Retroviral vector production.

Retroviral Vector Production

UNIT 12.5

A. Dusty Miller1 1

Fred Hutchinson Cancer Research Center and Department of Pathology, University of Washington, Seattle, Washington

ABSTRACT In this unit, the basic protocol generates stable cell lines that produce retroviral vectors that carry selectable markers. Also included are an alternate protocol that applies when the retroviral vector does not carry a selectable marker, and another alternate protocol for rapidly generating retroviral vector preparations by transient transfection. A support protocol describes construction of the retroviral vectors. The methods for generating virus from retroviral vector plasmids rely on the use of packaging cells that synthesize all of the retroviral proteins but do not produce replication-competent virus. Additional protocols detail plasmid transfection, virus titration, assay for replication-competent virus, and histochemical staining to detect transfer of a vector encoding alkaline phosphatase. C 2014 by John Wiley & Sons, Inc. Curr. Protoc. Hum. Genet. 80:12.5.1-12.5.22. Keywords: gene therapy r retroviral vectors r retrovirus packaging cells

INTRODUCTION Retroviral vectors promote efficient and stable gene transfer and expression in cultured cells, including primary cells from humans and other animals that are difficult to transfect using other techniques. Transferred genes are integrated at relatively random sites in the host cell genome with minimal alteration of host cell genomic DNA. These properties have led to the use of retroviral vectors as markers for cells transplanted into humans, with or without suicide genes to allow destruction of the transplanted cells when necessary, and for human gene therapy. Only replication-defective retroviral vectors that do not encode any viral proteins are considered in this unit, as they are the most useful for human genetic purposes. When using replication-defective vectors, the process of gene transfer and expression is called transduction to differentiate it from virus infection, which implies further virus replication and spreading. Numerous retroviral vector designs have been developed to address specific problems, discussion of which is beyond the scope of this unit (see Miller, A.D., 1997, for more information). To simplify the topic, the protocols described here are limited to vectors designed for transfer and constitutive expression of cDNAs encoding specific proteins. Production of a retroviral vector consists of two steps: constructing the vector in plasmid DNA form (see Support Protocol 1) followed by producing the virus from cultured cells—retrovirus packaging cell lines that express proteins required for viral replication (see Basic Protocol and Alternate Protocols 1 and 2). An overview of virus production techniques is shown in Figure 12.5.1. The method presented in the Basic Protocol is the method of choice for producing retroviral vectors that carry selectable markers; it results in the generation of stable cell lines that produce a virtually unlimited supply of genetically homogenous retroviral vectors. Alternate Protocol 1 provides a variation for use when the retroviral vector does not carry a selectable marker. Alternate Protocol 2 presents a method for rapidly generating retroviral vector preparations by transient transfection; this is particularly useful either when the gene carried by the vector is toxic to cells, thus precluding the generation of stable lines, or when many vector constructs are being screened for some property. Vectors for Gene Therapy Current Protocols in Human Genetics 12.5.1-12.5.22, January 2014 Published online January 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/0471142905.hg1205s80 C 2014 John Wiley & Sons, Inc. Copyright

12.5.1 Supplement 80

generation of stable vector-producing cell lines

transient transfection (Alternate Protocol 2)

vectors with selectable markers (Basic Protocol )

vectors without selectable markers (Alternate Protocol 1)

packaging cells

packaging cells

virus from transient transfection

transduce cells

isolate and screen clones (∼1 month)

harvest virus from clone(s) with highest titer

vector plasmid plasmid with selectable marker

transfect cells

isolate and screen clones (~1 month)

harvest virus from clone(s) with highest titer

packaging cells

vector plasmid

transfect cells

grow cells (2 days)

harvest virus

Figure 12.5.1 Production of virus from retroviral vector plasmids. Select suitable packaging cell combinations for the transient transfection and transduction steps (Table 12.5.3).

The methods for generating virus from retroviral vector plasmids rely on the use of retrovirus packaging cells that synthesize all of the retroviral proteins but do not produce replication-competent virus. Packaging cell lines that are most useful for mammalian cell gene transfer are listed in Table 12.5.1. The packaging cell lines are grouped by virus family (ecotropic or amphotropic retroviruses) or by the specific virus used to make the packaging line (e.g., GALV). Note that the retrovirus family designations ecotropic and amphotropic describe the host-range properties of murine retroviruses, ecotropic referring to viruses from mice that only infect mice and closely related rodent species, and amphotropic referring to viruses that infect both rodents and many other mammalian species. The range of cells that can be transduced by a retroviral vector is dependent on the retrovirus used to make the packaging cells. The most important determinant is the envelope or coat protein of the virus (Env), which binds to specific cell-surface receptors to promote vector entry (see Overbaugh et al., 2001 for a review of retrovirus receptors). The virus or virus family used to make the proteins for vector packaging into virions is referred to as the pseudotype of the vector. When proteins from several different retroviruses are incorporated into a vector, the pseudotype of the vector commonly refers to the virus from which the envelope protein was derived. For example, a vector produced by PG13 cells (Table 12.5.1) will contain Moloney murine leukemia virus (MoMLV) Gag and Pol proteins and a gibbon ape leukemia virus (GALV) Env protein, and thus by convention has a GALV pseudotype.


Packaging cells with a 10A1 pseudotype are generally the most useful of all the packaging cell lines because of their wide host range, and the protocols described below focus on production of vectors with this pseudotype, generated by using PT67 packaging cells (Miller, A.D. and Chen, 1996). The 10A1 virus is a variant of an amphotropic retrovirus that can infect cells using either the standard amphotropic retrovirus receptor, Pit2, or the


Current Protocols in Human Genetics

Table 12.5.1 Retrovirus Packaging Cell Lines

Maximum titerb

Drug resistance gene(s)c

Reference

gpt

Mann et al. (1983)

hph, gpt

Danos and Mulligan (1988)

gpt

Markowitz et al. (1988a)

tk

Miller, A.D. and Rosman (1989)

Pseudotypea

Name

Cell line origin

Ecotropic

ψ-2d

Mouse

107

Mouse

6

10

Mouse

4 × 10

ψCRE GP+E-86

Mouse

10

E

Mouse

? (high)

gpt

Morgenstern and Land (1990)

Mouse

5 × 10

neo

Takahara et al. (1992)

hph, gpt

Pear et al. (1993)

BOSC 23 PA317 ψCRIP e

FLYA13 GALV VSV

RD114 MDEV JSRV FeLV-C

PT67 PG13 GP7C-tTA-G10

6

7

Human

10

Mouse

4 × 10

Mouse

GP+envAm12 10A1

7

PE501 ampli-GPE Amphotropic

6

Mouse Human

7

tk

Miller, A.D. and Buttimore (1986)

6

hph, gpt

Danos and Mulligan (1988)

6

hph, gpt

Markowitz et al. (1988b)

7

bsr, ble

Cosset et al. (1995)

tk, dhfr*

Miller, A.D. and Chen (1996)

10 10 10

7

Mouse

10

Mouse

3 × 10

Mouse

6

10

tk, dhfr*

Miller, A.D. et al. (1991)

6

hph, pac

Yang et al. (1995)

6

hph, pac, dhfr*

Chen et al. (1996); also see UNIT 12.7

bsr, ble

Cosset et al. (1995)

293GP/tTAER/G-21

Human

10

FLYRD18e

Human

107

Mouse

2 × 10

tk, hph

Wolgamot et al. (1998)

Mouse

4 × 10

tk, hph

Rai et al. (2000)

Human

2 × 10

ble, hph

Doty et al. (2010)

PD223 PJ14 CatPac

5 5 6

a Abbreviations:

10A1, 10A1 murine leukemia virus; GALV, gibbon ape leukemia virus; VSV, vesicular stomatitis virus; RD114, RD114 endogenous cat retrovirus; MDEV, Mus dunni endogenous retrovirus; JSRV, jaagsiekte sheep retrovirus; FeLV-C, feline leukemia virus type C. b Highest reported titers (infectious units/ml). In some cases this value is from papers published after the initial report describing the cell line. c Drug resistance gene(s) that are already present in the indicated packaging cells. The packaging cells were made by cotransfection of these resistance genes with the retrovirus Gag, Pol, and Env expression plasmids. Selection for vectors carrying these markers cannot be performed in these packaging cells. Drug resistance genes: ble, a bacterial (Streptoalloteichus hindustanus) gene that confers resistance to bleomycin, phleomycin, and zeomycin (zeocin) in mammalian cells; bsr, a bacterial gene that confers resistance to blasticidin S; dhfr*, a mutant mammalian dihydrofolate reductase gene that confers resistance to methotrexate; gpt, xanthine-guanine phosphoribosyltransferase; hph, hygromycin phosphotransferase, confers resistance to hygromycin B; neo, neomycin phosphotransferase, confers resistance to G418; pac, puromycin N-acetyl phosphotransferase, confers resistance to puromycin; tk, a herpes simplex virus thymidine kinase gene. d ψ-2 packaging cells are one of the earliest packaging cell lines, but have a high propensity to produce helper virus (MoMLV) and thus are not recommended. e FLYA13 and FLYRD18 packaging cells are derived from HT-1080 cells, which express APOBEC3, and as a result, produce a high proportion of hypermutated retroviral vectors (Miller, A.D., and Metzger, 2011). Therefore, these packaging cell lines are not recommended for standard gene transfer or gene therapy uses, but may be useful for mutation studies.

