Retroviral Vector Production by Transient Transfection

Please cite as: CSH Protocols; 2008; doi:10.1101/pdb.prot4881

Protocol

Kenneth Cornetta, Karen E. Pollok, and A. Dusty Miller

This protocol was adapted from "Retroviral Vectors," Chapter 2, in Gene Transfer: Delivery and Expression of DNA and RNA (eds. Friedmann and Rossi). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2007. INTRODUCTION

This protocol describes vector production by transient transfection.The production of retroviral vectors requires a full-length copy of the vector RNA to be incorporated into virions. This is accomplished by coexpressing vector RNA and the viral proteins required for virion formation from expression plasmids. To avoid generation of replication-competent virus, the viral genes are carried by separate plasmids. Generally, the gag and pol genes are on one plasmid, and the viral envelope gene is on a second plasmid. The viral protein-coding regions can be expressed using various promoters to decrease homology and thereby decrease recombination. Because these plasmids do not contain the packaging() sequence, the viral genes are unlikely to be incorporated into virions.

RELATED INFORMATION

Retroviral Vectors for Gene Transfer (this issue) provides an overview of issues to consider when designing gene-transfer experiments involving retrovirus vectors. This issue of CSH Protocols also contains the related articles Transduction of Cell Lines by Retroviral Vectors and

Transduction of Primary Hematopoietic Cells by Retroviral Vectors.

MATERIALS

Reagents

CaCl2 (2.0 M)

D-10 medium

HEK 293T cells (ATCC, CRL-11268)

HEPES-buffered saline (HBS)

Phosphate-buffered saline (PBS)

Plasmids:

Packaging plasmid(s) containing retroviral gag, pol, and env genes

Vector plasmid

Prepare endotoxin-free plasmid stocks (e.g., by using QIAGEN Endotoxin-free Purification Kit) and determine plasmid DNA concentration.

Equipment

Biosafety cabinet

CO2 incubator

Pipettes (sterile disposable)

Sterilizing filters (0.22-m pore size)

Syringe filter (0.45-m pore size)

Tissue-culture centrifuge

Tissue-culture flasks (75 cm2)

METHOD

1. Prepare a single-cell suspension of HEK 293T cells, and seed5 x 106 cells in a 75-cm2 tissue-culture

flask. Incubate overnight.

2. Remove flasks from the incubator and aspirate the medium.Add 12 mL of fresh D-10 medium to

each flask and return flasks to the incubator.

3. Perform transfection as follows:

i.Sterilize all reagents before use by filtration through 0.22-m pore-size sterile filters.

ii. Prepare DNA for transfection by diluting plasmids in H2O to a total volume of 876 L.

iii. Add 124 L of 2.0 M CaCl2. Mix gently.

iv. Add DNA mix (1 mL) to 1 mL of HBS dropwise. A faint cloudiness should form. Let stand

for 30 min at room temperature.

v. Mix gently,and add 1.5 mL of DNA/HBS suspension to the flask.Incubate overnight.

4. Aspirate and discard the medium from the flasks. Add 5 mL of PBS and then aspirate. Add 12 mL

of medium to each flask,and return them to the incubator for 20-24 h.

5. Remove medium containing vector from the flasks. Filter through a 0.45-m syringe filter to

remove cells. Aliquot the vector based on anticipated needs and store at or below-70 C.

This vector may be used in the Transduction of Cell Lines by Retroviral Vectors and in the

Transduction of Primary Hematopoietic Cells by Retroviral Vectors.

Caution

CaCl2 (calcium chloride)

CaCl2 (calcium chloride) is hygroscopic and may cause cardiac disturbances. It may be harmful by inhalation, ingestion, or skin absorption. Do not breathe the dust. Wear appropriate gloves and safety goggles.

