinical trials from France and England demonstrated the efficacy of the gene therapy. Most of the patients who have received the gene therapy showed NK cell recovery and immunoglobulin production in addition to T cell reconstitution. However, a serious adverse effect was reported in three out of eleven patients treated in France. The complication was lymphoproliferative disease, which occurred about three years later after the gene therapy. Two of them were due to insertional mutagenesis of a proto-oncogene, LMO2 by retroviral vector. Therefore, development of a safer integrating vector is an urgent need. One of the approaches is to incorporate a suicide gene into a therapeutic retrovirus vector to offset the lymphoproliferative disease caused by the gene therapy. In this study, we constructed a retroviral vector encompassing a human |[gamma]|c chain cDNA and a suicide gene from the Herpes simplex virus thymidine kinase (HSVtk). For this purpose, a human |[gamma]|c cDNA was inserted into SFCMM3, a retroviral vector containing an HSVtk gene, which had already been applied in clinical trial. The resultant vector (SFCMM-gamma) was transfected into COS-7 cells and expression of the |[gamma]|c chain was confirmed. For recombinant retrovirus production, the SFCMM-gamma was transfected into an amphotropic packaging cell line, |[psi]|CRIP. 24 hours later after transfection, recombinant virus-producing |[psi]|CRIP cells were irradiated (30Gy) and then co-cultured with EBV transformed B cell lines established from X-SCID patients for 48 hours. The infection cycle was repeated twice. Just prior and a week after starting the co-culture, expression of the |[gamma]|c chain of the EBV-B lines was assessed by flow cytometric analysis. After confirmation of the |[gamma]|c chain expression on the transduced EBV-B lines, the cells were treated with 0.1, 1.0, 10 and 100 mM of ganciclovir (GCV) for 24, 48, 72 and 96 hours. At the end of GCV treatment, expression of the |[gamma]|c chain was analyzed. Cells expressing the transduced |[gamma]|c chain were dramatically reduced within 48 hours after the addition of the GCV, and the |[gamma]|c chain could not be detected on the cell surface after 96 hours with 10 or 100 mM GCV. In contrast, untreated cells expressed the |[gamma]|c chain constantly throughout the experiment. Concentration of GCV up to 10 mM did not affect the growth of the EBV-B cell lines. To test the efficacy and safety feature of the gene therapy in vivo, a murine model of X-SCID was treated dwith the vector. Currently, immunologic reconstitution and efficacy of the suicide gene in this murine model were under investigation.

480. Examination of the Relationship between the Level of Gene Transfer and the Number of Envelope Proteins That Are Incorporated Per Retrovirus Particle When High Doses of Purified Virus Stocks Are Used to Transduce Cells

An improved understanding of retrovirus-cell interactions is needed to achieve more predictable outcomes in gene transfer protocols. Transduction efficiency is determined largely by the interaction between the virus envelope proteins and their receptors. Nevertheless, little quantitative information is available about the relationship between gene transfer and envelope protein number. This motivated us to examine the relationship between the number of envelope proteins per retrovirus particle and the number of genes transferred when undiluted virus stocks are used, as is done in most human gene therapy clinical trials. We constructed a panel of several clonal amphotropic murine leukemia virus packaging cell lines, each of which were derived from the same parental cell line (TELCeB6 cells). These packaging cell lines (TELCeB6-A) differed only in the amount of envelope protein that they expressed. In agreement with the findings of others, we found that virus titer increased as the number of envelope proteins that were associated with the virus particles increased. In addition, we found that the virus stocks contained high levels of free envelope protein that strongly inhibited infection when undiluted virus stocks were applied directly to cells.