standard GALV receptor, Pit1 (Overbaugh et al., 2001). PT67 cells have the additional feature that the retroviral Gag protein in these cells is from MoMLV, and unlike the 10A1 virus, is not affected by the Fv1 restriction to infection of mouse cells (Stevens et al., 2004). Additional protocols are as follows. Support Protocol 2 details the calcium phosphate transfection method for transferring DNA into cells. Support Protocol 3 describes a method for titering vectors that carry a selectable marker. Support Protocol 4 describes a marker rescue assay for detection of replication-competent (helper) virus in retroviral vector preparations. Finally, Support Protocol 5 describes a histochemical method for titering vectors that express alkaline phosphatase.

Vectors for Gene Therapy

12.5.3 Current Protocols in Human Genetics

Supplement 80

BASIC PROTOCOL

GENERATION OF STABLE PACKAGING CELL LINES THAT PRODUCE VECTORS CARRYING SELECTABLE MARKERS Inclusion of a selectable marker in a retroviral vector allows facile selection of packaging cells that contain and produce the vector. In contrast to vector production by transient transfection (see Alternate Protocol 2), stable vector-producing cell lines can produce a virtually unlimited amount of virus without the need for repeated transfections. The vector can be directly transfected into the packaging cells using calcium phosphate– mediated transfection (see Support Protocol 2), and clones that produce the vector can be isolated. However, this approach can result in integration of multiple copies of the vector, including rearranged copies, and this can result in production of a mixture of intact and rearranged virus. The approach described in this protocol utilizes virus produced transiently following vector transfection into one packaging cell line to transduce another packaging cell line. The conditions of transduction involve infection at a low ratio of virions to cells (low multiplicity of infection, MOI), and clonal lines isolated using this approach almost always contain single integrated vectors that can be examined for the correct vector structure and DNA sequence. Note that there are restrictions on the ability of virus from one packaging cell line to infect the same or other packaging cell lines, because of a phenomenon known as virus interference. Virus interference is caused by binding of envelope protein made by a packaging cell to a specific retrovirus receptor, resulting in a block to infection by other viruses that use the same receptor for entry (see Overbaugh et al., 2001 for more information on retrovirus receptors). In addition, some retroviruses will not infect cells of the species used to make certain packaging cells (Table 12.5.2). For example, ecotropic virus will not infect packaging cells derived from human cells lines, such as the FLYA and FLYRD lines. A summary of virus pseudotypes that allow transduction of specific packaging cell types is given in Table 12.5.3, and this information should be considered in choosing packaging cell types for use in the following protocol.

Materials Two retrovirus packaging cell lines, in this example, PE501 and PT67 (see Table 12.5.1). PE501 cells are available from the author, and PT67 cells are available from the American Type Culture Collection (ATCC, http://www.atcc.org) cell line no. CRL-12284, or from Clontech Labs (http://www.clontech.com) cat. no. 631510. Other ecotropic packaging cell lines can be substituted for the PE501 cells, including GP+E-86 cells (Markowitz et al., 1988a), which are available as ATCC cell line no. CRL-9642. Dulbecco’s modified Eagle medium containing 4.5 g/liter glucose and 10% (v/v) FBS (DMEM/10% FBS; see APPENDIX 3G) Retroviral vector plasmid containing selectable marker (see Support Protocol 1) 4 mg/ml Polybrene (Sigma) in PBS (APPENDIX 2D) Drug appropriate for the selectable marker carried by the vector: e.g., 0.75 mg/ml G418 (active concentration), 4 mM L-histidinol, or 0.4 mg/ml hygromycin B Cell staining solution (see recipe) 6- and 10-cm tissue culture dishes 10-ml syringes 0.45-μm-pore-size low-protein-binding cellulose acetate syringe filters Cloning rings (UNIT 3.2)


Additional reagents and equipment for culturing and freezing mammalian cells (APPENDIX 3G), calcium phosphate–mediated transfection (see Support Protocol 2), isolating clones using cloning rings (UNIT 3.2), assaying vector titer (see Support Protocol 3), preparing genomic DNA (e.g., APPENDIX 3B), Southern blot hybridization (UNIT 2.7), and performing marker rescue assay for helper virus (see Support Protocol 4)



Table 12.5.2 Host Range of Retroviral Vectors

Cells that can be transduced Vector pseudotype

Mouse

Human

Ecotropic

Yes

No

Amphotropic

Yes

Yes

10A1

Yes

Yes

GALV

No

Yes

VSV

Yes

Yes

RD114

No

Yes

MDEV

Yes

Yes

JSRV

No

Yes

FeLV-C

No

Yes

Table 12.5.3 Packaging Cell Pseudotypes that Can Be Used for Infection of Other Packaging Cells

Target packaging cells Pseudotype

Species

Virus pseudotypes that allow efficient infection of the target packaging cellsa

Ecotropic

Mouse

Amphotropic, 10A1, VSV

Human

Amphotropic, 10A1, GALV, VSV, RD114, MDEV, FeLV-C

Mouse

Ecotropic, 10A1, VSV

Human

GALV, VSV, RD114, MDEV, FeLV-C

10A1

Mouse

Ecotropic, VSV

GALV

Mouse

Ecotropic, amphotropic, 10A1, VSV

VSV

Mouse

Ecotropic, amphotropic, 10A1

RD114

Human

Amphotropic, 10A1, GALV, VSV, MDEV, FeLV-C

MDEV

Mouse


JSRV

Mouse


FeLV-C

Human

Amphotropic, 10A1, GALV, VSV, RD114, MDEV

Amphotropic

a See

Table 12.5.1.

NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly. NOTE: All culture incubations are performed in a humidified 37°C, 10% CO2 incubator unless otherwise specified.

Transiently transfect PE501 cells 1. On day 1, seed PE501 retrovirus packaging cells at 5 × 105 cells per 6-cm dish in DMEM/10% FBS. 2. On day 2, replace the culture medium with 4 ml fresh tissue culture medium. Perform calcium phosphate–mediated transfection (see Support Protocol 2) using the retroviral vector plasmid containing a selectable marker. 3. On day 3, aspirate the medium from the transfected PE501 cells and add 4 ml fresh DMEM/10% FBS. Also, seed PT67 cells at 105 cells per 6-cm dish in DMEM/10%



Supplement 80

FBS. Two dishes of PT67 cells should be prepared for each dish of transfected PE501 cells.

Transduce PT67 cells 4. On day 4, replace the medium on the PT67 cells with DMEM/10% FBS containing 4 μg/ml Polybrene (1000-fold dilution of Polybrene stock solution). Polybrene (1,5-dimethyl-1,5-diazaundecamethylene polymethobromide) is a polycation that improves retrovirus infection rates in cultured cells, presumably by neutralizing the repulsive effects of negatively charged molecules on the surfaces of virions and cells.

5. Using 10-ml syringes, remove 3 ml virus-containing medium from each dish of transfected PE501 cells, and filter the medium through 0.45-μm-pore-size lowprotein-binding cellulose acetate syringe filters to remove live cells. Leave 1 ml medium on the dish to keep cells from drying out. 6. Infect one dish of PT67 cells with 1 ml filtered virus-containing medium from one dish of transfected PE501 cells, and another dish with 10 μl from the same PE501 dish. 7. Trypsinize and seed cells from each dish of transfected PE501 cells at a 1:20 dilution in 6-cm dishes containing DMEM/10% FBS supplemented with the drug appropriate for the selectable marker—e.g., 0.75 mg/ml G418 (active concentration), 4 mM Lhistidinol, or 0.4 mg/ml hygromycin B. 8. On day 5, trypsinize the infected PT67 cells and seed the cells from each dish at dilutions of 9:10 and 1:10 into 10-cm dishes containing 10 ml DMEM/10% FBS supplemented with the appropriate selective drug (see step 7). The 9:10 and 1:10 dilutions of PT67 cells infected with 1 ml or 10 μl of virus results in a 4-log range of dilutions, some of which should yield appropriate numbers of colonies for isolation of clonal cell lines.