Recipe

D-10 medium

Dulbecco’s modified Eagle’s medium (DMEM)

Fetal bovine serum (FBS; 10%)

L-glutamine (2 mM)

Penicillin (100 U/mL)

Streptomycin (100 g/mL)

Topic Introduction

Retroviral Vectors for Gene Transfer

Kenneth Cornetta, Karen E. Pollok, and A. Dusty Miller

Adapted from "Retroviral Vectors," Chapter 2, in Gene Transfer: Delivery and Expression of DNA and RNA (eds. Friedmann and Rossi). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2007. INTRODUCTION

Retroviral vectors from the -retrovirus genus were the first retroviral vectors to be developed. They have been called oncoretroviral vectors or simple retroviral vectors because of their derivation from oncogenic retroviruses having a simple gag-pol-env genome structure. Later additions to the retroviral vector family include the lentiviral and foamy viral vectors derived from more complex retroviruses that contain multiple accessory genes in addition to the standard gag-pol-env genes. This article describes the advantages and disadvantages of retroviral vectors for gene therapy. It also discusses the issues that must be considered in designing retroviral vectors and in choosing retroviral packaging cell lines.

RELATED INFORMATION

For specific protocols describing preparation of retroviral vectors and transduction of cell lines and primary hematopoietic cells, see the following CSH Protocols articles in this issue:Retroviral Vector Production by Transient Transfection, Generation of Stable Vector-Producing Cells for Retroviral Vectors,Transduction of Cell Lines by Retroviral Vectors, and Transduction of Primary Hematopoietic Cells by Retroviral Vectors. OVERVIEW

Retroviral vectors have several advantages for laboratory research and for clinical gene therapy applications. Unlike many viruses,retroviruses efficiently integrate into the genomes of infected cells. Transgenes carried by retroviral vectors also integrate into target cells such that the gene is copied to all progeny of the cell, making these vectors ideal for altering stem cells,progenitor cells, or other cells that are expected to expand in great number in vivo (e.g., T-cells responding to an immune response). The efficiency of retroviral gene transfer is significantly greater than that of nonviral gene transfer. Furthermore, vector production is easily performed in most research laboratories and is amenable to large-scale production, facilitating its use in clinical settings.

Retroviral vectors do have several disadvantages for gene therapy applications. First, they are generally not useful for systemic administration because of their inactivation by protein and cellular components of human blood and typically have been used to transduce cells ex vivo. Second, they require cell division for efficient integration. Third, integration has been associated with oncogene activation after transduction of hematopoietic cells, a rare and complex process that must be considered when calculating the risk/benefit ratio for this method of gene transfer.Despite these limitations, retroviral vectors are a well-defined system with many novel reagents resulting from the extensive experience with them during the past 20 years. They remain an attractive system for transducing target cells where integration of transgene sequences is required. RETROVIRUSES AS GENE DELIVERY VEHICLES

Retroviruses are attractive gene delivery vehicles because of certain unique aspects of their life cycle as well as their ability to efficiently integrate into target cell DNA. The viral genome is flanked by two regulatory regions, called long terminal repeats (LTRs), that contain promoter and enhancer functions and are required for integration. A packaging () sequence greatly facilitates the uptake of viral RNA into virions. There are three viral gene regions: gag, which encodes the viral structural proteins; pol, which encodes enzymatic proteins, most notably reverse transcriptase and integrase; and env, which generates an envelope glycoprotein that spans the lipid coat of the virus and mediates infection by targeting specific receptors on target cells. Within the viral capsid are two copies of the viral RNA genome along with reverse transcriptase and

integrase. Carrying the enzymatic genes allows the virus to infect a cell, make DNA copies from the RNA template, and integrate the DNA without expressing any of the viral genes. This allows for the deletion of the viral protein-coding genes (gag , pol , and env ) and substitution of this region with exogenous genes of interest (Fig. 1 ). The vector is rendered replication-defective, which presents a technical problem in packaging vector RNA into virions. This challenge can be met using transient transfection of plasmids expressing the viral genes along with a plasmid expressing the vector genome (Fig. 2 ) or, more commonly, through the use of retroviral packaging cell lines as discussed in detail below.