493. Concentration and Purification of Lentivirus Pseudotypes Bearing Alternative Envelope Glycoproteins

Current problems that impact the use of lentiviral (LV) vectors for applications in large animals include low titers, poor purity and difficulties related to their large-scale production. A number of useful protocols are available to concentrate and purify LV vector pseudotypes bearing the traditional vesicular stomatitis virus G (VSV-G) glycoprotein. These protocols include centrifugation-based approaches, ultrafiltration, anion exchange chromatography, and size exclusion chromatography. There is an increasing demand for alternative (i.e. non-VSV-G-based) LV pseudotypes and thus there are needs for improved concentration/purification protocols for such pseudotypes. We have investigated scaleable protocols for the capture and purification of alternative LV pseudotypes based on anion exchange membrane chromatography. In one approach, HIV-1-based LV vectors pseudotyped with a lyssavirus (rabies-related) glycoprotein were generated by transient transfection. In another approach, amphotropic LV pseudotypes were generated using the STAR-A amphotropic packaging cell line. Virus-containing cell culture supernatants were subsequently captured onto chromatography membranes bearing functional quarternary (Q) ammonium groups and eluted with high salt and then desalted and further concentrated by ultrafiltration. Using this procedure, up to 87 % of the virus applied (up to 1.8 |[times]| 108 transducing units) could be recovered from a single anion exchange membrane unit with a membrane bed volume of 0.18 ml. Our results indicate that these purification procedures are useful for concentrating LV vectors bearing alternative envelope glycoproteins with a minimal loss of infectivity. We expect these approaches to facilitate the preparation of lentiviral vectors for large-scale preclinical studies. They may ultimately be applicable to generate clinical-grade LV vectors.

Role of Rho/ROCK and p38 MAP Kinase Pathways in Transforming Growth Factor-β-mediated Smad-dependent Growth Inhibition of Human Breast Carcinoma Cells in Vivo

Role of Rho/ROCK and p38 MAP Kinase Pathways in Transforming Growth Factor-β-mediated Smad-dependent Growth Inhibition of Human Breast Carcinoma Cells in Vivo

182. Towards the Correction of the Genetic Defect in Corneal Keratinocytes from Patients with Macular Corneal Dystrophy Type II

Top of pageAbstract Macular Corneal Dystrophy (MCD) is an autosomal recessive hereditary disease in which progressive punctuate opacities in the cornea lead to bilateral loss of vision, eventually necessitating corneal transplantation. MCD is classified into two subtypes (I and II) defined by the respective absence and presence of sulphated Keratan (KS) in the patient serum, although both types are clinically indistinguishable. The carbohydrate sulfotransferase 6 gene (CHST6) encoding corneal N-acetylglucosamine 6-O-sulfotransferase (C-GlcNAc6ST) has been identified as a cause of MCD I and II. C-GlcNAc6ST transfers sulphate residues to keratan, a carbohydrate moiety of the major proteoglycans in the corneal stroma responsible for maintaining corneal transparency. In MCD type II, deletions and/or replacements in the upstream region of CHST6 lead to loss of cornea-specific expression of CHST6. A new packaging cell line producing a retroviral vector expressing CHST6 was developed. We constructed a pL(CHST6-IRES-GFP)SN plasmid by cloning in a pLXSN backbone the CHST6 cDNA and a cassette containing the Internal Ribosome Entry Site (IRES) and the reporter gene GFP. Since no antibodies against C-GlcNAc6ST are available the IRES sequence assures that transduced cells express both GFP and the CHST6 gene. The Am12/L(CHST6-IRES-GFP)SN producer cell line was generated by transfection of the GP+E86 cell line followed by infection of the amphotropic packaging cell line GP+env Am12. GFP(+)-Am12 cells were sorted by FACS and cloned by limiting dilution. Clones with higher viral titres were analysed for lack of re-arrangements and number of integrated proviruses (4.3 kb in size) by Southern blotting. We will show that keratinocytes from corneal biopsies of patients with MCD II plated onto a feeder layer composed of 3T3-J2 and Am12/L(CHST6-IRES-GFP)SN can be infected. Transduced GFP(+) keratinocytes will be further analysed and classified as holo-, mero- or paraclones for their growth potential. To evaluate whether GFP(+) keratinocytes also express the CHST6 gene, the ability of the C-GlcNAc6ST enzyme to transfer sulphate residues to KS will be analysed by (i) keratan sulphate ELISA or (ii) HPLC after treatment of the polysaccharide with specific enzymes. The latter allows to determine both the amount of sulphated keratan (sensitivity down to 100 ng with bovine corneal KS) and the position to which the sulphation process occurs on the keratan backbone. Analysis of the sulphation process is fundamental in the case of MCD II in which sulphation is not absent (as in MCD I) but only reduced. To our knowledge this is the first report of attempting gene correction of corneal keratinocytes stem cells for correction of corneal hereditary diseases. Following the tremendous progress achieved in restoring damaged corneal surfaces by cultivation of autologous limbal stem cells (Pellegrini et al., Lancet 1997; 349: 990–993; Rama et al., Transplantation 2001; 72: 1478–1485), application of gene transfer protocols to autologous limbal keratinocytes cultured in vitro could represent a challenging alternative therapy for patients with MCD II.