9. On day 9, aspirate the medium from the dishes of PE501 cells that were seeded into selective medium on day 4 and add 1.5 ml cell staining solution per dish. After 5 to 10 min, wash the stain off with water. Evaluate the dishes for colony formation as a measure of the efficiency of DNA transfection. A transfection efficiency of about 1000 colonies/μg plasmid DNA is typical.

Isolate vector-containing PT67 packaging cell clones 10. On day 10 to 15, after drug-resistant colonies of vector-transduced PT67 cells have formed, isolate clones from the dishes that contain small numbers of colonies using cloning rings (UNIT 3.2). Expand the clones in medium containing the selective drug to provide enough cells for freezing and clone analysis. Typically isolation of about ten colonies is sufficient to yield suitable clones that produce a vector at high titer.

Characterize vector-containing packaging cell clones 11. When the clonal cell lines are sufficiently expanded, freeze one or two vials of each clone as a backup in case the culture is accidentally lost during screening of clones for the best vector producer. In addition, the titer of vector produced by packaging cells can decrease during extended culture, so freezing some of the cells early can provide a continuous source of cells that produce high vector titers. Retroviral Vector Production

12. Assay the titer of the vector by measuring transfer of the selectable marker encoded by the vector (see Support Protocol 3).



13. Identify those clones that transfer drug resistance, as detected by the growth of the target cells in selective medium, and analyze for expression of the cDNA inserted into the vector. These two assays will reduce the number of candidate clones for further screening.

Analyze clone DNA 14. Prepare genomic DNA from the clones (e.g., APPENDIX 3B). Digest the DNA with a restriction endonuclease that cuts only in the viral LTRs and an endonuclease that cuts once in the vector and randomly in the surrounding genomic DNA. 15. Analyze the digested DNAs by Southern blot hybridization (UNIT 2.7) using a cDNA sequence probe. The DNA digested with the enzyme that cuts only in the LTR should show that the cDNA is carried by the vector and that the vector length is correct, and therefore that no deletions have occurred. DNA digested with an endonuclease that cuts once in the vector and randomly in the surrounding genomic DNA should reveal the number of integrated proviruses present. Clones with a single vector provirus will produce genetically homogeneous virus.

16. Sequence the cDNA insert. In cases where the exact sequence of the cDNA in the single vector copy integrated into the packaging cells is important, as might be the case for use of the vector in human gene therapy, primers should be designed to PCR amplify (UNIT 15.1) segments of the cDNA from the genomic DNA of the packaging cell line and determine the complete cDNA sequence.

Perform marker rescue assay for detection of helper virus production 17. Screen the clones with a single vector provirus for production of helper virus (see Support Protocol 4) and discard any clones that produce helper virus. Helper virus production from PT67 cells containing the vectors described here (Fig. 12.5.2) is expected to be very rare. Indeed, the author has never observed helper virus production from a vector-containing clone of PT67 cells in over 17 years of generating vector-producing packaging cell lines with the vectors described here. However, because of the possibility of helper virus production by retrovirus packaging cells, and the possibility of cross-contamination of cell lines with retroviruses from other cell lines carried in the lab, laboratories using this technology should have the capability of and experience with screening for helper virus, even if this assay is not always performed.

Expand the clone and prepare vector stocks 18. Once a clonal cell line is identified that has the desired characteristics, thaw one of the backup vials (step 11), expand the cells, and freeze multiple vials for future use. Most clones grown in continuous culture will produce the vector consistently for 2 months, and if vector titer declines, a new vial can be thawed for use.

19. To harvest vector from the cells, seed the cells in 10-cm tissue culture dishes in 10 ml medium and grow the cells until 1 day post confluence. Feed the cells with 10 ml fresh medium, harvest vector-containing medium 12 to 24 hr later, and store at –70°C. The half-life of the vector at 37°C is 4 hr, so harvests made at longer intervals are counterproductive in that defective virions are likely to accumulate in the medium. At least three 12-hr harvests can be made sequentially. Large amounts of the vector-containing medium can be frozen at –70°C indefinitely for future use. Vector stocks should be frozen and thawed quickly to preserve vector activity.



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GenBank accession number name

M28245

LN

Ψ+ LTR

LTR Ψ+

M28246

M28247

LNSX

pA

neo SV

LTR

Ψ+ LNCX

pA

neo

LTR

LTR

(Bam HI) StuI Avr II Hind III ClaI pA neo CMV LTR (Bam HI) Hind III HpaI ClaI

Ψ+ M28248

LXSN

pA

neo SV

LTR

LTR

EcoRI Hpal Xhol Bam HI

M64754

Ψ+ LHDCX

CMV

LTR (Bam HI)

LXSHD

LTR

LTR

(Bam HI) Hind III

Ψ+ M64753

pA

hisD

pA

hisD SV

LTR

EcoRI Hpal Xhol Bam HI

M77239

LXSH

Ψ+

pA

hph SV

LTR

LTR

Hpal Xhol Bam HI

LAPSN

Ψ+ LTR

AP

neo SV

pA LTR

1 kb


Figure 12.5.2 Retroviral vectors. Retroviral vectors that contain selectable drug markers— neomycin phosphotransferase (neo), histidinol dehydrogenase (hisD), or hygromycin phosphotransferase (hph)—are shown with their GenBank accession numbers for the complete vector sequences. Vector names are composed of the abbreviations for the genetic elements they contain. The coding regions of these genes are shaded. Connecting lines indicate other viral sequences, and arrows indicate the cap sites of promoters and the direction of transcription. Restriction sites for cDNA insertion are indicated. Restriction sites in parentheses are discussed in the text. The vectors carrying neo and hisD have been described (Miller, A.D. and Rosman, 1989; Stockschlaeder et al., 1991), LXSH was made from LXSN by replacement of the neo insert with hph (M. E. Emerman and J. V. Garcia, unpub. observ.), and LAPSN has been described (Miller, D.G. et al., 1994). Abbreviations in vector names: C, cytomegalovirus (CMV) immediate early promoter; H, hygromycin-B-phosphotransferase (hph); HD, hisD gene; L, long terminal repeat (LTR); N, neomycin (neo) gene; pA, polyadenylation signals; S, SV40 early promoter; X, cloning site; + , extended retroviral packaging signal.



Confluent layers of PT67 and other packaging cell lines can detach from cell culture dishes if shocked by cooling or rough handling. Feed these cells only with culture medium prewarmed to 37°C, and keep them out of the incubator for as little time as possible.

GENERATION OF STABLE PACKAGING CELL LINES THAT PRODUCE VECTORS WITHOUT SELECTABLE MARKERS

ALTERNATE PROTOCOL 1

This procedure relies on the process of cotransfection to select for cells that contain a vector that does not carry a selectable marker (see Fig. 12.5.1). In this technique, a small amount of a plasmid that carries a selectable marker is transfected along with an excess of the vector plasmid. Cells are selected for the presence of the selectable marker. Cells with the selectable marker often contain the unselected plasmid that was present in excess during transfection. In this procedure the pSV2neo plasmid is used for selection, but plasmids that express other selectable markers work equally well. PT67 packaging cells are used in this example, but other packaging cell lines can be substituted.

Materials Retrovirus packaging cells, in this example, PT67 cells (see Table 12.5.1) Dulbecco’s modified Eagle medium containing 4.5 g/liter glucose and 10% (v/v) FBS (DMEM/10% FBS; see APPENDIX 3G) Retroviral vector plasmid DNA (see Support Protocol 1) Plasmid DNA with selectable marker: e.g., pSV2neo 0.75 mg/ml G418 (active concentration) or other selective drug appropriate for the plasmid with selectable marker 6-cm tissue culture dishes Cloning rings (UNIT 3.2), sterile Additional reagents and equipment for culturing and freezing mammalian cells (APPENDIX 3G), calcium phosphate–mediated transfection of cells (see Support Protocol 2), isolating clones using cloning rings (UNIT 3.2), and analyzing clones (see Basic Protocol 1, steps 14 to 18, and UNIT 2.7) NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly. NOTE: All culture incubations are performed in a humidified 37°C, 10% CO2 incubator unless otherwise specified.

