GB2254424A - Indicator cells and retroviruses for the detection of gene sequenes - Google Patents

Abstract

An indicator cell which may be used to test for the presence of a virus carrying a particular gene capable of expressing an identifying protein in the indicator cell wherein the indicator cell comprises a genome coding for (1) a marker gene which can express a marker product in the indicator cell; and (2) control sequences which respond to the presence of the identifying protein in the indicator cell by switching expression of the marker gene between expressing and non-expressing states, is described. There is also provided a process for the detection of a specific gene in a cell sample comprising infecting the cells with a recombinant retrovirus comprising a vector construct having the features defined in (1) and (2) above.

Description

Descristion RECOMBINANT RETROVIRUSES Technical Field The present invention relates generally to retroviruses, and more specifically, to recombinant retroviruses which are capable of delivering vector constructs to susceptible target cells. These vector constructs are typically designed to express desired proteins in target cells, for example, proteins which stimulate immunogenic activity or which are conditionally active in defined cellular environrents.

Background of the Invention Although bacterial diseases are, in general, easily treatable with antibiotics, very few effecti@ treatmentws or prophylactic measures exist for ran viral, cancerous, and other nonbacterial diseases, including genetic diseases. Traditional attests treat these diseases have employed the use of chemical drugs. In general, these drugs have laced specificity, exhibited high overall toxicity, and thus have Lean therapeuticaly ineffective.

Another classic technique for treating a numper of nonbacterial diseases involves the elicitation of an immune response to a pathogenic agent, such as a virus, through the administration or a noninfectious forr.. of the agent, such as a killed virus, thereb providing antigens from the pathogenic agent which woul act as an immunostimulant.

A t:ore recent approach for treating viral diseases, such as acquired immunodeficiency syndrome (A@DS) and related disorders, involves blocking r@@@@@@@s on cells susceptible to infection by fro::: rec@@@@ing or forming a complex with viral envelope proteins. For example, @@@@@@ et @@. (Science @@@:@@@@- 1127, 1986) demonstrated that anticodies to CD4 @4 receptors inhibited cell fusion (syncytia, between infected and noninrected CD4 presenting cells in vitro.

A similar CD4 blocking effect using moncclonal antibodies has been suggested by McDougal et al.

(Science 231:382-385, 1986). Alternatively, Pert et al.

(Proc. natl. Acad. Sci. USA 83:9254-9258, 1986) have reported the use of synthetic peptides to bind T4 receptors and block HIV infection of human T-cells, while Lifson et al. (J. Exp. Med, 164:@101, 1986) have reported blocking both syncytia and virus/T4 cell fusion by using a lectin which interacts with a viral envelope glycoprotein, thereby blocking it from being received by CD4 receptors.

A fourth, recently suggested technique for inhibiting a pathogenic agent, such as a virus, whi@@ transcribes RNA is to provide antisense RNA whi@@ complements at least a portion or the transcribed RNA, and binds theret@, so as to inhibit translation @@ et al., Mol. Cell. Biol. 6:788, 1986).

However, a major shortcoming of the techniques described above is that they do not readily lend themselves to control as to the time, location or extent to which the drug, antigen, blocking agent or antisense RNA are utilised. In particular, since the above techniques require exogenous aplication of the treatment agent (i.e., exogenous to the sample in an in vitro situation., they are not directly responsive to the presence of the pathogenic agent. For example, it may be desirable to have an immunostimulant expressed in increased amcunts immediately following infection by the pathogenic agent.In iddition, in the case of antisense RNA, large amounts would be required for useful therapy in an animal, which under current techniques would be administered without regard to the location at which it is actually needed, that is, at the cells infected b@ the pathogenIc agent.

As an alternative to exogenous application, techniques have been suggested for producing treatment agents endogenously. More specifically, prcteins expressed from viral vectors based on DNA viruses, suon as adenovirus, simian virus 40, bovine papilloma, and vaccinia viruses, have been investigated. By way of example, Panicali et al. (Proc. Natl. Acad. Sci. USA 80:5364, 1983) introduced influenza virus hemagglutinin and hepatitis B surface antigens into the vacoinia genome and infected animals with the virus particles produced from such recombinant genes. Following infection, the animals acquired immunity to both the vaccinia virus and the hepatitis B antigen.

