Advances in recombinant retroviruses for gene delivery

Advanced Drug Deliver)' Reviews, 12 (1993) 143 158

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© 1993 Elsevier Science Publishers B.V. All rights reserved. / 0169-409X/93/$24.00

A D R 00130

Advances in recombinant retroviruses for gene delivery Jeffrey R. Morgan a, Ronald G. Tompkins a and Martin L. Yarmush a'b aSurgical Services, Massachusetts General Hospital, Shriners Burns Institute, Boston, MA and bDepartment o]" Chemical and Biochemical Engineering, Rutgers University, Piscataway, N J, USA (Received September 22, 1992) (Accepted O c t o b e r 10, 1992)

K e y words: G e n e therapy; R e c o m b i n a n t retrovirus; G e n e transfer

Contents Summary .................................................................................................................

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I. I n t r o d u c t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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il. Life cycle o f replication-competent wild-type retrovirus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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lIl. D e v e l o p m e n t o f safe r e c o m b i n a n t retroviruses for gene transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. P a c k a g i n g sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. P a c k a g i n g cell lines for the p r o d u c t i o n o f r e c o m b i n a n t retrovirus . . . . . . . . . . . . . . . . . . . . . . . . . . 3. H o s t range o f r e c o m b i n a n t retrovirus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. C o n f i g u r a t i o n s o f r e c o m b i n a n t retroviral vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Steps to retroviral-mediated gene transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

146 146 147 147 147 149

IV. G e n e s expressed by retroviral-mediated gene transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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V. Tissues modified using retroviral-mediated gene transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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VI. Safety issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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VII. F u t u r e directions for retroviral-mediated gene transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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VIII. C o n c l u s i o n s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Correspondence to: Jeffrey R. Morgan, Ph.D., Shriners Burns Institute Research Center, Building 1400, Cambridge, MA 02142, USA.

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Summary A naturally occurring murine retrovirus has been adapted to serve as a safe and effective vehicle for the transfer of genes to human cells. This process of gene transfer, known as "retroviral-mediated gene transfer", facilitates the efficient introduction and expression of stable gene copies in human diploid cells from a variety of tissues. Using this approach, numerous genes have been introduced into human cells and attention has been focused on the use of this technology to achieve therapeutic goals. Gene transfer technology has the potential for the correction of genetic diseases as well as the development of novel solutions to the problems involved with delivery of therapeutic proteins.

I. Introduction Recent technological advances in gene transfer coupled with a precise understanding of the molecular basis of many diseases have provided the tools necessary for a new approach to the treatment of both inherited and acquired diseases. This approach, called "gene therapy", has the potential of providing longterm and cost-effective treatment for some inherited diseases which have few other therapeutic options, as well as providing novel methods for the delivery of therapeutic proteins for the treatment of many acquired diseases. To date, as many as 17 gene therapy clinical protocols have been approved [reviewed by Freeman, S.M. et al., this issue]. These pioneering protocols are the beginning of what many believe will be numerous clinical applications of gene transfer technologies. The gene transfer method of the majority of these approved protocols, and much of the current research devoted to gene therapy, is based on the use of recombinant retroviruses (retroviral-mediated gene transfer). While there are a variety of viral-mediated and physical/chemical methods for gene transfer into mammalian cells (most of which are reviewed in subsequent articles in this issue), retroviral-mediated gene transfer has some distinct advantages which have to date made it the method of choice. Recombinant retroviruses (1) are able to introduce genes into many cell types, including primary cells, (2) are stably integrated into chromosomal DNA, (3) can faithfully transmit encoded genes without rearrangements, and (4) are able to simultaneously introduce genes into large numbers of cells as would be required for many clinical applications. Potential disadvantages of retroviral-mediated gene transfer are that recombinant retroviruses; (1) are able to introduce genes only into cells which are dividing; and (2) are limited to the size of gene that can be packaged. Other methods of gene transfer without these specific limitations have been developed and are being applied in areas where these limitations preclude the use of recombinant retroviruses. The purpose of this review is to outline the key events in the development of recombinant retroviruses for gene transfer, starting with an overview of the retroviral life cycle. The fundamental technology of retroviral-mediated gene transfer and the current areas of applications will be described. Lastly, future directions to the technology of retroviral-mediated gene transfer will be discussed.