Cotransfect packaging cells 1. On day 1, seed PT67 retrovirus packaging cells at 5 × 105 cells per 6-cm tissue culture dish in 4 ml DMEM/10% FBS. 2. On day 2, replace the culture medium with 4 ml fresh medium. Using calcium phosphate–mediated transfection (see Support Protocol 2), transfect the cells with 10 μg retroviral vector plasmid DNA and either 0.1 or 0.2 μg pSV2neo plasmid DNA. 3. On day 3, aspirate the medium from the transfected PT67 cells and add 4 ml fresh medium.

Isolate cotransfected packaging cell clones 4. On day 4, trypsinize the transfected cells and seed at dilutions of 1:2 and 1:10 in 10-cm dishes containing DMEM/10% FBS supplemented with 750 μg/ml G418 (active concentration). 5. On day 9 to 14, isolate drug-resistant colonies from dishes containing small numbers of colonies by using cloning rings (UNIT 3.2). Expand the clones.



Supplement 80

Because 50% of the clones may contain vector sequences, it may be necessary to screen about 50 clones to find a high-titer vector-producing cell line.

Characterize vector-containing packaging cell clones 6. When the clones are sufficiently expanded, freeze one or two vials of each as a backup in case the cells are accidentally lost during screening of clones for the best vector producer. In addition, the titer of vector produced by packaging cells can decrease during extended culture, so freezing the clones early will provide a continuous source of cells that produce high vector titers.

7. If possible, test the clonal lines for production of the protein encoded by the cDNA included in the vector. This screening will eliminate many clones that do not contain the vector or that contain rearranged copies of the vector. It may not be possible to screen the packaging cells directly for the desired protein product if the cells already make the protein; in that case, virus must be harvested from the clones and used to infect cells that will allow detection of the desired protein.

8. Prepare genomic DNA from the clones (e.g., APPENDIX 3B) and analyze the DNA by restriction endonuclease digestion and Southern blot hybridization (UNIT 2.7). It may be useful to select clones containing single vector proviruses for further use because they will produce genetically homogeneous virus. However, cotransfection often results in the integration of multiple concatemerized copies of the vector, and as long as none of these produce rearranged vectors, the vector produced by the clone will be suitable for use.

Titer the vector 9. Determine the vector titer. Often the titer of vectors that do not carry selectable markers cannot be determined directly. However, it may be possible to develop single-cell assays for expression of the protein encoded by the vector to allow direct determination of the vector titer. For example, if antibodies to the vector-encoded protein are available, it may be possible to develop a fluorescence-activated cell sorter (FACS) assay for expression of the protein in single cells. It would then be possible to infect cells and directly measure the number of cells transduced to determine the vector titer. If such assays are not feasible, vector transfer from the packaging cells to the target cells can be measured by Southern blot hybridization (UNIT 2.7) or PCR quantitation of vector copy number, or by bulk assay for the protein carried by the vector.

10. Screen for helper virus using the marker rescue assay (see Support Protocol 4), expand the clones, and harvest the vector (see Basic Protocol, steps 17 to 19). ALTERNATE PROTOCOL 2

PRODUCTION OF VECTOR BY TRANSIENT TRANSFECTION This procedure is used to rapidly produce virus from the plasmid form of a retroviral vector. The virus can then be used directly for gene transfer. Virus produced by transient transfection can also be used to generate stable vector-producing cell lines (see Basic Protocol). The use of calcium phosphate for plasmid transfection is described below because this procedure is simple and inexpensive, but other methods, such as the use of cationic lipids for DNA transfection, can readily be substituted.

Materials Retroviral Vector Production

Retrovirus packaging cells (see Table 12.5.1) and appropriate tissue culture medium Retroviral vector plasmid DNA (see Support Protocol 1)



6-cm tissue culture plates 10-ml syringes 0.45-μm-pore-size low-protein-binding cellulose acetate syringe filters Additional reagents and equipment for culture of mammalian cells (APPENDIX 3G) and calcium phosphate–mediated transfection (see Support Protocol 2) NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly. NOTE: All culture incubations are performed in a humidified 37°C, 10% CO2 incubator unless otherwise specified. 1. On day 1, seed the retrovirus packaging cells at 5 × 105 per 6-cm dish in 4 ml of the appropriate tissue culture medium. 2. On day 2, introduce the retroviral vector plasmid DNA into the packaging cells by calcium phosphate–mediated transfection (see Support Protocol 2). 3. On day 3, replace the medium with fresh culture medium. 4. On day 4, after 12 to 24 hr, using a 10-ml syringe, harvest the culture medium and filter it through a 0.45-μm-pore-size low-protein-binding cellulose acetate syringe filter to remove cells and debris. Use the vector-containing medium immediately to infect recipient cells or store it at −70°C indefinitely. Several harvests of vector can be made at 12 hr intervals by refeeding the cells after each harvest. Alternatively, centrifuge the medium 5 min at 3000 × g (5000 rpm in a Sorvall HB-4 rotor) at 4°C, to pellet cells and debris; however, this method may not completely remove all of the cells. Confluent layers of PT67 and other packaging cell lines can detach from cell culture dishes if shocked by cooling or rough handling. Feed these cells only with culture medium prewarmed to 37°C, and keep them out of the incubator for as little time as possible.

CONSTRUCTION OF RETROVIRAL VECTORS A variety of retroviral vectors suitable for expression of inserted cDNAs are available from academic laboratories and biotechnology companies. High-titer virus has been prepared from vectors with cDNA inserts of up to 4.6 kb in the LXSN retroviral vector (Kaleko et al., 1990), but the upper limit for vector size has not been established. For most experimental purposes, a retroviral vector that also carries a selectable marker is desirable, although for human gene therapy purposes, vectors that carry only the therapeutic gene may be required to avoid possible immune reactions to selectable marker proteins. Figure 12.5.2 depicts a set of retroviral vectors that contain selectable markers and unique cloning sites for insertion of cDNAs (Miller, A.D. and Rosman, 1989; Miller, A.D. et al., 1993). The vectors are named according to the order of genetic elements in the vector: L, long terminal repeat (LTR); N, neomycin gene (neo); S, SV40 early promoter; C, human cytomegalovirus (CMV) immediate early promoter; HD, hisD gene; H, hygromycin-B-phosphotransferase (hph); and X, cloning site. With the exception of LN, the vectors contain two promoters, one of which drives expression of the selectable marker and the other expression of the inserted DNA. Transcription of inserted cDNAs is driven by strong viral promoters—either the retroviral LTR (LXSN, LXSHD, and LXSH), an immediate early promoter from human cytomegalovirus (LNCX, LHDCX), or the SV40 early promoter (LNSX). In general, the LTR and CMV promoters are very strong promoters in human cells, and the SV40 promoter is weaker. Vectors containing

SUPPORT PROTOCOL 1



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either of three different dominant selectable markers are shown. Selection for each of the markers is independent of the presence or absence of the other markers, allowing sequential use of vectors carrying different selectable markers to transfer multiple genes into cells.

Materials cDNA of interest Retroviral vector in a bacterial plasmid Additional reagents and equipment for constructing a plasmid (e.g., Struhl, 1991), transforming E. coli cells (e.g., Seidman et al., 1997), and preparing DNA minipreps (UNIT 5.3 or, e.g., Engebrecht et al., 1991) 1. Scan the cDNA of interest for the presence of polyadenylation signals (AATAAA) in the 3 untranslated region. Trim the 3 untranslated end of the cDNA to remove the polyadenylation signal. The inserted cDNAs should not contain polyadenylation signals, as these will cause premature termination of transcription and reduce the levels of full-length vector RNA. Removal of the 3 untranslated region from the cDNA can also be helpful if this deletes sequences such as those located in the 3 untranslated regions of hematopoietic growth factor genes that promote RNA degradation and result in short mRNA half-life (Shaw and Kamen, 1986). Although sequences that reduce the amount of full-length vector RNA can be included in retroviral vectors, these sequences will reduce the titer of virus and may lead to frequent deletion of the inserted sequences because there is a strong selection for recombinant vectors that lack the offending sequences.