However, a number of difficulties have been experienced to date with viral vectors based on D"A viruses. These difficulties include (a) the product: of other viral proteins which may lead to pathogenesis or the suppression of the desired protein; (b) the capacity of the vector to uncontrollably replicate in the host, and the pathogenic effect of such uncontrolled replication; (c) the presence of wild-type virus which say lead to viremia; and (d) the transitory nature co expression in these systems.These difficulties have virtually precluded the use of viral vectors based on DNA viruses in the treatment of viral, cancerous, other nonbacterial diseases, including genetic diseases.

Due to the nontransitory nature of their expression in infected target cells, retroviruses have een suggested as a useful vehicle for the treatment of genetic diseases (for example, see F. Ledley, The Journal of Pediatrics 110:1, 1987). However, in view of a number of problems, the use of retroviruses in the treatment or- genetic diseases has not been attempted.

Such problems relate to (a) the apparent need to infect a are nuroer or cells in inaccessible tissues (e.g., brain) ; (b) the need to cause these vectors to express in a very controlled and permanent fashion; (c) the lack of cloned genes; (d) the irreversible damage to tissue and organs due to metabolic abnormalities; and (e) the availability of other partially effective therapies certain instances.

In additIon to genetic diseases, other researchers have contemplated using retroviral vectors to treat nongenetic diseases (see, for example, EP 243,204 - Cetus Corporation; Sanford, J. Theor.

Biol.130:469, 1988; Tellier et al., Nature 318:414, 1985; and Bolognese er al., Cancer Res. 45:4700, 1985).

Tellier et al, suggested protecting T-cell clones by apparently infecting stem cels with "defective" HIV having a genome which could express antisense RNA to HIV RNA. Bolognesi et al. have suggested the concept of generating a nonvirulent HIV strain to infect stem cells so that T4 cells generated therefrom would carry interfering, nonvirulent forms of virus and thereby protect those cells from infection by virulent HIV.However, it would appear that the "attenuated" or "defective" HIV viruses used in both of the foregoing papers could reproduce (i.e., are not replication defective) such that the resulting viruses could infect other cells, with the possibility of an increased risk of recombination with previously present HIV or other sequences, leading to loss of attenuat@ Non-nonreplicative forms would necessitate a defective helper or packaging line for HIV. However, since tne control of HIV gene expression is complex, such cells have to date not been constructed. Furthermore, as the infecting attenuated or defective virus is not chimeric (a "nonchimeric" retrovirus being one with substantially all of its vector from the same retrovirus species? even if they were made replication defective, for example, by deletion from their genomes of an essential element, there still exists a significant possibility for recombination within the host cells with resultant production of Infectious viral particles.

Although Sanford (J. Theor. Biol. 130:469, 1983) has also proposed using a genetic cure for HIV, he notes that due to the potential that exists for creating novel virulent viruses via genetic recombination between natural AIDS virus and therapeutic retroviral vectors carrying anti-HIV genes, retroviral gene therapy or AIDS may not be practical. Similarly, while McCormick @ Kriegler (EP 243,204 A2) have proposed using retroviral vectors to deliver genes for proteins, such as tumor necrosis factor (TNF), the techniques they describe suffer from a number of disadvantages.

Sumrarv of the Invention Briefly stated, the present invention provi recombinant retroviruses carrying a vector construct capable of preventing, inhibiting, stabilizing or reversing Infectious, cancerous, auto-lmmune cr immune diseases. Such diseases include HIV infection, melanoma, diabetes, graft vs. host disease, Alone imer's disease, and heart disease.

The present invention is directed, in part, toward methods for (a) stimulating a specific in response, either ucral or cell-mediated, to an antigen or pathogenic antigen; (b) inhibiting a function pathogenic agent, such as a virus; and (c) inhibiting the interaction of an agent with a host cell receptor, through the use of recombinant retroviruses.

More specifically, within one aspect of the present invention a method for stimulating a specific: immune response is provided, comprising infecting susceptible target cells with recombinant retroviruses carrying a vector construct that directs the express Ion of an antigen or modified form thereof In infected target cells.Where an immune response is to be stimulated to 3 pathogenic antigen, the recombinant retrovirus is preferably designed to express a modifie form of the antigen whion will stimulate an immun response and which has reduced pathogenicity relative t the native antigen. An Immune response can also be achieved by transferring to an appropriate immune ce (such as a T lymphocyte) the gene for the specific cell receptor which recognizes the antigen of interest in the context of an appropriate MHC molecule cr or an immunoglobulin which recognizes the antigen of | Interest.