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II. Life cycle of replication competent wild-type retrovirus Most retroviral-mediated gene transfer systems are based on a modification of the Moloney murine leukemia virus (Mo-MLV) and what follows is a brief description of the life cycle of wild type, replication-competent virus [see Ref. 1 for a more detailed description). Mo-MLV is an enveloped virus composed of several types of viral encoded proteins including: gag, a group of polypeptides involved in many functions including virus assembly; pol, a multi-functional protein with enzymatic activities important for reverse transcription and integration; and env, a glycoprotein on the surface of virions that determines the host range of the virus through a specific interaction with a receptor on the surface of the target cells. Encapsulated in the virion is an R N A genome. The life cycle of Mo-MLV begins when the virion adsorbs to cells via the specific

A.

5'

An

5'

B.

.- . .

-'''--.

An

"CAAT""TATA"Poly A site

enhancer

7272 I

I

I

U3

R

5'~

U5

_--

_-------'----"

5'

A

5'

n

An

Fig. 1. (A) Diagram of the structure of an integrated copy of a wild-type retroviral genome. Shown are the locations of the long terminal repeat sequences (LTR), the sequences encoding the gag, pol, and env proteins and the (q0) sequences required for packaging. Also shown are the major R N A transcripts from the retroviral genome, the packaged full-length R N A from which the gag-pol proteins are translated and the unpackaged spliced R N A which encodes the env protein. (B) Diagram of a typical recombinant retroviral vector and the genetic elements found in the LTR. Shown is an expanded view of the U3, R and U5 regions of the L T R and the location of the enhancer sequences, the C A A T and T A T A sequences of the promoter and the start of transcription. Also shown is the poly A site which is most active at the 3'-LTR. Diagram of a typical vector depicts the location of two inserted genes and a promoter fragment as well as the predicted R N A s transcribed from the vector, a packaged full-length transcript from which gene 1 is translated and the shorter transcript starting at the internal promoter from which gene 2 is translated.

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interaction of the env protein of the virion and a receptor on the cell surface. Within minutes, the virion is internalized, uncoated and the RNA genome converted to double-stranded D N A by the reverse transcriptase activity of the pol protein enzyme found in the virion. This DNA genome is then integrated at random sites in transcriptionally-active chromatin domains. The integrated genome, also called the "provirus", is capable of expressing the viral proteins necessary for virion production (i.e., gag-pol-env). Two major RNAs are transcribed from the integrated provirus, a full-length transcript encoding gagpol-env from which only the gag and pol proteins are translated and a spliced RNA encoding only env sequences from which the env protein is translated (Fig. 1A). The full-length RNA transcript which encodes all the viral proteins is also the genomic RNA and thus is packaged into the assembling virion. These full-length RNA molecules are packaged as dimers by means of specific interactions of the gag protein(s) with a packaging sequence in the RNA. In addition to the two RNA molecules and gag proteins, the virion contains approximately 50 molecules of the pol protein as well as env proteins which protrude from a lipid bilayer derived from the plasma membrane. Newly assembled infectious virus buds from the plasma membrane without adversely affecting cell function. III. Development of safe recombinant retroviruses for gene transfer

Central to the development of retroviruses as safe vehicles for gene transfer was the identification of sequences necessary for the selective packaging of RNAs and the development of packaging cell lines. These two breakthroughs provided the means by which stocks of recombinant retroviruses, free of wild-type virus or infectious helper virus, could be produced. These recombinant retroviruses are replication defective but still able to transmit a gene of interest to a target cell. III.1. Packaging sequences

The packaging of retroviral RNAs into virions is mediated by specific c/s-acting packaging sequences also known as "(q0-sequences" [2,3]. The deletion of these sequences from a retroviral genome prevented the packaging of this RNA into virions. Conversely, the addition of these sequences to a recombinant vector promoted the packaging of recombinant RNAs into virions. Originally, the 4sequences were mapped to a 350-base-pair site (Bal I to Pst I) located between the splice donor site and the start codon of the gag protein. Deletion of this sequence blocked the packaging of the RNA, and the inclusion of this sequence in a recombinant retroviral vector resulted in the selective packaging of recombinant RNAs into virions [2]. These q~-sequences could be inserted at various sites of the retroviral genome and still promote the packaging of recombinant RNAs [4]. Later reports have demonstrated that the optimal packaging sequence extends outside this region, and a fragment which overlaps with the amino terminal sequences of the gag protein has been shown to promote the most efficient packaging of recombinant RNAs [5]. To block the translation of the gag protein from RNAs transcribed from the recombinant vector, point mutations have been incorporated into these extended packaging sequences [5,6].