2. Insert the cDNA of interest into the cloning site of the retroviral vector using standard molecular biology techniques for plasmid construction (e.g., Struhl, 1991). 3. Transform competent E. coli cells with the resulting cDNA ligation mixture (Seidman et al., 1997). 4. Prepare miniprep DNA from individual bacterial colonies (UNIT 5.3 or Engebrecht et al., 1991). 5. Digest the plasmids with restriction endonuclease(s) to identify those plasmids that contain the insert in the correct orientation. 6. Prepare large-scale preparation of the recombinant vector plasmid DNA for transfection. For efficient transfection of cultured cells, the plasmid preparation must be relatively pure, e.g., by banding in CsCl (see UNIT 5.3) or by passage over a Qiagen resin column (e.g., Heilig et al., 1998). Use of miniprep DNA typically leads to poor transfection efficiency in cultured cells. SUPPORT PROTOCOL 2

CALCIUM PHOSPHATE–MEDIATED TRANSFECTION OF CULTURED CELLS Coprecipitates of calcium, phosphate, and DNA can be taken up by cultured cells, after which many cells will express genes carried by the DNA. Peak expression of transfected genes occurs a few days after transfection. Transfected DNA can also be integrated into the genome of a small proportion of the cells, resulting in long-term persistence and expression of the genes.

Materials Retroviral Vector Production

Retroviral vector plasmid DNA (see Support Protocol 1) 10 mM Tris·Cl, pH 7.5 (APPENDIX 2D)



Retrovirus packaging cells (see Table 12.5.1) Dulbecco’s modified Eagle medium containing 4.5 g/liter glucose and 10% (v/v) FBS (DMEM/10% FBS; see APPENDIX 3G) 2.0 M CaCl2 500 mM HEPES, pH 7.1 2.0 M NaCl 150 mM sodium phosphate buffer, pH 7.0 (APPENDIX 2D) Sterile H2 O 12 × 75–mm clear polystyrene tubes (Falcon) Additional reagents and equipment for selection or screening of transfected cells (see Basic Protocol or Alternate Protocol 1 or 2) NOTE: All solutions should be sterilized by filtration through 0.22-μm-pore-size sterile filters; if the plasmid DNA is not already sterile because it was ethanol precipitated during preparation, it should also be filtered. NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly. NOTE: All mammalian cell culture incubations are performed in a humidified 37°C, 10% CO2 incubator unless otherwise specified. 1. Resuspend the purified retroviral vector plasmid DNA in 10 mM Tris·Cl, pH 7.5, to a final concentration of 1 μg/μl. It is important that the DNA be relatively pure and free of salts and EDTA.

2. On day one, seed retrovirus packaging cells at 5 × 105 cells in 6-cm tissue culture dishes containing DMEM/10% FBS. 3. On day 2, feed the retrovirus packaging cells with fresh medium. 4. For each plasmid, prepare a DNA/CaCl2 solution:

25 μl 2.0 M CaCl2 10 μg plasmid DNA in 10 mM Tris·Cl, pH 7.5 Sterile H2 O to 200 μl. Mix. 5. Prepare fresh precipitation buffer:

100 μl 500 mM HEPES, pH 7.1 125 μl 2.0 M NaCl 10 μl 150 mM sodium phosphate buffer, pH 7.0 Sterile H2 O to 1 ml. Mix. 6. Add the 200 μl DNA/CaCl2 solution dropwise with constant agitation to 200 μl precipitation buffer in a clear 12 × 75–mm polystyrene tube. Incubate 30 min at room temperature. A faint cloudiness in the solution should be immediately apparent. If the mixture remains clear or a precipitate consisting of large clumps develops, something is wrong. A new precipitation solution should be prepared using freshly prepared plasmid and precipitation solutions.

7. Check that the precipitate in the tube is fine, not clumpy. Add the fine precipitate to a 6-cm dish of retroviral packaging cells and swirl the dish to distribute the precipitate.



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Incubate and proceed to select or harvest virus from the cells (see Basic Protocol or Alternate Protocol 1 or 2). SUPPORT PROTOCOL 3

ASSAY TO TITER VECTORS CARRYING SELECTABLE MARKERS The titer of vectors carrying selectable markers can be determined by this procedure, which can be adapted to vectors carrying histochemical markers (e.g., alkaline phosphatase or β-galactosidase) by reducing the plating density on day 1 to 105 per 6-cm dish, eliminating the trypsinization and drug selection step on day 3, and staining the cells for the histochemical marker on day 4. It is more difficult to determine vector titer for vectors that do not carry marker genes—such titer determinations can involve Southern blot hybridization (UNIT 2.7) of DNA from cells transduced with the vector to be assayed and cells transduced with another otherwise comparable vector that carries a selectable marker that allows direct determination of titer.

Materials Target cells susceptible to the vector pseudotype to be tested (e.g., NIH 3T3 or HeLa cells) and appropriate tissue culture medium 4 mg/ml Polybrene (Sigma) Retroviral stock to be tested (e.g., from Basic Protocol or Alternate Protocol 1 or 2) Selective drug: 0.75 mg/ml G418 (active concentration), 0.4 mM hygromycin B, or 4 mM L-histidinol Cell staining solution (see recipe) 6-cm tissue culture dishes Additional reagents and equipment for culture and passaging of mammalian cells (APPENDIX 3G) NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly. NOTE: All culture incubations are performed in a humidified 37°C, 10% CO2 incubator unless otherwise specified. 1. On day 1, seed target cells susceptible to vector pseudotype at 5 × 105 cells per 6-cm dish in the appropriate tissue culture medium. 2. On day 2, change the medium on the cells to DMEM/10% FBS containing 4 μg/ml Polybrene and add varying amounts of retroviral vector stock to separate plates. Depending upon the titer of the retroviral stock, it is useful to test 0.01 μl to 100 μl of the retroviral vector stock.

3. On day 3, trypsinize and seed the cells at a 1:20 dilution into 6-cm plates containing DMEM/10% FBS and a selective drug at the appropriate concentration. A final concentration of 0.75 mg/ml G418 (active concentration) is used for cells infected with vectors carrying the neo gene, 4 mM L-histidinol for cells infected with vectors carrying the hisD gene, or 0.4 mg/ml hygromycin B for cells infected with vectors carrying the hph gene. Concentrations may need to be adjusted depending on the cell line.

4. On day 8 to 11, after colonies have formed, aspirate the culture medium from the plates, add 1.5 ml cell staining solution to each dish for 5 to 10 min, wash off stain with water, and count colonies. Calculate the virus titer.


Virus titer in colony-forming units per milliliter (CFU/ml) is calculated by dividing the number of colonies by the volume (in milliliters) of undiluted virus stock used for infection and multiplying by 20 to correct for the 1:20 cell dilution.



MARKER RESCUE ASSAY FOR HELPER VIRUS The marker rescue assay for helper virus detection measures the ability of virus that may contaminate a retrovirus vector stock to rescue or mobilize a retroviral vector from cells that contain but do not produce the vector (nonproducer cells). A good vector to use in this assay is one that encodes alkaline phosphatase (AP) because of the ease and speed of detection techniques for AP (see Support Protocol 5). It should be remembered that the ability of this assay to detect a given helper virus depends on whether the helper virus can infect the cells used in the assay. For example, gibbon ape leukemia virus (GALV) helper virus cannot infect mouse cells but can infect human cells (see Table 12.5.2); thus, a human cell line should be used for GALV detection. Although this assay is somewhat time consuming, it is reliable and very sensitive.

SUPPORT PROTOCOL 4

Materials LAPSN vector plasmid or virus (see Fig. 12.5.2) Na¨ıve target cells that are susceptible to the pseudotype of the retrovirus vector stock to be tested, and that do not express high levels of endogenous heat-stable AP: e.g., NIH 3T3 or HT-1080 cells Retrovirus stock to be tested (see Basic Protocol or Alternate Protocol 1 or 2), filtered through 0.45-μm-pore-size low-protein-binding cellulose acetate syringe filter 4 mg/ml Polybrene (Sigma) in PBS (APPENDIX 2D) Positive control virus: amphotropic replication-competent “helper” virus 6-cm tissue culture dishes 10-ml syringes 0.45-μm-pore-size low-protein-binding cellulose acetate syringe filters Additional reagent and equipment for culturing and passaging mammalian cells (APPENDIX 3G), transient or stable transfection of cells (see Basic Protocol or Alternate Protocol 1 or 2), and staining for alkaline phosphatase activity (see Support Protocol 5) NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly. NOTE: All culture incubations are performed in a humidified 37°C, 10% CO2 incubator unless otherwise specified.