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Figure 1. Retroviral vector design. (A ) Schematic representation

of the Moloney murine leukemia virus vector. The three viral

gene regions, gag , pol , and env are flanked by the LTRs, which contain promoter and enhancer functions. The psi () sequence

is required for efficient packaging of viral RNA into virions. (B ) A vector construct retains the LTRs and sequence with deletion of

the majority of the viral gene region. The promoter in the 5' LTR

is used to express the cDNA of interest. (C ) More than one gene

product or sequence can be expressed by use of an internal

ribosome entry site (IRES) sequence (I) or introduction of a

second promoter (P). (D ) When the sequence to be expressed

contains introns or other sequences that may interfere with

production of a full-length vector transcript, a gene can be

created and inserted in the opposite orientation. This allows the

full-length transcript to be incorporated into virions, and the gene

of interest will be expressed after integration into target cells.

Arrows indicate the orientation of the gene with respect to the 5'

LTR. View larger version (17K):

[in this window]

[in a new window]

Figure 2. Vector production by transient transfection. Plasmids expressing the vector, the viral gag and pol genes, and the viral envelope are introduced into a cell that transfects with high efficiency (e.g., HEK 293T cells) using calcium phosphate, lipofection, or electroporation. Vector supernatant is harvested after 48-72 h and can be used immediately or frozen at -70 C for later use.

CONSIDERATIONS IN RETROVIRAL VECTOR DESIGN

A number of important issues must be considered when constructing retroviral vectors. Simplistically, the vector can be thought of as containing two major components: the vector backbone and the transgene cassette. As depicted in Figure 1, the vector backbone contains those sequences derived from the parent retroviruses.The majority of initial studies used vectors derived from the Moloney murine leukemia virus (Mo-MLV). The promoter and enhancer regions within the Mo-MLV LTR drive expression in most cell lines and in differentiated primary cells. A limitation of this LTR is poor expression in a variety of primitive cells such as preimplantation embryo cells, embryonic stem cells, and primitive hematopoietic progenitor cells (Jahner et al. 1982; Challita and Kohn 1994).It has subsequently been shown that the Mo-MLV LTR and primer binding site contains at least four silencer elements. The mechanisms by which vectors are silenced is complex and not completely understood but has prompted a variety of investigators to develop novel vectors using components of other viruses or mutation of silencer sequences (for review, see Pannell and Ellis 2001).During the past 15 years, a significant number of vector backbones have been generated with LTRs derived from alternative retroviruses or with engineered LTRs that have been shown to improve expression in specific cell types (e.g., hematopoietic cells of myeloid lineage) and may be less likely to undergo in vivo silencing due to methylation or other cellular mechanisms (for review,see Hawley 2001). Investigators seeking to express vectors in primitive cell types now have a variety of novel backbones that decrease, but do not completely eliminate, retroviral vector silencing.

Once a vector backbone has been selected, the transgene cassette must be inserted. The most simplistic design uses the LTR promoter to express the transgene. Generally, the transgene sequence lacks introns to prevent splicing during RNA processing. In situations where intron sequences are important for transgene expression, or where tissue-specific promoters are preferred to the nonspecific expression associated with the viral LTR, the transgene cassette can be placed in reverse orientation(Fig. 1).

Most recently, the documented ability of retroviral vectors to cause malignancy by insertional mutagenesis has led to further considerations of vector design. Insertional mutagenesis occurs when retroviral regulatory sequences (most commonly the enhancer)integrate near susceptible oncogenes, leading to overexpression of the oncogene. Insertional mutagenesis is believed to require alterations in multiple oncogenes and/or tumor suppressor genes.T-cell lymphomas that arise from infection with the Mo-MLV have multiple viral integrations per cell. The ability of replication-competent-retroviruses to cause malignancy has been shown

in nonhuman primates (Donahue et al. 1992; Cornetta et al. 1993), but the risk of insertional mutagenesis with a single integration (as typically occurs with retroviral vectors) was believed to be very low (Cornetta 1992; Li et al. 2002). Clinical trials using retroviral vectors had not reported insertional mutagenesis until recently, when a single vector integration near the LMO2gene was associated with leukemia in at least two of 11 subjects participating in a gene therapy trial for X-linked severe combined immunodeficiency disease