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111.2 Packaging cell lines for the production of recombinant retrovirus Another important milestone in the development of retroviruses for safe gene transfer was the generation of packaging cell lines which provide in trans the viral proteins necessary for the assembly of virions. These packaging cell lines, derived from NIH-3T3 fibroblasts, provide the gag-pol-env proteins necessary to form virions but are incapable of packaging into virions the transcript s encoding these proteins. The first attempts to create this type of packaging cell line utilized a wildtyp6 proviral genome that was deleted of its V-sequence [2,3]. Stable transfection of this crippled wild-type genome into NIH-3T3 cells led to the isolation of cell lines that produce virions. As predicted, the virions were void of viral RNA and were shown to be able to package other transcripts from recombinant retroviral vectors that contained the q~-sequences. Most importantly, recombinant retroviruses, produced by the packaging cell lines, were shown to be replication-defective, free of wild-type virus, and able to transmit the recombinant vector to target cells. One problem with these first packaging cell lines was that from time to time wildtype replication virus appeared in some packaging cell lines. The pathway for the generation of this wild-type replication-competent virus is not clear but probably occurred by homologous recombination event(s). Improved packaging cell lines have been made and the chances of homologous recombination reduced by expressing the gag, pol and env genes on separate expression plasmids with additional mutations in the regulatory sequences in the 3' long terminal repeats [68]. These packaging cell lines and recombinant virus produced by these cell lines are now widely used and have been approved for clinical trials [9,10]. 111.3. Host range of recombinant retrovirus The ability of retroviruses to infect the cells of certain species and not others is known as the "host range" and is classified into host range subgroups known as "tropism" [1]. Three basic classes of virus tropism have been exploited for gene transfer. Ecotropic viruses are able to infect only murine cells. Xenotropic viruses are unable to infect murine cells, but able to infect cells from a wide variety of other mammalian species. Amphotropic viruses are able to infect murine cells as well as ceils from a wide variety of mammalian species including human cells. Viruses of each tropism infect cells via separate and distinct cellular receptors, and the tropism of particular retroviruses is determined solely by its env protein. Accordingly, packaging cell lines which produce recombinant retroviruses with either ecotropic, xenotropic or amphotropic host ranges have been developed by transfecting the appropriate env gene into the packaging cell line [6,7,11]. For clinical applications, the amphotropic recombinant retroviruses are most commonly used since they can infect human cells [9,10]. III.4. Configurations of recombinant retroviral vectors In its simplest form a retroviral vector is a modified version of the wild-type provirus in which the sequences encoding gag, pol and env are removed and c D N A sequences encoding a gene of interest and/or promoter sequences are inserted in a

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cloning site [12]. The backbone of the recombinant retroviral vector contains the genetic elements necessary for the transcription, packaging, reverse transcription, integration, and expression of the inserted gene of interest. Many of these functions are encoded by the repeated sequences at each end of the retroviral vector called "long terminal repeats" (LTR)(Fig. 1B). Within the 594-bp LTRs, there are three distinct regions: (a) a U3 region which encodes a promoter with " C A A T " and " T A T A " boxes and two 72-bp repeat enhancers; (b) an R-region which encodes the polyadenylation signal; and (c) a U5 region with sequences unique to the 5'-end. Just downstream from the 5'-LTR are the t R N A primer binding sites critical for reverse transcription and the ql-packaging sequences which include a portion of the g a g gene sequences and have been termed "q~ + " [5]. A configuration of a retroviral vector commonly used and one of the first developed is shown in Fig. lB. This vector expresses two genes, one from an R N A transcribed from the LTR and the other from an R N A transcribed from a promoter/enhancer located in the middle of the vector. Typically, these first vectors were used to express the gene of interest and a dominant selectable marker gene such as the n e o gene (neomycin phosphotransferase) which confers resistance to the antibiotic, G418. After transduction of a population of cells with the recombinant retrovirus, the cells were grown in selective media (containing G418) so that only those cells expressing the recombinant retrovirus survived. This population of drugresistant cells also expressed the gene of interest. Other dominant selectable marker genes used include the herpes virus thymidine kinase gene [13,14], a mutant form of the dihydrofolate reductase gene [15], the hygromycin B phosphotransferase gene [16] and the histidinol dehydrogenase gene [17]. More recently, several variations of this vector configuration have been developed. The gene of interest has been expressed from either the LTR or the internal promoter and varied promoter/enhancer combinations have been used to express the internal gene. In addition, vectors which express only the gene of interest and no selectable marker gene have been developed [18]. The advantage of these vectors, particularly in clinical applications, is that the vector encodes only the therapeutic gene of interest and not a selectable marker gene which could be a source of antigenic protein in transplanted cells. Other configurations of retroviral vectors have been developed. "Reverse orientation" vectors have a transcriptional unit with the gene of interest, including a promoter/enhancer and a polyadenylation site, inserted in the vector in the opposite orientation of transcription from the LTR. Reverse orientation vectors have been used to introduce genomic clones of genes (complete with introns) into cells and have exhibited tissue-specific gene expression [19]. "Double copy" vectors have been developed in which the gene of interest is inserted within the U3 region of the 3'-LTR [20]. Additional modifications have been made which disable the vector once it is inserted into the target cells. These vectors called "suicide vectors" or "selfinactivating vectors" have deletions in the promoter/enhancer region of the 3'-LTR of the vector [21]. During reverse transcription, the sequences of the 3'-LTR are used as a template for both the 3'- and 5'-LTRs of the newly formed proviral D N A that integrates into the chromosomal DNA. Therefore, deletions or mutations in the 3'LTR of the retroviral vector D N A will be copied to both LTRs during reverse