Generate nonproducer cells 1. Produce a virus stock containing the LAPSN vector (Fig. 12.5.2), which encodes alkaline phosphatase (AP), from retrovirus packaging cells that are transiently transfected with the LAPSN vector plasmid (see Alternate Protocol 2), or from stable LAPSN-vector-producing cells (see Basic Protocol or Alternate Protocol 1). 2. Transduce na¨ıve target cells with the virus. Passage the cells for 2 weeks to allow potential helper virus (which should not be present) to spread. 3. Assay the cells for vector production by exposing parental cells that do not contain the vector to medium from the putative non-producer cells and staining for transfer of the LAPSN vector, as described in steps 7 to 10, below. Multiple aliquots of those cells that contain but do not release the LASPN vector (nonproducer cells) should be preserved in liquid nitrogen for use in future marker rescue assays. Nonproducer cells need to be generated only once. Vectors for Gene Therapy


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Perform marker rescue assay 4. On day 1, seed nonproducer cells containing the LAPSN vector at 5 × 105 cells per 6-cm tissue culture dish in the appropriate medium. 5. On day 2, expose nonproducer cells to 1 ml filtered retrovirus stock to be tested, 3 ml tissue culture medium, and 4 μg/ml Polybrene. As a positive control, infect some dishes with a small amount of amphotropic replication-competent helper virus plus 4 ml tissue culture medium. A suitable positive control would be 1 μl of virus produced by NIH 3T3 cells transfected with pAM-MLV plasmid (Miller, A.D. and Buttimore, 1986) and passaged for 2 weeks to allow complete infection of the cells, or other helper virus capable of replicating in the nonproducer cells. Polybrene (1,5-dimethyl-1,5-diazaundecamethylene polymethobromide) is a polycation that improves retrovirus infection rates in cultured cells, presumably by neutralizing the repulsive effects of negatively charged molecules on the surfaces of virions and cells.

6. Beginning on day 3, incubate the cells 2 weeks to allow helper virus spread. Two to three times a week, trypsinize the cells and seed them at 1:10 to 1:40 dilutions. Cells should be kept at relatively high density to facilitate virus spread. Take care not to cross-contaminate the cultures, some of which may begin to make helper virus at very high titer.

7. On day 16, seed na¨ıve target cells susceptible to the vector pseudotype at 105 cells per 6-cm dish. Also, change the medium on the confluent dishes of nonproducer cells. The target cells should be the same type of cells as those used to construct the nonproducer cell line. The “nonproducer” cells may be producing virus.

8. On day 17, harvest medium from the nonproducer cells and filter through a 0.45-μm-pore-size low-protein-binding syringe filter to remove cells and debris. 9. Infect na¨ıve target cells using 1-ml samples of the medium, 3 ml tissue culture medium, and 4 μg/ml Polybrene. 10. On day 19, stain the infected target cells for alkaline phosphatase activity (see Support Protocol 5). A positive staining reaction indicates transfer of virus from the nonproducer vectorcontaining cells due to the presence of helper virus in the tested retroviral vector stock. SUPPORT PROTOCOL 5

STAINING CULTURED CELLS FOR ALKALINE PHOSPHATASE ACTIVITY This protocol for staining for alkaline phosphatase activity can be used to titer virus; it is modified from Fields-Berry et al. (1992). It is written for cells cultured in 6-cm dishes, but the conditions can easily be adjusted for dishes of other sizes. Alkaline phosphatase cleaves phosphate from 5-bromo-4-chloro-3-indoyl phosphate (BCIP); the product from this reaction reacts with nitroblue tetrazolium (NBT) to produce a purple reaction product.

Materials



Cell cultures transduced with alkaline phosphatase–encoding vector (e.g., LAPSN) in 6-cm tissue culture dishes 0.25% (v/v) glutaraldehyde in PBS (APPENDIX 2D) PBS (APPENDIX 2D) Alkaline phosphatase staining buffer (see recipe) Alkaline phosphatase staining solution (see recipe) Current Protocols in Human Genetics

1. Aspirate the medium from cell cultures transduced with an alkaline phosphatase– encoding vector. Fix cells with 3 ml of 0.25% glutaraldehyde for 5 to 10 min at room temperature. It is not necessary to wash the cells with PBS prior to fixation. Untransduced cells are used as a negative control and cells known to be producing alkaline phosphatase are used as a positive control.

2. Wash the cells twice with 2 to 3 ml PBS each wash. Add 2 ml PBS and heat 30 min at 65°C to inactivate cellular alkaline phosphatase activity. The vector-encoded alkaline phosphatase is heat stable, but cellular alkaline phosphatases are relatively heat labile in most cells types, with the notable exception of HeLa cells.

3. After the dishes have cooled, aspirate the PBS and wash once with 2 ml alkaline phosphatase staining buffer. This wash removes phosphates, which can inhibit alkaline phosphatase activity.

4. Add 1.5 ml alkaline phosphatase staining solution. Incubate 4 hr at room temperature. The duration of the incubation depends on the cell type and background alkaline phosphatase activity.

5. Count alkaline phosphatase–positive foci of cells.

REAGENTS AND SOLUTIONS Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see APPENDIX 2D; for suppliers, see SUPPLIERS APPENDIX.

Alkaline phosphatase staining buffer 100 mM NaCl 50 mM MgCl2 100 mM Tris·Cl, pH 8.5 (APPENDIX 2D) Store indefinitely in glass container at room temperature Alkaline phosphatase staining solution Prepare a solution containing 1× NBT solution (see recipe) and 1× BCIP solution (see recipe) in alkaline phosphatase staining buffer (see recipe). Prepare just before use. BCIP solution, 100× Dissolve 5-bromo-4-chloro-3-indoyl phosphate (BCIP) in water to give a final concentration of 10 mg/ml. Store in foil-wrapped glass container (i.e., in the dark) at –20°C (stable for months). Cell staining solution 1 g/liter Coomassie brilliant blue G 40% (v/v) ethanol 10% (v/v) acetic acid 50% (v/v) H2 O Store indefinitely at room temperature NBT solution, 100× 50 mg/ml nitroblue tetrazolium 70% (v/v) dimethylsulfoxide (DMSO)


continued Current Protocols in Human Genetics


100 mM Tris·Cl, pH 8.5 (APPENDIX 2D) Store in foil-wrapped glass container (i.e., in the dark) at −20°C (stable for months) COMMENTARY Background Information A few eukaryotic viruses integrate into the host cell genome as part of their normal life cycle. This process is very efficient for retroviruses and provides a useful tool for gene transfer applications. Retrovirus integration is specific with respect to the virus genome, resulting in a characteristic structure with terminal repeats flanked by cellular DNA. Integration occurs at nearly random sites in the host genome with only a minor alteration at the site of integration, consisting of a short duplication of 5 bases at the site of integration. Retroviral vectors have been developed to take advantage of the efficient entry and integration properties of retroviruses. Early vectors were propagated in the presence of replication-competent or “helper” retroviruses, but this allowed vector and helper virus to spread after infection; because many retroviruses are pathogenic, this prevented use of these vectors for human gene transfer. These problems were solved by the development of retrovirus packaging cell lines that provide all of the viral proteins required for vector transmission but do not produce helper virus (Miller, A.D., 1990; Miller, A.D., et al., 1993). Packaging lines still have the potential to produce helper virus by recombination events between the vector, helper virus sequences in the packaging cells, and endogenous retroviral elements, but the frequency of helper virus production in the current generation of packaging cells is very low to nonexistent. No viral proteins need be synthesized during vector entry and integration into target cells, and thus all viral protein–coding regions can be removed from retroviral vectors without affecting gene transfer rates.



Human gene therapy applications Retroviral vector use for human gene therapy is generally limited to modification of human somatic cell ex vivo, followed by return of the cells to the human subject. This limitation is due to the fact that retroviral vectors only transduce replicating cells (Miller, D.G., et al., 1990), and many cells in the human body replicate slowly or not at all. Examples of cell types that have been modified ex vivo and returned to human subjects include T cells and hematopoietic stem cells. Other suitable targets are skin fibroblast and skin keratinocytes, which can readily be grown and transduced ex

vivo and returned to human subjects as skin grafts. The techniques described here have been used to generate vector stocks for human gene therapy. Additional work required prior to vector use in humans involves extensive testing of the vector-producing cells for the absence of adventitious agents, thorough testing for the absence of helper virus production, and testing of the vector in animals to detect potential toxicity, which can be due to the particular cDNA insert used. Standard laboratory culture practices can yield cell lines that pass these additional tests for human use. For example, the PA317 packaging cell line (Table 12.5.1) used for vector production to treat adenosine deaminase (ADA) deficiency in humans (Blaese et al., 1995; Kohn et al., 1995) was made using the Basic Protocol under standard laboratory conditions (Hock et al., 1989). Only one clonal cell line was tested for human use and it passed all United States Food and Drug Administration (FDA) testing requirements. Ultimately, however, vector stocks for use in humans must be produced using current Good Manufacturing Practices (cGMP) established by the FDA and similar international regulatory agencies, which require specialized facilities and procedures. Several examples of successful use of retroviral vectors for treatment of human genetic disease are available. For example, sustained correction of X-linked severe combined immunodeficiency (SCID), due to defects in the common γ (γc) cytokine receptor subunit, has been achieved by ex vivo gene therapy using a retroviral vector carrying the γc gene (Cavazzana-Calvo et al., 2000; Hacein-BeyAbina et al., 2002). In this study, a retroviral vector encoding the γc protein, produced using ψCRIP amphotropic packaging cells (Table 12.5.1), was used to transduce autologous CD34+ bone marrow stem cells ex vivo, which were then returned to the patient. Four of five patients treated showed dramatic clinical improvement. Unfortunately, T cell leukemias were later found in four of nine successfully treated patients, which correlated with integration of the gene therapy vector near proto-oncogenes, including LMO2 (Hacein-Bey-Abina, 2003, 2008). Three of the affected patients subsequently responded to chemotherapy to ablate the leukemic cells, while one died. Current Protocols in Human Genetics

