Purification and Injection of Retroviral Vectors.

Retroviral vectors are powerful tools for genetic manipulation. This protocol discusses the production, purification, and use of replication-deficient retroviral vectors based on Moloney murine leukemia virus and lentivirus. It also describes the injection of a retroviral vector into the dentate gyrus of young adult mice to fluorescently label live murine brain tissue.

Preparation and Use of Retroviral Vectors for Labeling, Imaging, and Genetically Manipulating Cells.

Retroviral vectors are a powerful technology for achieving long-term genetic manipulation. This introduction provides some background on replication-deficient retroviral vectors based on Moloney murine leukemia virus and lentivirus. Details, examples, and associated protocols are provided for using these vectors to fluorescently label, genetically alter, and image both live and fixed murine brain tissue.

Gene therapy for chronic granulomatous disease.

Mutations in phagocyte NADPH oxidase cause CGD, resulting in recurrent infections and granulomatous inflammation. Hematopoietic stem cell (HSC) transplant can cure CGD, but most patients lack a suitable donor. We conducted a clinical trial of ex vivo autologous HSC gene transfer as salvage therapy for three patients with X-linked CGD (X-CGD) who had incurable infection. Patients received nonmyeloablative busulfan conditioning and then were infused with amphotropic MFGS-gp91phox murine retrovirus vector-transduced autologous HSC, resulting in early gene marking and high-level oxidase function correction of 24%, 5%, and 4% of circulating neutrophils. Subjects #1 and #3 fully resolved infection and have maintained gene marking at 5 years at 0.7% and 0.03% oxidase-normal neutrophils. Subject #2 lost gene marking by 4 weeks and at 6 months succumbed to his infection. The two surviving subjects have normal blood count and bone marrow exam, with no evidence for clonal dominance of vector inserts. We conclude that gene therapy salvage treatment for severe infection unresponsive to conventional therapy can provide life-saving clinical benefit to CGD patients lacking a suitable donor. We are developing lentivectors for our next generation gene therapy of CGD. We are also exploring novel alternate approaches to gene therapy using zinc finger nuclease-mediated gene targeting of induced pluripotent stem cells derived from CGD patients.

Identification of host proteins associated with retroviral vector particles by proteomic analysis of highly purified vector preparations.

The Moloney murine leukemia virus (MMLV) belongs to the Retroviridae family of enveloped viruses, which is known to acquire minute amounts of host cellular proteins both on the surface and inside the virion. Despite the extensive use of retroviral vectors in experimental and clinical applications, the repertoire of host proteins incorporated into MMLV vector particles remains unexplored. We report here the identification of host proteins from highly purified retroviral vector preparations obtained by rate-zonal ultracentrifugation. Viral proteins were fractionated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in-gel tryptic digested, and subjected to liquid chromatography/tandem mass spectrometry analysis. Immunogold electron microscopy studies confirmed the presence of several host membrane proteins exposed at the vector surface. These studies led to the identification of 27 host proteins on MMLV vector particles derived from 293 HEK cells, including 5 proteins previously described as part of wild-type MMLV. Nineteen host proteins identified corresponded to intracellular proteins. A total of eight host membrane proteins were identified, including cell adhesion proteins integrin beta1 (fibronectin receptor subunit beta) and HMFG-E8, tetraspanins CD81 and CD9, and late endosomal markers CD63 and Lamp-2. Identification of membrane proteins on the retroviral surface is particularly attractive, since they can serve as anchoring sites for the insertion of tags for targeting or purification purposes. The implications of our findings for retrovirus-mediated gene therapy are discussed.

Complexation with chondroitin sulfate C and Polybrene rapidly purifies retrovirus from inhibitors of transduction and substantially enhances gene transfer.

Using amphotropic retrovirus stocks produced by TELCeB6-A cells that encode the Escherichia coli lacZ gene, we found that complexation with chondroitin sulfate C (CSC) and Polybrene (PB) is an effective means to purify retrovirus. Virus stocks contained high levels of inhibitory activity that blocked amphotropic, but not ecotropic, retrovirus transduction. When virus stocks were brought to 80 microg/mL each of CSC and PB, complexes of CSC and PB formed. These complexes incorporated more than 70% of the virus particles but less than 0.4% of all other proteins and no detectable inhibitory activity. Purified virus transduced NIH 3T3 murine fibroblasts 21 to 186-fold more efficiently than virus that was not purified. In addition, virus purification significantly altered the dose response of transduction. When virus that had not been purified was used to transduce cells, the relationship between transduction and virus concentration was highly non-linear. In contrast, when purified virus was used, transduction increased monotonically and was linearly proportional to virus concentration, except when high doses of virus were used. Interestingly, when high doses of virus were used gene transfer reached a maximum plateau level, most likely because particle-associated amphotropic envelope proteins had saturated the cellular receptors for the virus. Our findings illustrate that retrovirus purification increases the maximum number of genes that can be transferred, reduces the amount of virus required to achieve a given level of gene transfer, and reduces uncertainties about the relationship between the amount of virus used and the number of genes transferred.