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transcription. Deletion of the control elements in both LTRs of the integrated vector reduces the transcription of full-length RNAs which could be repackaged into virions. In addition, the potential for transcriptional activation of cellular oncogenes near the integrated retrovirus is greatly reduced since the control elements in the LTRs are disabled. 111.5. Steps to retroviral-mediated gene transfer The production of recombinant retrovirus transmitting a gene is a multi-step process beginning with the insertion of the gene of interest into a cloning site in the recombinant retroviral vector DNA. This construct is then introduced into the packaging cell line by calcium phosphate transfection and stable transfectants are selected. The recombinant retroviral vector D N A in these cell lines is transcribed to generate a full-length genomic R N A initiated from the promoter in the U3 region of the 5'-LTR and terminating in the polyadenylation site in the R-region of the 3'LTR. This genomic R N A is packaged by the retroviral proteins provided by the packaging cell line to form virions that bud from the plasma membrane. These transfected packaging cell lines continuously shed recombinant retrovirus into the media and multiple stocks of recombinant retrovirus can be harvested, frozen, and tested. To effect gene transfer, a stock of recombinant retrovirus is added to cultures of the target cells wherein the virus is rapidly adsorbed on the cell surface by a specific receptor. This virus/receptor interaction is the basis by which recombinant retroviruses can simultaneously introduce genes into large numbers of cells since all cells have the receptor and each cell can have as many as 1 × 105 receptors/cell [1]. In addition, receptors for amphotropic virus are found on nearly all cell types which accounts for the ability of recombinant retrovirus to introduce genes into a wide variety of tissues. Polycations, such as polybrene (1,5-dimethyl-l,5-diazaundecamethylene polymethobromide) and protamine have been shown to promote the adsorption of virus as much as tenfold and are commonly used [12]. Soon after adsorption, the recombinant retrovirus is internalized, uncoated, its packaged R N A genome reverse-transcribed, and the resulting proviral D N A integrated into random sites of the chromosomal D N A of the target cell. Integrated recombinant retroviral D N A and its encoded gene of interest are stable and transferred to daughter cells as any other autosomal genes [1]. In addition, because the integration of the retroviral vector molecule is mediated by virion-associated pol protein acting on specific sequences in the LTRs, integration always occurs at the same location on the vector molecule, thus preventing disruption of the sequences of the gene of interest [1]. This site specificity accounts for the ability of recombinant retroviruses to faithfully transmit genes without rearrangements. Once integrated, the recombinant retroviral D N A is transcribed and the encoded gene(s) expressed in the target cell. Since the recombinant vector does not encode the gag, pol and env proteins, the viral life cycle is stopped and there is no further production of virus. Recombinant retroviruses can transduce only those cells which are replicating. Attempts to transduce cells in stationary phase have been unsuccessful even if the stationary cells are stimulated to grow 6 hours after infection [22]. On the other

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TABLE I GENES EXPRESSED U S I N G R E C O M B I N A N T RETROVIRUSES Gene