A better outcome was observed in another study of gene therapy treatment for γcdeficient SCID (Gaspar et al., 2011a). In this study, a retroviral vector encoding the γc protein, produced using PG13 packaging cells (Table 12.5.1), was used to transduce autologous CD34+ bone marrow stem cells ex vivo, which were then returned to the patient. Ten of ten treated patients showed clinical improvement, but one of these developed an acute T cell leukemia, again associated with vector insertion near the LMO2 proto-oncogene (Howe et al., 2008). While some view the adverse leukemic events in this and the previous study of gene therapy for γc-deficient SCID as an argument against further use of retroviral vectors for human gene therapy, it seems that an 18 out of 20 patient clinical benefit rate for this severe disease, with many complete cures, is actually a rather exciting result, and further work is underway to reduce the incidence of uncontrolled cell growth. Another example of successful use of retroviral vectors for treatment of human genetic disease is for treatment of SCID due to defects in the adenosine deaminase (ADA) gene. In a recent study (Aiuti et al., 2009), ADA-deficient patients were treated with nonmyeloablative conditioning followed by infusion of autologous CD34+ bone marrow cells transduced with a retroviral vector–carrying gene encoding ADA. Here, the vector used was the LXSN vector (Fig. 12.5.2) into which a human ADA cDNA was inserted, such that both ADA and neomycin phosphotransferase are expressed, and the vector was made using GP+envAm12 retrovirus packaging cells (Table 12.5.1). All of the ten patients treated showed clinical benefit with no evidence for abnormal lymphoproliferation. In another recent study of gene-therapy treatment for ADA-deficient SCID (Gaspar et al., 2011b), ADA-deficient patients again received CD34+ bone marrow cells transduced with a retroviral vector carrying a gene encoding ADA. Here, the vector contained only an ADA gene, and the vector was made by using PG13 retrovirus packaging cells (Table 12.5.1). All four patients who showed bone marrow reconstitution, of a total of six treated patients, also showed clinical benefit with no evidence of abnormal lymphoproliferation.

perform a parallel procedure with the “empty” vector. This can reveal inefficiencies in production due to insertion of particular cDNAs into retroviral vectors. For example, insertion of the clotting factor VIII cDNA into a retroviral vector reduces the titer of the virus produced by 100-fold (Lynch et al., 1993). This effect has been found to be due not simply to the large size of the factor VIII cDNA (a vector with another insert of similar size was transmitted efficiently), but also to sequences within the coding region that decrease RNA accumulation. The purity of the DNA used is critical for the calcium phosphate–mediated DNA transfection steps. A clumpy DNA precipitate is one sign of impure DNA. CsCl-gradient or Qiagen-column DNA purification yields adequately pure DNA for transfection. It can be difficult to generate stable packaging cell lines that produce vectors carrying toxic genes or genes that inhibit cell growth. In this case the best method for vector production is by transient transfection (see Alternate Protocol 2). A key step in generating stable vectorproducing cell lines is Southern blot hybridization analysis (UNIT 2.7) of clones for the presence of unrearranged vector sequences. This should be performed by cutting the cellular DNA with restriction endonucleases that cut in the vector long terminal repeats (LTRs) to show the size of the integrated provirus. Because retroviruses go through an RNA intermediate, unanticipated splicing events can result in deletion of sequences. This and other problems involving vector rearrangement are revealed by Southern blot hybridization. Northern blot hybridization can also be performed to determine if the transcription pattern is as planned. Lastly, direct sequencing of the vector integrated into the genomic DNA packaging cells can be performed to confirm the exact sequence of the integrated vector. In cases where the desired protein is not expressed or a predicted phenotype is not observed following vector transfer, these analyses can determine the nature of the problem. It is important to freeze clonal cell lines in liquid nitrogen soon after isolation, because packaging function (retroviral titer) can decrease with passage of the packaging cells.

Critical Parameters and Troubleshooting

Anticipated Results

When applying the techniques described in this chapter to the production of retroviral vectors with inserted genes, it is always helpful to

Transient transfection of retroviral vectors into packaging cell lines generally results in vector titers between 103 and >105



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transducing units/ml. Stable cell lines typically produce vector titers of 105 to 107 transducing units/ml.

Doty, R.T., Sabo, K.M., Chen, J., Miller, A.D., and Abkowitz, J.L. 2010. An all-feline retroviral packaging system for transduction of human cells. Hum. Gene Ther. 21:1019-1027.

Time Considerations

Engebrecht, J., Brent, R., and Kaderbhai, M. A. 1991. Minipreps of plasmid DNA. Curr. Protoc. Mol. Biol. 15:1.6.1–1.6.10.

The time required for generation of stable vector-producing clones is typically about a month. Producing virus by transient transfection of vector plasmids into packaging cells takes 3 days from the time the packaging cells are seeded. Cloning a cDNA in a retroviral vector requires a few days. Preparing the precipitate for calcium phosphate–mediated transfection requires 1 hr. Titering vectors carrying selectable markers takes 8 to 10 days. The marker rescue assay requires 2 weeks to generate nonproducer cells and an additional 3 weeks for the assay. Staining cultured cells for alkaline phosphatase activity requires 5 hr.

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Danos, O. and Mulligan, R.C. 1988. Safe and efficient generation of recombinant retroviruses with amphotropic and ecotropic host ranges. Proc. Natl. Acad. Sci. U.S.A. 85:6460-6464.

Fields-Berry, S.C., Halliday, A.L., and Cepko, C.L. 1992. A recombinant retrovirus encoding alkaline phosphatase confirms clonal boundary assignment in lineage analysis of murine retina. Proc. Natl. Acad. Sci. U.S.A. 89:693-697. Gaspar, H.B., Cooray, S., Gilmour, K.C., Parsley, K.L., Adams, S., Howe, S.J., Al Ghonaium, A., Bayford, J., Brown, L., Davies, E.G., Kinnon, C., and Thrasher, A.J. 2011a. Long-term persistence of a polyclonal T cell repertoire after gene therapy for X-linked severe combined immunodeficiency. Sci. Transl. Med. 3:97ra79. Gaspar, H.B., Cooray, S., Gilmour, K.C., Parsley, K.L., Zhang, F., Adams, S., Bjorkegren, E., Bayford, J., Brown, L., Davies, E.G., Veys, P., Fairbanks, L., Bordon, V., Petropoulou, T., Kinnon, C., and Thrasher, A.J. 2011b. Hematopoietic stem cell gene therapy for adenosine deaminase-deficient severe combined immunodeficiency leads to long-term immunological recovery and metabolic correction. Sci. Transl. Med. 3:97ra80. Hacein-Bey-Abina, S., Le Deist, F., Carlier, F., Bouneaud, C., Hue, C., De Villartay, J.P., Thrasher, A.J., Wulffraat, N., Sorensen, R., Dupuis-Girod, S., Fischer, A., Davies, E.G., Kuis, W., Leiva, L., and Cavazzana-Calvo, M. 2002. Sustained correction of X-linked severe combined immunodeficiency by ex vivo gene therapy. N. Engl. J. Med. 346:1185-1193. Hacein-Bey-Abina, S., Von Kalle, C., Schmidt, M., McCormack, M.P., Wulffraat, N., Leboulch, P., Lim, A., Osborne, C.S., Pawliuk, R., Morillon, E., Sorensen, R., Forster, A., Fraser, P., Cohen, J.I., de Saint Basile, G., Alexander, I., Wintergerst, U., Frebourg, T., Aurias, A., StoppaLyonnet, D., Romana, S., Radford-Weiss, I., Gross, F., Valensi, F., Delabesse, E., Macintyre, E., Sigaux, F., Soulier, J., Leiva, L.E., Wissler, M., Prinz, C., Rabbitts, T.H., Le Deist, F., Fischer, A., and Cavazzana-Calvo, M. 2003. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 302:415-419. Hacein-Bey-Abina, S., Garrigue, A., Wang, G.P., Soulier, J., Lim, A., Morillon, E., Clappier, E., Caccavelli, L., Delabesse, E., Beldjord, K., Asnafi, V., MacIntyre, E., Dal Cortivo, L., Radford, I., Brousse, N., Sigaux, F., Moshous, D., Hauer, J., Borkhardt, A., Belohradsky, B.H., Wintergerst, U., Velez, M.C., Leiva, L., Sorensen, R., Wulffraat, N., Blanche, S., Bushman, F.D., Fischer, A., and Cavazzana-Calvo, M. 2008. Insertional oncogenesis in 4 patients after retrovirusmediated gene therapy of SCID-X1. J. Clin. Invest. 118:3132-3142. Heilig, J., Elbing, K. L., and Brent, R. 1998. Largescale preparation of plasmid DNA. Curr. Protoc. Mol. Biol. 41:1.7.1-1.7.16.