A novel human packaging cell line with hematopoietic supportive capacity increases gene transfer into early hematopoietic progenitors.

The hematopoietic stem/progenitor cell (HSPC) represents the ideal target for gene therapy of disorders of the hematopoietic system, but still faces problems related to ex vivo manipulation and gene transfer efficiency. We demonstrate that soluble factors from the human endothelial-like cell line ECV 304/T24 support the growth of human CD34(+) progenitor cells as primary human bone marrow stroma and increase the rate of gene transfer into progenitor cells up to 5-fold. ECV 304/T24 was used to generate split-function amphotropic packaging cell lines (named APEX) with the purpose of combining, in the same cells, hematopoietic support and gene transfer vehicle functions. The APEX cell lines were negative for the presence of replication-competent retroviruses and produced complement-resistant vector particles. When mobilized peripheral blood or umbilical cord blood CD34(+) cells were exposed once to APEX supernatants, the level of gene transfer was equivalent to that observed with GP + Am12, in spite of the lower titer of the APEX producers. More importantly, APEX supernatants gave rise reproducibly to a 2-fold increase in transduction of early progenitors (long-term culture-initiating cells), reaching on average 50% gene transfer. This novel packaging cell represents a significant advance in HSPC genetic modification technology, combining both a beneficial hematopoietic supportive effect and the gene transfer vector function in a human-based system.

Telomerase Can Inhibit the Recombination-based Pathway of Telomere Maintenance in Human Cells

Telomere length can be maintained by telomerase or by a recombination-based pathway. Because individual telomeres in cells using the recombination-based pathway of telomere maintenance appear to periodically become extremely short, cells using this pathway to maintain telomeres may be faced with a continuous state of crisis. We expressed telomerase in a human cell line that uses the recombination-based pathway of telomere maintenance to test whether telomerase would prevent telomeres from becoming critically short and examine the effects that this might have on the recombination-based pathway of telomere maintenance. In these cells, telomerase maintains the length of the shortest telomeres. In some cases, the long heterogeneous telomeres are completely lost, and the cells now permanently contain short telomeres after only 40 population doublings. This corresponds to a telomere reduction rate of 500 base pairs/population doubling, a rate that is much faster than expected for normal telomere shortening but is consistent with the rapid telomere deletion events observed in cells using the recombination-based pathway of telomere maintenance (Murnane, J. P., Sabatier, L., Marder, B. A., and Morgan, W. F. (1994) EMBO J. 13, 4953-4962). We also observed reductions in the fraction of cells containing alternative lengthening of telomere-associated promyelocytic leukemia bodies and extrachromosomal telomere repeats; however, no alterations in the rate of sister chromatid exchange were observed. Our results demonstrate that human cells using the recombination-based pathway of telomere maintenance retain factors required for telomerase to maintain telomeres and that once the telomerase-based pathway of telomere length regulation is engaged, recombination-based elongation of telomeres can be functionally inhibited.

Development of a suspension packaging cell line for production of high titre, serum-resistant murine leukemia virus vectors.