In the particular case of disease caused by HIV infection, where immunostimulation is desired, the antigen generated from the recombinent retroviral genome is of a form which will elicit either or both an HL HLA class I- or class II-restricted Immune response.In the case of HIV envelope antigen, or example, the is preferably selected from gp 160, gp 120, and gp 41, which have been modified to reduce their pathogenicity, In particular, the antigen selected is modifi reduce the possibility of syncvtia. Antigens from other HIV genes, such as gag, pol, vif, nef, etc., may also provide protection in particular cases.

In another aspect or the present invention, methods for inhibiting a function of a pathogenic agent necessary for disease, such as ' teases caused by infections, cancers or immunological abnormalities, are disclosed. Such inhibition is accomplished by means which include expressing a palliative that is toxic for a diseased cell, expressing a palliative that selectively inhibits the expression or the effects of pathogenic genes, expressing antisense RNA, or by inserting a sequence into a pathogenic genome so as to disrupt its function.

More specifically, the present invention provides recombinant retrovlral aenomes which. expresses a defective structural protein of a pathogenic agent, leading to inhibition of assembly of the pathogeni agent, e.g., expression of a defective structure protein of viral particles, leading to inhibitIon assembly 0= V al particles.

there a toxic palliative is to be produced V cells containing the recombinant viral genome, either it can be produced from precursors existing within the cells or, additionally, through exogenously providing a precursor to a toxic agent. In the latter case, the viral construct codes for a product which converts the precursor to a toxic agent. In either case, the toxic agent would only be localized to cells containing the viral construct and another agent associated with the pathogenic condition. For example, the other agent might be a protein produced through transcription and translation of a pathogenic viral genome present in the cell.Specificity for the pathogenic condition can also be attained or further enhanced through targeting of the entr': of the recombinant retroviruses to the cells affected by the pathogenic condition. It should be understood in the foregoing discussion, and throughout this application, that when reference is made to the viral construct "expressing" or "producing" any substance in a cell, or the like, this in fact refers to the action of the resulting provirus following reverse transcription of the viral RNA in the cell.

Within a related aspect, the present invent@ also provides methods for diminishing or eliminating an unwanted or deleterious immune response. Immune supcressicn, where appropriate, can be achieved by targeting expression of immune suppressive genes, such as the viral derived E3 gene of adenovirus.

Within another aspect of the present invention, methods are disclosed or inhibiting the interaction of viral particles with cells, cells with cells, or cells with factors. The methods generally comprise infecting susceptible cells with a recombinant, replication defective retrovirus which directs the expression cf a blocking element in infected cells, the blocking element being capable of binding with a cell receptor (preferably the h@st cell receptor, while the receptor is intercellular or cn the cell surface, or alternatively, by binding with the agent.

In either event, the interact on is s blocked.

Regardless of the means by which recombinant retrovirus exerts its immunogenic inhibitory action as described above, it is preferred that the retroviral genome be ?1replication defective" (i.e., incapable of reproducing in cells infected with it). Thus, there will be only a single stage o= infection in either an in vitro or in vivo application, thereby substantially reducing the possibility of insertlonal mutagenesis. Preferably, to assist in this end, the recombinant retrovirus lacks at least one the gag, pol, or env genes.Further, the recombi viral vector is preferably chimeric (that is, the gene which is to produce the desired result is from a different source than the remainder of the retrovirus).

A chimeric construction further reduces the possibility of recombination events within cells Infected with the recombinant retrovirus, which could produce a genome that can generate viral particles.

Within another @spect of the present invention, recombinant retroviruses which are use=ui executing the above methods as well as delivering @@@ therapeutic genes are disclosed. The present invent- also provides a method for producing such recom retroviruses in which the retrovlral genome is packaged in a capsid and envelope, preferably through the use a packaging call.The packaging cells are provided with viral protein-coding sequences, preferably in the of two plasmids, which produce all proteins necessary for production of viable retroviral particles, an viral construct which will carrv te desired gene along with a packaging signal which will direct packaging the RNA into the retroviral particles.

The present invention additionally provides a number of techniques for producing recombinant retroviruses which can facilitate: i) the production of higher titres from packaging cells; ii) packaging of vector constructs by means not involving the use of packaging cells; iii) the production of recombinant retroviruses which can be targeted for preselected cell lines; and iv) the integration of the proviral construct Into a preselected site or sites in a cell's genome.