Purification and characterization of retrovirus vector particles by rate zonal ultracentrifugation.

Sucrose equilibrium density ultracentrifugation remains the most widely used technique for retrovirus purification. However, purified virus preparations obtained by this routine method usually contain considerable amounts of contaminating cell membrane vesicles. In addition, sucrose solutions are highly viscous and hyperosmotic which jeopardizes the integrity and functionality of the retrovirus particle. In order to overcome these limitations, an alternative purification technique using rate zonal ultracentrifugation and iodixanol as gradient medium was developed. Recombinant retrovirus particles were produced by 293-GPG packaging cells grown in suspension in the presence of 10% FBS. Concentrated supernatants were purified by rate zonal sedimentation on a 10-30% continuous iodixanol gradient. Virus particles were recovered intact and active from the central fractions of the gradient. By using this strategy, high levels of purification were achieved, with no evident contamination with cell membrane vesicles as indicated by subtilisin treatment studies. The level of purity of the retrovirus preparation is over 95% as shown by SDS-PAGE analysis and size-exclusion chromatography. Purified particles appear homogenous in size and morphology according to negative stain electron microscopy. In addition, large amounts of defective retrovirus particles produced by 293-GPG packaging cells can be separated from functional retrovirus particles using this purification strategy.

Affinity recovery of Moloney Murine Leukaemia Virus.

Lipid enveloped retroviruses such as Moloney Murine Leukaemia Virus (MoMuLV) are commonly used gene therapy vectors. Downstream processing protocols used for their purification are time consuming and a potentially generic, single step capture method for the recovery of retroviral particles is proposed that exploits streptavidin-biotin affinity chromatography. The ability of four conventional adsorbent solid phases, Fractogel, Sepharose, Magnespheres and STREAMLINE immobilised with streptavidin, to capture and recover biotinylated Moloney Murine Leukaemia Virus was studied. MoMuLV can be biotinylated whilst retaining infectivity and the biotinylated virus can be adsorbed to Streptavidin Magnespheres yielding a 2298-fold increase in titre. For optimal virus biotinylation purification using Fractogel streptavidin can yield a 1896-fold increase in cfu/mg of protein and a 1191-fold decrease in DNA/cfu. Infectious virus can be recovered from Fractogel streptavidin with a maximum recovery of 16.7%.

Affinity capture of a biotinylated retrovirus on macroporous monolithic adsorbents: towards a rapid single-step purification process.

A streptavidin derivitised macroporous monolith was developed to enable single-step capture of chemically biotinylated Moloney Murine Leukaemia Virus (MoMuLV) from crude, unclarified cell culture supernatant. Monoliths were prepared by aqueous cryopolymerisation of acrylamide with N,N''-methylene-bis (acrylamide) and glycidyl methacrylate (Arvidsson et al. [2003] J Chrom A 986:275-290). Streptavidin was immobilised to the epoxy functionalised monoliths. Particulate-containing cell culture supernatant was passed through the monolith without preclarification of the feedstock and adsorption capacities of 2 x 10(5) cfu/ml of adsorbent were demonstrated (cf. Fractogel streptavidin, at 3.9 x 10(5) cfu/ml of adsorbent). The specific titre of the recovered fraction was increased by 425-fold; however, recoveries of less than 8% were achieved. Adsorption of nonbiotinylated MoMuLV on the streptavidin-coated monolith was not observed.

Vertical transmission of a murine retrovirus, ts1.