To date, only adherent cell lines have been used for the generation of packaging cells for the production of type C retrovirus vectors. The large-scale production of high titre retrovirus vectors could benefit from the development of packaging cells growing in suspension. Here, we describe the ability of two different lymphoid cell lines, one B- and one T-lymphoblastoid cell line (Namalwa and CEM, respectively), to produce MLV-based vectors. Upon transfection with a third generation packaging construct, the virus particle production by Namalwa cells was characterised by low RT-activity, and by CEM cells as high RT activity as previously established adherent packaging cells. An amphotropic packaging cell line (CEMFLYA) was therefore established from CEM cells. Upon introduction of a lacZ vector genome, the novel packaging cell line produced vector particles routinely in the region of 10(7) infectious units/ml. The vectors were helper-free and highly stable in fresh human serum. The potential for scaled up vector production was demonstrated by continuous culture of the new packaging cells for 14 days in a 250 ml spinner flask. These suspension packaging cells should be applicable to large bioreactor systems to bulk produce high titre, complement-resistant retrovirus vectors for gene therapy.

Lack of superinfection interference in retroviral vector producer cells.

Vesicular stomatitis virus G protein (VSV-G)-pseudotyped retroviral vectors have become more feasible for clinical gene transfer protocols since stable tetracycline (tet)-regulated packaging cell lines have become available. Here, we analyzed superinfection interference in VSV-G-pseudotyped and classic amphotropic packaging cell lines. No superinfection interference was observed in VSV-G-pseudotyped packaging cell lines. Thus, integrated retroviral vector genomes accumulated during culture. Similar results were obtained with the amphotropic packaging cells, but to a lesser degree. In addition, VSV-G packaging cells were susceptible to infection with vector particles devoid of envelope proteins, which are produced by these cells in high titers when VSV-G expression is suppressed by tetracycline. For both packaging systems, superinfection could be blocked by azidothymidine (AZT). With regard to safety, this study suggests that in clinical protocols amphotropic producer clones should be tested for superinfection interference and VSV-G packaging cells should always be cultured in the presence of AZT.

Using GFP retrovirus to label tumor cells and vascular endothelia cells

To prepare retrovirus which carry GFP gene and are able to label living cells simply and rapidly. The recombinant retroviral vector pLNCX-GFP was constructed by inserting 780?bp GFP cDNA fragment into the MCS site of retroviral plasmid pLNCX. Both ecotropic packaging cell line ΦX-Eco and amphotropic packaging cell line ΦX-Ampho and PA317 were transfected by pLNCX-GFP with liposome. The supernate collected from transfected packaging cells was used to infect a variety of tumor cell lines and vascular endothelia cell lines. When packaging cells were transfected by retroviral vector pLNCX-GFP, the GFP expression could be observed in 25%-40% of cells and GFP retrovirus then could be detected, however G418 resistant clones showed more stable GFP expression and higher retrovirus titer. The GFP retrovirus from different packaging cell line showed variant ability to infect tumor cell lines and vascular endothelia cell lines and the tumor cells infected by GFP retrovirus showed stable GFP expression in vitro. GFP transduced tumor cells could grow in syngenic animal and continue expressing GFP. Using GFP retrovirus to label target cells represent an important advantage over conventional plasmid because they can efficiently transfer GFP gene into target cells and GFP can be stably expressed in target cells no matter in vitro or in vivo.

Delivery of a Retroviral Vector Expressing Human β-Glucuronidase to the Liver and Spleen Decreases Lysosomal Storage in Mucopolysaccharidosis VII Mice