Abbreviation

Origin*

Reference

Adenine phosphoribosyl transferase Adenosine deaminase Adenovirus early region 1 Alpha-hemoglobin Alpha-l-antitrypsin Arginnosuccinate synthetase Arylsulphatase A Arylsulphatase B

APRT ADA E1A ~-globin alpha 1AT AS ASA ASB

M M V M M M M M

47 29,48 49 50 51,52 53 54 55

Basic fibroblast growth factor Beta chorionic gonadotropin Beta-hemoglobin Beta-tryptophan synthase Beta-galactosidase Beta-glucuronidase

bFGF b-hCG fl-globin trpB lacZ

M M M B B M

56 18 28,57 17 58 59

CD-5 CD-18 Chloramphenicol acetyltransferase Clotting factor IX Clotting factor VIII Collagen Cystic fibrosis transmembrane Conductance regulator Cytochrome P3-450

CD-5 CD-18 CAT factorlX factor VIII CFTR P450

M M B M M M M M

60 61 37 31,62 25 63 64 65

Dihydrofolate reductase

DHFR

M

15

EB virus nuclear antigen Epidermal growth factor receptor Erythropoietin Erythropoietin receptor Estrogen receptor

EBNA EGFR EPO EPOR

V M M M M

66 67 68 69 70

Fibronectin Firefly luciferase

FN

M I

71 72

Gamma-interferon Glucocerebrosidase Glucocorticoid receptor Glucose transporter Granulocyte-macrophage colony stimulating factor Growth hormone

7-IFN

M M M M M M

39 73 74 75 76 32

GM-CSF GH

*Abbreviations used for origin of genes: M = mammalian: B = bacterial; V = viral; I = insect.

hand, it is possible to increase the frequency of transduction by stimulating target cells to divide with growth factors prior to infection [23].

IV. Genes expressed by retroviral-mediated gene transfer A wide variety of genes have been expressed using retroviral-mediated gene transfer. A list of genes expressed and representative publications is given in Table I. Most of the genes expressed to date are the normal alleles of genes of wellcharacterized genetic diseases such as adenosine deaminase for severe combined immunodeficiency. The obvious therapeutic intent is to correct the genetic disease by

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TABLE I

(continued) Gene

Abbreviation

Origin*

Reference

Hepatitis B antigen Hepatitis delta virus structural antigen Herpes thymidine kinase Histidinol dehydrogenase Histocompatability antigens Human immunodeficiency virus glycoprotein 120 Hygromycin B phosphotransferase Hypoxanthine phosphoribosyl transferase

HBAg HDAg tK hisD HLA gpl20 hygro HPRT

V V V B M V B M

77 78 13,14 17 79 80 16 81

Immunoglobulin light chain Influenza hemagglutinin Influenza virus nucleoprotein gene Insulin Interleukin-2 Interleukin-5 Interleukin-2 receptor Interleukin-3 Interleukin-6

IgG HA NP IL-2 IL-5 FL-2R IL-3 IL-6

M V V M M M M M M

82 83 84 85 86 87 88 89,90 91

Low-density lipoprotein receptor Lymphocyte antigen

LDLR Leu-1

M M

35 92

Multi-drug resistance Myelin-associated glycoprotein

MDR MAG

M M

93 94

Neomycin phosphotransferase

neo

B

30

O6-alkylguanine-DNA alkyltransferase Ornithine transcarbamylase

B

OCT

M

95 93

Parathyroid hormone Phenylalanine hydroxylase Pro-opiomelanocortin Purine nucleoside phosphorylase

PTH PAH POMC PNP

M M M M

96 97 98 99

Soluble CD-4 Somatostatin

CD-4 SRIF

M M

100 24

Tissue plasminogen activator Trans-activator genes Transferrin receptor Transforming growth factor alpha Tumor necrosis factor Tumor suppressor gene p53 Tyrosine hydroxylase

tPA TAT TGF-c~ TNF p53 TH

M V M M M M M

101 102 92 103 38 104 105

Xanthine-guanine phosphoribosyltransferase

XGPT

B

106

the introduction of the normal allele into the affected tissue. The preponderance of genes from genetic diseases demonstrates the bias that this field has had from the onset, namely the correction of genetic disease. However, more recently, other potential clinical applications of this technology have emerged and genes such as tumor necrosis factor and soluble CD4 have been expressed in hopes of developing therapeutic treatments for cancer and AIDS, respectively. These applications represent a new direction for gene therapy and can be considered methods for either the local or systemic delivery of therapeutic proteins. Some of the potential