(SCID) (Cavazzana-Calvo et al. 2000;Hacein-Bey-Abina et al. 2002, 2003a,b). The reasons these children have developed leukemia is complex, but preliminary evidence suggests that the transgene in this study (the common cytokine receptor -chain) is also acting as an oncogene (Berns 2004;Dave et al. 2004). Emerging data suggest that different LTRs may have different potentials for causing malignancy, and new vectors are being developed that eliminate the enhancer sequence using self-inactivating vector design (Kraunus et al. 2004).In theory, these vectors should provide a higher safety profile,but they do require other regulatory regions to drive transgene expression. These concerns have also led to evaluation of insulator sequences, matrix attachment regions, and locus control regions.Such sequences have the potential to prevent undesired activation of surrounding genetic sequences and may also protect the transgene cassette from silencing due to positional effects related to the site of integration.

CONSIDERATIONS IN RETROVIRAL PACKAGING CELL LINE CHOICE

Many retroviral packaging cell lines have been made since the first such cell lines were described (Mann et al. 1983; Watanabe and Temin 1983).The key considerations are (1) the range of cell types that can be transduced, which is primarily determined by the Env protein produced by the cells; (2) the propensity of the cells to generate replication-competent virus (also called helper virus), which was a problem with early packaging cell lines but has been largely resolved with newer designs; (3) the susceptibility of vector produced by the packaging cells to inactivation by serum from humans, which is sometimes important for gene therapy applications; and (4) copackaging of endogenous retroviral sequences into virions, especially from packaging cells derived from mouse cells, which is of concern for gene therapy applications.

Table 1 provides a list of some commonly available packaging cells. For standard laboratory usage, a typical choice is a packaging line that produces vectors that can transduce a broad range of mammalian and avian cell lines, such as the PT67 cells.Alternatively, for transfer of oncogenes that represent a potential hazard, the investigator might choose a cell line that produces vectors capable of transducing only rodent cells but not human cells, such as the GP+E-86 cells. For genetic studies, the PG13cell line has the useful property that vectors produced from these cells cannot reinfect the packaging cells, unlike many other packaging cells that undergo reinfection with time of cultivation.

For generation of stable vector-producing packaging cells, the best approach is to transfect one packaging cell line and use the virus from these cells to transduce a second packaging cell line. The second packaging cell line is then selected for the presence of the vector, and clonal isolates can be analyzed for the presence of an intact, single copy of the vector. Vector stocks generated from such cells are likely to be as genetically homogeneous as possible, because the genomic RNA in the vector virions all originates from a single stable integrated provirus.In contrast, vector produced from stably or transiently transfected packaging cells is more heterogeneous because vector RNA can arise from multiple vector copies, some of which can be rearranged(for a detailed protocol, see Retroviral Vector Production by Transient Transfection).Stable

vector-producing cells can be produced by using PE501packaging cells for transient transfection, followed by transduction of the PT67 packaging cells (see Generation of Stable Vector-Producing Cells for Retroviral Vectors).It is important to use a packaging cell line for transfection that will result in virus capable of transducing the second packaging cells. The Env protein made in packaging cells can bind to and block the receptor(s) used by this Env for cell entry, but it will not block other cell surface receptors used by other Env proteins. For example, the 10A1 Env protein made by PT67 cells binds to Pit1 and Pit2 receptors, but it does not bind to the CAT-1 receptor used by virus produced by the GP+E-86 packaging cells. For a broader discussion of these virus classes, see Overbaugh et al. (2001).

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