Hock, R.A., Miller, A.D., and Osborne, W.R.A. 1989. Expression of human adenosine deaminase from various strong promoters after gene transfer into human hematopoietic cell lines. Blood 74:876-881. Howe, S.J., Mansour, M.R., Schwarzwaelder, K., Bartholomae, C., Hubank, M., Kempski, H., Brugman, M.H., Pike-Overzet, K., Chatters, S.J., de Ridder, D., Gilmour, K.C., Adams, S., Thornhill, S.I., Parsley, K.L., Staal, F.J., Gale, R.E., Linch, D.C., Bayford, J., Brown, L., Quaye, M., Kinnon, C., Ancliff, P., Webb, D.K., Schmidt, M., von Kalle, C., Gaspar, H.B., and Thrasher, A.J. 2008. Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene therapy of SCID-X1 patients. J. Clin. Invest. 118:31433150. Kaleko, M., Rutter, W.J., and Miller, A.D. 1990. Overexpression of the human insulinlike growth factor I receptor promotes liganddependent neoplastic transformation. Mol. Cell. Biol. 10:464-473. Kohn, D.B., Weinberg, K.I., Nolta, J.A., Heiss, L.N., Lenarsky, C., Crooks, G.M., Hanley, M.E., Annett, G., Brooks, J.S., El-Khoureiy, A., Lawrence, K., Wells, S., Moen, R.C., Bastian, J., Williams-Herman, D.E., Elder, M., Wara, D., Bowen, T., Hershfield, M.S., Mullen, C.A., Blaese, R.M., and Parkman, R. 1995. Engraftment of gene-modified umbilical cord blood cells in neonates with adenosine deaminase deficiency. Nat. Med. 1:1017-1023.

Miller, A.D. and Metzger, M.J. 2011. APOBEC3mediated hypermutation of retroviral vectors produced from some retrovirus packaging cell lines. Gene Ther. 18:528-530. Miller, A.D. and Rosman, G.J. 1989. Improved retroviral vectors for gene transfer and expression. Biotechniques 7:980-990. Miller, A.D., Garcia, J.V., von Suhr, N., Lynch, C.M., Wilson, C., and Eiden, M.V. 1991. Construction and properties of retrovirus packaging cells based on gibbon ape leukemia virus. J. Virol. 65:2220-2224. Miller, A.D., Miller, D.G., Garcia, J.V., and Lynch, C.M. 1993. Use of retroviral vectors for gene transfer and expression. Methods Enzymol. 217:581-599. Miller, D.G., Adam, M.A., and Miller, A.D. 1990. Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection. Mol. Cell. Biol. 10:4239-4242. Miller, D.G., Edwards, R.H., and Miller, A.D. 1994. Cloning of the cellular receptor for amphotropic murine retroviruses reveals homology to that for gibbon ape leukemia virus. Proc. Natl. Acad. Sci. U.S.A. 91:78-82. Morgenstern, J.P. and Land, H. 1990. Advanced mammalian gene transfer: High titer retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucl. Acids Res. 18:3587-3596.

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Markowitz, D., Goff, S., and Bank, A. 1988b. Construction and use of a safe and efficient amphotropic packaging cell line. Virology 167:400406. Miller, A.D. 1990. Retrovirus packaging cells. Hum. Gene Ther. 1:5-14. Miller, A.D. 1997. Development and applications of retroviral vectors. In Retroviruses (J.M. Coffin, S.H. Hughes, and H.E. Varmus, eds.) pp. 437473. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Miller, A.D. and Buttimore, C. 1986. Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production. Mol. Cell. Biol. 6:2895-2902. Miller, A.D. and Chen, F.C. 1996. Retrovirus packaging cells based on 10A1 murine leukemia virus for production of vectors that use multiple receptors for cell entry. J. Virol. 70:5564-5571.

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Struhl, K. 1991. Subcloning of DNA fragments. Curr. Protoc. Mol. Biol. 13:3.16.1-3.16.2. Takahara, Y., Hamada, K., and Housman, D.E. 1992. A new retrovirus packaging cell for gene transfer constructed from amplified long terminal repeat-free chimeric proviral genes. J. Virol. 66:3725-3732. Wolgamot, G., Rasko, J.E., and Miller, A.D. 1998. Retrovirus packaging cells expressing the Mus dunni endogenous virus envelope facilitate transduction of CHO and primary hematopoietic cells. J. Virol. 72:10242-10245. Yang, Y., Vanin, E.F., Whitt, M.A., Fornerod, M., Zwart, R., Schneiderman, R.D., Grosveld, G., and Nienhuis, A.W. 1995. Inducible, high-level production of infectious murine leukemia retroviral vector particles pseudotyped with vesicular stomatitis virus G envelope protein. Hum. Gene Ther. 6:1203-1213.




Ping-pong amplification of a retroviral vector achieves high-level gene expression: human growth hormone production.

Large-scale clinical-grade retroviral vector production in a fixed-bed bioreactor.

Improved gene expression by a U3-based retroviral vector.

Stimulation of retroviral vector infection of murine hematopoietic progenitors.

Optimization of retroviral vector-mediated gene transfer into endothelial cells in vitro.

Retroviral vector-mediated gene transfer into hematopoietic cells: prospects and issues.

Efficient gene expression in mammalian cells from a dicistronic transcriptional unit in an improved retroviral vector.

Restoration of arylsulphatase A activity in human-metachromatic-leucodystrophy fibroblasts via retroviral-vector-mediated gene transfer.

Development of a retroviral vector for inducible expression of transforming growth factor beta 1.

Investigation of the mutagenic specificity of X-rays using a retroviral shuttle vector in CHO cells.

Correction of alpha-L-fucosidase deficiency in fucosidosis fibroblasts by retroviral vector-mediated gene transfer.

Use of a retroviral vector for expressing antisense RNA of proopiomelanocortin in AtT-20 cell line.

Radial mobility and cytotoxic function of retroviral replicating vector transduced, non-adherent alloresponsive T lymphocytes.

In vivo gene transfer with retroviral vector-producer cells for treatment of experimental brain tumors.

A self-inactivating gamma-retroviral vector reduces manifestations of mucopolysaccharidosis I in mice.

A spleen necrosis virus-based retroviral vector which expresses two genes from a dicistronic mRNA.

Optimization of lentiviral vector production using polyethylenimine-mediated transfection.

Leukemia inhibitory factor improves survival of retroviral vector-infected hematopoietic stem cells in vitro, allowing efficient long-term expression of vector-encoded human adenosine deaminase in vivo.

Enhanced lentiviral vector production in 293FT cells expressing Siglec-9.

Retroviral superantigens.

Maintaining therapeutic activity in the operating room: compatibility of a gamma-retroviral replicating vector with clinical materials and biofluids.

Clonal Dominance With Retroviral Vector Insertions Near the ANGPT1 and ANGPT2 Genes in a Human Xenotransplant Mouse Model.

Stable RNA secondary structure in a retroviral vector insert terminates reverse transcriptase elongation in vitro but not in cultured cells.

Restoration of arylsulphatase B activity in human mucopolysaccharidosis-type-VI fibroblasts by retroviral-vector-mediated gene transfer.