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advantages of this approach to protein delivery are sustained delivery and sitespecific delivery without the need for recombinant protein purification. Sizes of the genes expressed vary from somatostatin gene at approximately 300 bp [24] to a truncated clotting factor VIII gene at 4.5 kbp [25]. Although the maximum size of the entire retrovirus has been estimated to be about 8 kbp [26], the molecular events which limit insert size as well as the mechanisms of R N A packaging during viral assembly are as yet unclear. This insert size limitation is clearly a disadvantage of retroviral-mediated gene transfer when compared to other means of gene transfer such as lipofection and receptor-mediated gene targeting in which size does not appear to be a limitation. V. Tissues modified using retroviral-mediated gene transfer Retroviral-mediated gene transfer has been used to modify a wide variety of tissues. One of the first somatic tissues to be modified and transplanted to an animal model was mouse bone marrow stem cells [27]. In these pioneering studies, a nontherapeutic gene (neo gene) was transferred to explanted bone marrow cells and the modified cells were transplanted to lethally irradiated syngeneic mice. D N A extracted from spleen colonies 2 weeks after transplantation was shown to contain the new genetic information, thus demonstrating the engraftment of cells with new genetic information. These studies illustrate the basic protocol for gene therapy using retroviralmediated gene transfer, which has also been called "ex vivo gene therapy". Ex vivo gene therapy is a multi-step process whereby a tissue specimen is obtained, its component cells grown in tissue culture, the new genetic information efficiently introduced into this population of cells, and ultimately the genetically augmented cells transplanted back to the patient from which they were obtained. All the currently approved gene therapy protocols utilizing retroviral-mediated gene transfer are considered ex vivo gene therapy procedures. Using animal models of transplantation, many tissues have been shown to be amenable to ex vivo gene therapy procedures. These tissues include: bone marrow [19,28,29], lymphocytes [30], fibroblasts [31], keratinocytes [32], endothelial cells [33], myoblasts [34], hepatocytes [35], tracheal epithelial cells [36] and neural cells [37]. Numerous applications of the technology with diverse therapeutic goals are being pursued. Enhancement of the anti-tumor activity of lymphocytes by over-expression of lymphokines [38], systemic delivery of therapeutic proteins by genetically modified skin grafts [31,32], enhancement of tumor recognition by expression of lymphokines [39], correction of liver-based genetic diseases [35], and a novel chemotherapeutic treatment of inoperable brain cancers [13] are but a few of the applications being pursued. VI. Safety issues At least two safety issues are unique to the use of recombinant retroviruses [40-42]. Foremost is the assurance that stocks of recombinant retrovirus are free of replicationcompetent wild-type virus. Sensitive assays have been developed and used to screen stocks of frozen recombinant retrovirus as well as the packaging cell lines [6].

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Another safety issue is the potential for activation of a proto-oncogene in the modified cells by insertional activation. Rare insertional activation events have been clearly shown to occur for replication-competent wild-type virus which undergoes repeated rounds of replication and spread to m a n y cells [1]. As for recombinant retroviruses which do not undergo repeated rounds of replication, there has yet to be a clear documentation of insertional activation of a proto-oncogene in a population of modified cells. However, the potential for insertional activation does exist, and what is needed are new assays to measure the frequency (however low) of such events. In the event that the frequency is unacceptably high, the vector can be modified and further disabled as has been done with the "suicide vectors" [21]. VII. Future directions for retrovirai-mediated gene transfer

Improvements to retroviral-mediated gene transfer will come from advancement in our understanding of the basic and applied aspects of recombinant retroviruses. More fundamental understanding of the molecular events which control maximum insert size and the strict requirement of cell division for successful transduction could lead to retroviral vectors with greater capacity and/or vectors which can deliver genes to non-dividing cells and tissues. Molecular cloning of the receptor for amphotropic virus and an understanding of its interaction with the e n v protein could lead to methods for even more efficient gene transfer. Other areas of improvements already underway will also continue, such as improvements to vector design for increased gene expression and improved safety as well as improved packaging cell lines which produce higher titers of recombinant retrovirus. The targeting of recombinant retroviruses to specific cells and the in vivo delivery of recombinant retroviruses are exciting new directions for retroviral-mediated gene transfer. One group using an antibody bridge between the e n v protein and a cell membrane marker demonstrated the feasibility of targeting recombinant retroviruses to specific cells in vitro [43]. Another group chemically modified the virion by addition of galactose residues and showed the redirection of virus targeting to the asialoglycoprotein receptor unique to hepatocytes [44]. A similar approach to targeting might be achieved by modifying the amino acid sequence of the e n v protein or by incorporating another receptor/ligand into the virion. The feasibility of the in vivo delivery of recombinant retrovirus to cells of the regenerating liver has also been demonstrated [45,46]. For these studies, animals were subjected to partial hepatectomy in order to stimulate hepatocyte cell division and 2448 hours after surgery recombinant retrovirus was infused into the liver circulation. Although the frequency of gene transfer was low in comparison with ex vivo procedures, there are distinct advantages to direct gene delivery in vivo. The ability to deliver recombinant retroviruses in vivo and possibly to target them to specific tissues would greatly expand the potential clinical applications of this technology. VIII. Conclusions