Mechanism of maternal retroviral transmission remains an unsolved problem. The current investigation is a part of our ongoing research on vertical transmission of MoMuLV-TB ts1 in BALB/c mice. A total of 270 adult mice and 165 fetuses were used. Forty-four experimental mice were injected with 0.1 mL of 4.0 x 10(6) ffu/mL of ts1 virus at 72 h after birth; 24 controls were injected with DMEM. Almost half of the females went through two rounds of pregnancies. In the first round, 135 experimental and 57 control pups were produced. Forty-three experimental and 20 control pups were followed until they developed clinical symptoms. The second round of pregnancy produced a total of 46 mid-gestational and 119 full-term fetuses. PCR, and light and electron microscopy were performed to evaluate viral transmission. Overall, 99% vertical transmission occurred in pups of infected mothers. Twelve percent of mid-gestational and 39% full-term fetuses were PCR positive. We have established that, if mothers are infected with ts1 virus at 72 h after birth, then nearly 100% vertical transmission occurs, via in utero, intrapartum, or breast milk. Thirty-nine percent transmission occurred in utero alone. This is an excellent model to study the transplacental and post-gestational transmission of retroviruses, such as ts1.

Characterization of recombinant nonecotropic murine leukemia viruses from the wild mouse species Mus spretus.

The wild mouse species most closely related to the common laboratory strains contain proviral env genes of the xenotropic/polytropic subgroup of mouse leukemia viruses (MLVs). To determine if the polytropic proviruses of Mus spretus contain functional genes, we inoculated neonates with Moloney MLV (MoMLV) or amphotropic MLV (A-MLV) and screened for viral recombinants with altered host ranges. Thymus and spleen cells from MoMLV-inoculated mice were plated on Mus dunni cells and mink cells, since these cells do not support the replication of MoMLV, and cells from A-MLV-inoculated mice were plated on ferret cells. All MoMLV-inoculated mice produced ecotropic viruses that resembled their MoMLV progenitor, although some isolates, unlike MoMLV, grew to high titers in M. dunni cells. All of the MoMLV-inoculated mice also produced nonecotropic virus that was infectious for mink cells. Sequencing of three MoMLV- and two A-MLV-derived nonecotropic recombinants confirmed that these viruses contained substantial substitutions that included the regions of env encoding the surface (SU) protein and the 5' end of the transmembrane (TM) protein. The 5' recombination breakpoint for one of the A-MLV recombinants was identified in RNase H. The M. spretus-derived env substitutions were nearly identical to the corresponding regions in prototypical laboratory mouse polytropic proviruses, but the wild mouse infectious viruses had a more restricted host range. The M. spretus proviruses contributing to these recombinants were also sequenced. The seven sequenced proviruses were 99% identical to one another and to the recombinants; only two of the seven had obvious fatal defects. We conclude that the M. spretus proviruses are likely to be recent germ line acquisitions and that they contain functional genes that can contribute to the production of replication-competent virus.

A small animal model for mother-to-fetus transmission of ts1, a murine retrovirus.

Infection with a murine retrovirus, MoMuLV-TB, ts1 in BALB/c mice has been established as a small animal model for retroviral neurodegenerative disease as shown with infections such as HIV. However, mother-to-pup transmission has never been demonstrated in this model. The current investigation examines vertical transmission of ts1 in this mouse model. A total of 15 females were used to produce 59 pups (16 were used for control, and 43 were used as experimental animals). For experiment 1, 24 5-day-old mice were injected with [0.2 mL of 2.0 x 10(6) ffu/mL ts1] virus. For experiment 2, 19 48-h-old mice were injected with [0.1 mL of 4 x 10(6) ffu/mL ts1] virus. Control groups were injected with DMEM only. PCR and electron microscopy were performed to determine the presence of virus. All mice from experiment 1 injected with ts1 showed viral infection, and retained 100% reproductive capacity. Three out of 102 pups produced by these infected females were infected with ts1. Nine percent of the pups from experiment 2 injected with ts1 retained normal reproductive capacity, and two out of eight (25%) pups had viral infection. Vertical transmission of this unique retrovirus occurs and is dependent, in part, on the timing of maternal infection.

Determination of infectious retrovirus concentration from colony-forming assay with quantitative analysis.