Mucopolysaccharidosis VII (MPS VII) is caused by beta-glucuronidase (beta-gluc) deficiency and results in lysosomal storage due to the inability to degrade glycosaminoglycans. Transfer of a beta-gluc gene into the liver reduces hepatic pathology as well as storage in other organs via uptake of secreted protein. A Moloney murine leukemia-based retroviral vector expressing the human beta-gluc cDNA was injected intravascularly into MPS VII mice during hepatocyte replication, which was induced with im injection of an adenoviral vector that transiently expressed hepatocyte growth factor (Ad.CMV. HGF). This procedure resulted in transduction of approximately 1% of hepatocytes, 1% of normal liver enzyme activity, and a reduction in lysosomal storage in the liver at 3.5 months. Surprisingly, controls that received retroviral vector without HGF had transduction of nonparenchymal cells in the liver, significant levels of enzyme and RNA in the liver at 2 but not 3.5 months, and reduced lysosomal storage at 3.5 months. Transduction was also achieved in the replicating cells of the spleen, where lysosomal storage was reduced. An approach using a retroviral vector without a growth factor might temporarily reduce lysosomal storage in the liver and spleen in humans. Addition of HGF might be used to augment and prolong gene transfer.

Generation of Retroviral Packaging and Producer Cell Lines for Large-Scale Vector Production and Clinical Application: Improved Safety and High Titer

For many applications, human clinical therapies using retroviral vectors still require many technological improvements in key areas of vector design and production. These improvements include higher unprocessed manufacturing titers, complement-resistant vectors, and minimized potential to generate replication-competent retrovirus (RCR). To address these issues, we have developed a panel of human packaging cell lines (PCLs) with reduced homology between retroviral vector and packaging components. These reduced-homology PCLs allowed for the use of a novel high multiplicity of transduction ("high m.o. t.") method to introduce multiple copies of provector within vector-producing cell lines (VPCLs), resulting in high-titer vector without the generation of RCR. In a distinct approach to increase vector yields, we integrated manufacturing parameters into screening strategies and clone selection for large-scale vector production. Collectively, these improvements have resulted in the development of diverse VPCLs with unprocessed titers exceeding 2 x 10(7) CFU/ml. Using this technology, human Factor VIII VPCLs yielding titers as high as 2 x 10(8) CFU/ml unprocessed supernatant were generated. These cell lines produce complement-resistant vector particles (N. J. DePolo et al., J. Virol. 73: 6708-6714, 1999) and provide the basis for an ongoing Factor VIII gene therapy clinical trial.

Stable expression of the ecotropic retrovirus receptor in amphotropic packaging cells facilitates the transfer of recombinant vectors and enhances the yield of retroviral particles

Retroviral vectors are used widely as gene transfer vehicles. Vector particles are generated by packaging cell lines, which supply the structural proteins gag, pol and env needed to package the retroviral vector RNA. The most efficient way to introduce the vector genome into the packaging cell line is cross-infection with a retroviral vector. Since the infection of a packaging cell line by the produced virus is blocked due to the down regulation of the retrovirus receptor by the envelope glycoprotein, the vector genome should be introduced by a virus with a host tropism different from the one of the packaging cell line. The murine ecotropic retrovirus receptor was expressed in the human amphotropic packaging cell line FLYA13 to generate a cell line which can be infected by murine ecotropic retroviruses. Vector transfer can now be facilitated by cross-infection with the appropriate ecotropic retroviral vectors and provides a simple and efficient method for the generation of amphotropic packaging lines.

Amphotropic Retroviral Vectors Displaying Hepatocyte Growth Factor-Envelope Fusion Proteins Improve Transduction Efficiency of Primary Hepatocytes

The development of retroviral vectors with cell-specific targeting capabilities will be an important step toward successful in vitro gene therapy. This article describes the generation of a retroviral vector with enhanced binding abilities for cells bearing the c-Met receptor: the Madin-Darby canine kidney (MDCK) cell line and primary hepatocytes. The human hepatocyte growth factor (HGF) was displayed on murine amphotropic retroviral vectors by fusion to the viral transmembrane envelope glycoprotein (TM). The resulting chimeric envelope HGF-TM was expressed in an amphotropic packaging cell line producing viral particles that display both HGF-TM and the wild-type envelope. These modified viral particles had a titer equivalent to that of unmodified particles. Modified particles infected MDCK cells more efficiently than did unmodified amphotropic retrovirus. Adding anti-HGF antibodies to the viral vector particle supernatant prior to infection confirmed that the increased infection was mediated by the HGF moiety. The chimeric viruses also infected primary mouse and nonhuman primate fetal hepatocytes more effectively. Furthermore, these cells could be induced to proliferate by the modified HGF-TM viruses. Since exogenous HGF is primarily taken up by the liver, these results may have implications for retroviral vector design for liver-directed human gene therapy.