Retroviral-mediated gene transfer provides a means for the efficient introduction and expression of stable gene copies into large numbers of cells. These properties are

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the result of distinct features of the retroviral life cycle and it was through the identification of sequences which direct the packaging of retroviral RNAs and the subsequent development of packaging cell lines which produce stocks of recombinant retrov.irus free of wild-type virus, that retroviral-mediated gene transfer became a safe and clinically useful method for gene transfer. Originally, retroviral-mediated gene transfer was developed as a means for the correction of genetic diseases by the introduction of a normal gene copy into the cells of the diseased tissue. Much work is in progress in this area and clinical trials have commenced for some applications. More recent work using retroviral-mediated gene transfer has broadened the areas of application to include therapeutic goals for the treatment of acquired diseases such as cancer and AIDS. Novel means for the delivery of therapeutic proteins have been developed and these are being tested in experimental models and limited clinical trials. References 1 Weiss, R., Teich, N., Varmus, H. and Coffin, J. (1982) Molecular biology of tumor viruses. In: R N A Tumor Viruses, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 2 Mann, R.S., Mulligan, R.C. and Baltimore, D. (1983) Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus. Cell 33, 871 879. 3 Watanabe, S. and Temin, H.M. (1983) Construction of a helper cell line for avian reticuloendotheliosis virus cloning vetors. Mol. Cell. Biol. 3, 2241 2249. 4 Mann, R. and Baltimore, D. (1985) Varying the position of a retrovirus packaging sequence results in the encapsidation of both unspliced and spliced RNAs. Virology 54, 401~,07. 5 Bender, M.A., Palmer, T.D., Gelinas, R.E. and Miller, A.D. (1987) Evidence that the packaging signal of Moloney murine leukemia virus extends into the gag region. J. Virol. 6, 1639-1646. 6 Danos, O. and Mulligan, R.C. (1988) Safe and efficient generation of recombinant retroviruses with amphotropic and ecotropic host ranges. Proc. Natl. Acad. Sci. USA 85(17), 6460~. 7 Miller, A.D., Trauber, D.R. and Buttimore, C. (1986) Factors involved in production of helper virusfree retrovirus vectors. Somat. Cell Mol. Genet. 12(2), 175-83. 8 Markowitz, D., Goff, S. and Bank, A. (1988) A safe packaging line for gene transfer, separating viral genes on two different plasmids. J. Virol. 62, 1120-1124. 9 Anonymous (1990) T N F / T I L human gene therapy clinical protocol. Hum. Gene Ther. 1,441~480. 10 Anonymous (1990) The N2-TIL human gene transfer clinical protocol. Hum. Gene Ther. 1, 73 92. 11 Miller, A.D., Garcia, J.V., von, S.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(5), 22202224. 12 Miller, A.D. and Rosman, G.J. (1989) Improved retroviral vectors for gene transfer and expression. Biotechniques 7, 980-982. 13 Culver, K.W., Ram, Z., Wallbridge, S., Ishii, H., Oldfield, E.H., and Blaese, R.M. (1992) In vivo gene transfer with retroviral vector-producer cells for treatment of experimental brain tumors. Science 256, 1550-1552. 14 Freeman, S.M., Whartenby, K.A., Koeplin, D.S., Moolten, F.L., Abboud C.N. anql Abraham, G.N. (1992) Tumor regression when a fraction of the tumor mass contains the HSV-TK gene. J. Cell Biol. 16F., 47. 15 Hock, R.A. and Miller, A.D. (1986) Retrovirus-mediated transfer and expression of drug resistance genes in human haematopoietic progenitor cells. Nature 320(6059), 275-277. 16 Yang, Z., Korman, A.J., Cooper, J., et al. (1987) Expression of H L A - D R antigen in human class II mutant B-cell lines by double infection with retrovirus vectors. Mol. Cell Biol. 7, 3923-3928. 17 Hartman, S.C. and Mulligan, R.C. (1988) Two dominant-acting selectable markers for gene transfer studies in mammalian cells. Proc. Natl. Acad. Sci. USA 85(21), 8047-8051. 18 Morgan, J.R. and Eden, C.A. (1991) Retroviral-mediated gene transfer into transplantable human epidermal cells. Prog. Clin. Biol. Res. 365.