The colony formation assay is the most commonly used titration method for defining the concentration of replication-incompetent murine leukemia virus-derived retroviral vectors. However, titer varies with target cell type and number, transduction time, and concentration of polycation (e.g., Polybrene). Moreover, because most of the viruses cannot encounter target cells due to Brownian motion, their short half-lives, and the requirement for target cell division for activity, the actual infectious retrovirus concentration in the collected supernatant is higher than the viral titer. Here we correlate the physical viral particle concentration with the infectious virus concentration and colony formation titer with the help of a mathematical model. Ecotropic murine leukemia retrovirus supernatant, collected from the GP+E86/LNCX retroviral vector producer cell line, was concentrated by centrifugation and further purified by a sucrose density gradient. The physical concentration of purified viral vectors was determined by direct particle counting with an electron microscope. The concentrations of fresh and concentrated supernatant were determined by a quantitative reverse transcriptase activity assay. Titration of all supernatants by neomycin-resistant colony formation assay was also performed. There were 767 +/- 517 physical viral particles per infectious CFU in the crude viral supernatant. However, the infectious viral concentration determined by mathematical simulation was 143 viral particles per infectious unit, which is more consistent with the concentration determined by particle counting in purified viral solution. Our results suggest that the mathematical model can be used to extract a more accurate and reliable concentration of infectious retrovirus.

Purification of a functional gene therapy vector derived from Moloney murine leukaemia virus using membrane filtration and ceramic hydroxyapatite chromatography.

The ability of membrane ultra- and diafiltration and two chromatography media, Matrex Cellufine Sulfate (Millipore) and Macro-Prep ceramic hydroxyapatite (Bio-Rad), to adsorb, elute, and purify gene therapy vectors based on Moloney murine leukaemia virus (MoMuLV) carrying the 4070A amphotropic envelope protein was studied. Membrane ultra- and diafiltration provided virus concentration up to 160-fold with an average recovery of infectious viruses of 77 +/- 14%. In batch experiments, Macro-Prep ceramic hydroxyapatite (type 2, particle size 40 microm) proved superior to Matrex Cellufine Sulfate for MoMuLV vector particle adsorption. Furthermore, functional vector particles could be eluted using phosphate buffer pH 6.8 (highest titres from >or=300 mM phosphate) from the Macro-Prep adsorbent, with higher specific titres (cfu/mg protein) than the starting material. Similar results were obtained when this ceramic hydroxyapatite was packed into a column and used in a liquid chromatography system. Recovery of transduction-competent virus was between 18 and 31% for column experiments and 32 and 46% for batch experiments.

Breeding of retroviruses by DNA shuffling for improved stability and processing yields.

Manufacturing of retroviral vectors for gene therapy is complicated by the sensitivity of these viruses to stress forces during purification and concentration. To isolate viruses that are resistant to these manufacturing processes, we performed breeding of six ecotropic murine leukemia virus (MLV) strains by DNA shuffling. The envelope regions were shuffled to generate a recombinant library of 5 x 106 replication-competent retroviruses. This library was subjected to the concentration process three consecutive times, with amplification of the surviving viruses after each cycle. Several viral clones with greatly improved stabilities were isolated, with the best clone exhibiting no loss in titer under conditions that reduced the titers of the parental viruses by 30- to 100-fold. The envelopes of these resistant viruses differed in DNA and protein sequence, and all were complex chimeras derived from multiple parents. These studies demonstrate the utility of DNA shuffling in breeding viral strains with improved characteristics for gene therapy.

A colorimetric reverse transcriptase assay optimized for Moloney murine leukemia virus, and its use for characterization of reverse transcriptases of unknown identity.

A non-radioactive reverse transcriptase (RT) assay, reported as useful for lentivirus RTs, was optimized for the measurement of Moloney murine leukemia virus (MMuLV) RT. The optimized assay could detect 0.3 microU of MMuLV RT. The specificities of the MMuLV and lenti RT assays were demonstrated using the RTs of human immunodeficiency virus type 1, simian immunodeficiency virus, feline immunodeficiency virus (FIV), visna virus, human T-cell lymphotropic virus type 1, MMuLV and feline leukemia virus (FeLV). An RT activity blocking antibody (RTb-ab) assay was standardized for Mn2+ dependent MuLV-related RTs. The assay was used to demonstrate the distinct antigenic properties of RTs from mammalian MuLV-related retroviruses and lentiviruses. Cross-reactivity between MMuLV RTb-ab and FeLV RT but not between MMuLV RTb-ab and e.g. FIV RT was demonstrated. An RT activity found in the murine myeloma cell line SP2/0 was found to have similar assay preferences as MMuLV RT, and the MMuLV-RT hyperimmune sera reacted strongly against this RT, indicating the RT to be of MuLV-related etiology. The use of the RT and RTb-ab assays for detection and characterization of RTs of known or unknown identity is discussed.

Efficient recovery and regeneration of integrated retroviruses.