Amphotropic retroviral vectors displaying hepatocyte growth factor-envelope fusion proteins improve transduction efficiency of primary hepatocytes.

The development of retroviral vectors with cell-specific targeting capabilities will be an important step toward successful in vitro gene therapy. This article describes the generation of a retroviral vector with enhanced binding abilities for cells bearing the c-Met receptor: the Madin-Darby canine kidney (MDCK) cell line and primary hepatocytes. The human hepatocyte growth factor (HGF) was displayed on murine amphotropic retroviral vectors by fusion to the viral transmembrane envelope glycoprotein (TM). The resulting chimeric envelope HGF-TM was expressed in an amphotropic packaging cell line producing viral particles that display both HGF-TM and the wild-type envelope. These modified viral particles had a titer equivalent to that of unmodified particles. Modified particles infected MDCK cells more efficiently than did unmodified amphotropic retrovirus. Adding anti-HGF antibodies to the viral vector particle supernatant prior to infection confirmed that the increased infection was mediated by the HGF moiety. The chimeric viruses also infected primary mouse and nonhuman primate fetal hepatocytes more effectively. Furthermore, these cells could be induced to proliferate by the modified HGF-TM viruses. Since exogenous HGF is primarily taken up by the liver, these results may have implications for retroviral vector design for liver-directed human gene therapy.

Increased gene transfer into human CD34+ progenitor cells using retroviral vectors produced by a canine packaging cell line

Using retroviral supernatants derived from the amphotropic murine packaging cell line PA317 and the amphotropic canine packaging cell line (DA), cord blood and mobilized peripheral blood CD34+ cells were transduced with the vector LN (neomycin resistance) and the vector L-TR/TAT neo (neomycin resistance in conjunction with a double-hammerhead ribozyme conferring anti-HIV activity). Different multiplicities of infection (MOI) were applied in the setup according to vector titrations on NIH-3T3 cells. PA317-based supernatants were tested at MOI of 10 and 30. Purified concentrated DA-derived vector preparations were tested at MOI of 10, 30, 100, and 300. Immediately after transduction, CD34+ cells were plated into colony assays in the presence and absence of G418 to evaluate the amount of gene transfer and potential toxic effects of the vectors on colony growth. The remaining cells were subjected to G418 selection in liquid culture for 12 days and subsequently challenged with HIV-1JR-FL to test for efficacy of the anti-HIV gene in macrophages derived from transduced CD34+ cells. Transduction by the PA317-packaged vectors was maximal at the lowest MOI used and did not increase with increasing MOI. In contrast, transduction by the DA-packaged vectors could be progressively increased using increased MOI. The net transduction efficiency per unit of reverse transcriptase activity in the DA vector preparations was 8.7-fold higher than in the PA317 vector supernatants. HIV-1 challenge of the cells transduced by the ribozyme vector derived from the PA317 packaging cells resulted in a 1.5 log inhibition of p24 output compared with the control cells containing neomycin resistance only. A 2.5 log inhibition of p24 output could be observed in the cell population transduced with DA-packaged vector supernatants. Compared with retroviral supernatants from PA317 packaging cell lines, DA packaging line-derived vector preparations demonstrated higher transduction efficiency into CD34+ cells, particularly at higher MOI, and increased efficacy of the transferred anti-HIV gene when challenged with HIV-1JR-FL. The increase in transduction efficiency may be due to a higher ratio of intact vs. defective vector particles in the DA-derived vector preparations.Biol Blood Marrow Transplant 1998;4(3):119-27.