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71 Schwarzbauer, J.E., Mulligan, R.C. and Hynes, R.O. (1987) Efficient and stable expression of recombinant fibronectin polypeptides. Proc. Natl. Acad. Sci. USA 84, 754-758. 72 Garber, E.A., Rosenblum, C.I., Chute, H.T., Scheidel, L.M. and Chen, H. (1991) Avian retroviral expression of luciferase. Virology 185, 652-660. 73 Choudary, P.V., Barranger, J.A., Tsuji, S., et al. (1986) Retrovirus-mediated transfer of the human glucocerebrosidase gene to Gaucher fibroblasts. Mol. Biol. Med. 3, 293-299. 74 Cook, P.W., Swanson, K.T., Edwards, C.P. and Firestone, G.L. (1988) Glucocorticoid receptordependent inhibition of cellular proliferation in dexamethasone-resistant and hypersensitive rat hepatoma cell variants. Mol. Cell Biol. 8, 1449 1459. 75 Gould, G.W., Derechin, V., James, D.E., et al. (1989) Insulin-stimulated translocation of the HepG2/ erythrocyte-type glucose transporter expressed in 3T3-LI adipocytes. J. Biol. Chem. 264, 218~2184. 76 Gonda, T.J., Ramsay, R.G. and Johnson, G.R. (1989) Murine myeloid cell lines derived by in vitro infection with recombinant c-myb retroviruses express myb from rearranged vector proviruses. EMBO J. 8, 1767-1775. 77 Raney, A.K., Milich, D.R., Hughes, J.L., et al. (1989) Retroviral-mediated transfer and expression of hepatitis B e antigen in human primary skin fibroblasts and Epstein-Barr virus-transformed B lymphocytes. Virology 168, 31 39. 78 Macnaughton, T.B., Gowans, E.J., Reinboth, B., Jilbert, A.R. and Burrell C.J. (1990) Stable expression of hepatitis delta virus antigen in a eukaryotic cell line. J. Gen. Virol. 71, 1339-1345. 79 Shafer, G.E., Emery, D.W., Gustafsson, K., et al. (1991) Expression of a swine class II gene in murine bone marrow hematopoietic cells by retroviral-mediated gene transfer. Proc. Natl. Acad. Sci. USA 88, 9760 9764. 80 Sosa, M.A., DeGasperi, R., Fazely, F. and Ruprecht, R.M. (1989) Human cell lines stably expressing HIV env and tat gene products. Biochem. Biophys. Res. Commun. 161, 305-311. 81 Chang, S.M., Wager, S.K., Tsao, T.Y., et al. (1987) Construction of a defective retrovirus containing the human hypoxanthine phosphoribosyltransferase cDNA and its expression in cultured cells and mouse bone marrow. Mol. Cell Biol. 7, 854 863. 82 Cone, R.D., Reilly, E.B., Eisen, H.N. and Mulligan, R.C. (1987) Tissue-specific expression of functionally rearranged lambda l Ig gene through a retrovirus vector. Science 236, 954~957. 83 Eager, K.B., Hackett, C.J., Gerhard, W.U., et al. (1989) Murine cell lines stably expressing the influenza virus hemagglutinin gene introduced by a recombinant retrovirus vector are constitutive targets for MHC class I- and class II-restricted T lymphocytes. J. Immunol. 143, 2328 2335. 84 Fetten, J.V., Roy, N. and Gilboa, E. (1991) A frameshift mutation at the NH2 terminus of the nucleoprotein gene does not affect generation of cytotoxic T lymphocyte epitopes. J. Immunol. 147, 2697 2705. 85 Perkins, A.S., Kirschmeier, P.T. and Weinstein, I.B. (1989) Transduction of the human insulin gene

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