We report a rapid and efficient PCR-based rescue procedure for integrated recombinant retroviruses. Full-length proviral DNA is amplified by long-range PCR using a pair of primers derived from the long terminal repeats (LTR), and virus is regenerated by transfecting retrovirus-packaging cells with the PCR-derived provirus. The viral yield from the PCR product is similar to that from the retroviral plasmid vector, and the representation of different inserts is accurately maintained in the recovered retroviral population. This procedure is suitable for expression cloning from retroviral libraries and should be applicable to the analysis of natural retrovirus populations.

Functional domains of Moloney murine leukemia virus integrase defined by mutation and complementation analysis.

Retroviral integrases perform two catalytic steps, 3' processing and strand transfer, that result in the stable insertion of the retroviral DNA into the host genome. Mutant M-MuLV integrases were constructed to define the functional domains important for 3' processing, strand transfer, and disintegration by in vitro assays. N-terminal mutants had no detectable 3' processing activity, and only one mutant which lacks the HHCC domain, Ndelta105, had strand transfer activity. Strand transfer mediated by Ndelta105 showed preference for one site in the target DNA. Disintegration activity of N-terminal mutants decreased only minimally. In contrast, all C-terminal mutants truncated by more than 28 amino acids had no integration or disintegration activity. Activity on a single-strand disintegration substrate did not require a functional HHCC domain but did require most of the C-terminal region. Complementation analysis found that the HHCC region alone was able to function in trans to a promoter containing only the DD(35)E and C-terminal regions and to enhance integration site selection. Increasing the reducing conditions or adding the HHCC domain to Ndelta105 reaction mixtures restored the wild-type strand transfer activity and range of target sites. The reducing agent affected Cys-209 in the DD(35)E region. The presence of C-209 was required for complementation of Ndelta105 by the HHCC region.

A rapid and quantitative assay to estimate gene transfer into retrovirally transduced hematopoietic stem/progenitor cells using a 96-well format PCR and fluorescent detection system universal for MMLV-based proviruses.

The polymerase chain reaction (PCR) is an extremely sensitive assay that has many uses in retroviral-mediated gene transfer protocols. Because the majority of retroviral vectors used in current gene transfer protocols are based on the Moloney-murine leukemia virus (MMLV), we have designed primers which amplify a region of the psi packaging sequence from all MMLV retroviruses tested. This assay detects gene transfer by all MMLV-based vectors and is especially useful for the laboratory that routinely screens a number of different retroviruses for their gene transfer efficiency. Furthermore, we present here a novel technique for harvesting single colonies derived from hematopoietic stem/progenitor cells growing in methylcellulose medium that expedites and substantially improves the resulting quantitative estimates of retroviral transduction frequencies. This technique utilizes a conventional 96-well format and, when coupled with a fluorescence-based post-PCR detection system, makes it unnecessary to run agarose gels to visualize the PCR product. This system of PCR product detection, which uses the 5'-->3' exonuclease activity of Taq DNA polymerase to cleave a fluorescently labeled probe during each round of PCR amplification, is fast, convenient, and at least as sensitive as an ethidium bromide-based detection system when used in conjunction with our universal PCR assay.

Quantitative micro P30 and reverse transcriptase assays for Moloney murine leukemia virus.

An anti-P30 immunohistochemical and a reverse transcriptase assay for Moloney murine leukemia virus (MoMLV) are adapted to 96-well plates. The assay results are shown to be directly proportional to the number of infectious particles, and can therefore be used to estimate the infective titers of a virus preparation. The micro P30 assay yields a direct estimate of infectious centers, and the reverse transcriptase assay quantitates progeny from a single cycle of replication. The semi-automated nature of these assays is well suited to the analysis of a large number of samples and therefore permits the examination of the efficiency of the process of retroviral/MoMLV infection under varied times or conditions.

A simple method for obtaining highly viable virus from culture supernatant.

Traditionally density-gradient methods are used to purify viruses. However, these procedures are not only time consuming and cumbersome, recovery of viable viruses are often quite low. In this report, a single-step concentration technique was used to concentrate a mutant of Moloney murine leukemia virus (ts1) virus from culture supernatants by ultrafiltration. A special ultrafiltration unit with a 100,000 mol wt cut-off was able to concentrate viruses about 30-fold without losing any infectivity. In comparison, traditional sucrose density gradient purified viruses lost a significant portion of their infectivity. This technique could be used for concentrating other viruses for many useful purposes where more viable viruses are needed, e.g., study of virus-cell binding.