One technique for producing higher titres from packaging cells takes advantage of the discovery that of the many factors which can limit titre from a packaging cell, one of the most limiting is the level of expression of the packaging proteins, namely, the gao, pol, and env proteins, as well as the level of expression of the retroviral vector RNA from the proviral vector.This technique allows the selection of packaging cells which have higher levels of expression (i.e., produce higher concentrations) of the foregoing packaging proteins and vector construct RNA. More specifically, this technique allows selection of packaging cells which produce high levels of what 5 referred to herein as a "primary agent," which is either a packaging protein (e.g., gag, pol, or env proteins) or a gene of interest to be carried into the genome arget cells (typically a vector construct) .This is accomplished by providing in packaging cells a genome carrying a gene (the "primary gene") which expresses the primary agent in the packaging cells, along with a selectable gene, preferably downstream from the primary ee. The selectable gene expresses a selectable protein in the packaging cells, preferably one which conveys resistance to an otherwise cytotoxic drug. The cells are then exposed to a selecting agent, preferably the cytotoxic drug, which enables identification those cells which express the selectable protein at critical level (i.e., in the case of a cytotoxic drug, by killing those cells which do not produce a level resistance protein required for survival).

Preferably, in the technique briefly describe above, the expressions of both the selectable ad primary genes is controlled by the same promoter.

this regard, it may be preferable to utilize a retroviral 5' LTR. In order to maximize titre of a recombinant retrovirus from packaging cells, this technique is first used to select packaging cells expressing high levels of all the required packaging proteins, and then is used to select which of these cells, following transfection with the desired proviral construct, produce the highest titres of the recombinant retrovirus.

Techniques are also provided for packaging vector constructs Sv means not involving the use packaging cells. These techniques make use of DNA viruses such as baculovirus, adenovirus, or vaccini virus, preferably adenovirus.These viruses are known to express relatively hI n levels of proteins exogenous genes provir'ea therein. or such DNA vIrus vectors, recombinant DNA viruses can oe produced in vivo recombination in tissue culture between viral DNA and plasmids carrying retroviral or retrovi vector genes. The resultant DNA viral vectors carrying either sequences coding for retroviral proteins or retroviral vector RNA are purl fled into high titre stocks. Alternatively, the constructs can ze constructed in vitro and subsequently transfected int@ cells which provide in trans viral functions missing from the DNA vectors. Regardless of the method of production, high titre (107 to 10 units/ml) stocks can Se prepared that will, upon Infection of susceptible cells, cause high level expression of retrovira proteins (such as gag, pol, and env) or RNA retroviral vector genomes, or both.Infection of cells in culture with these stocks, singly or in combination, will lead to high-level production of retroviral vectors, if the stocks carry the viral protein and viral vector genes.

This technique, when used with adenovirus or other mammalian vectors, allows the use of primary cells (e.g., from tissue explants or cells such as WI38 used in production of vaccines) to produce recombinant retroviral vectors.

In an alternative to the foregoing technique, recombinant retroviruses are produced by first generating the gag/pol and env proteins from a cell line infected with the appropriate recombinant DNA virus in manner sImilar to the preceding techniques, excerpt tnat the cell line is not infedted with a DNA virus carryin the vector construct.Subsequently, the proteins purified and contacted with the desired viral vector RNA made in vitro, transfer RNA (tRNA) , liposomes, ar.d a cell etract to process the env protein ito the liposomes, such that recombinant retroviruses carrying the viral vector RNA are produced. Within this technique, it may be necessary to process the env protein Into the liposomes prior to contacting them with the remainder of the foregoing mixture. The gag/pol an env proteins may also be made after plasmid mediated transfection in eukaryotic cells, in yeast, or bacteria.

The technique for producing recombinant retroviruses which can be targeted for preselected cell lines utilizes recombinant retroviruses having an env gene comprised of a cytoplasmic segment of a first retroviral phenotype, and an extracellular binding segment exogenous to the first retroviral phenotype.

@he being segment is from a second viral phenotype or from another protein with desired binding properties which is selected to be expressed as a peptide whi will bind to the desired target Techniques for integrating a retroviral genome at a specific site in the DNA of a target cell involve the use of homologous recombination, or alternatively, the use of a modified integrase enzyme which will recognize a specific site on the target cell gene.

Suc site-specific insertion allows genes to be inserted at sites on the target cells' DNA, which will minimi the chances of insertional mutagenesis, minimite interference from other sequences on the DNA, and @llow insertIon of sequences at specific target sites so as reduce or eliminate the expression of an undesirable gene (such as a viral gene) in the DNA of the target cell.

It will be appreciated that any of the above- described techniaues may be used independently of the others in particular situations, or can be used conjunction with one or more of the remainder of the techniques.

These and other aspects of the present Invention will become evident upon reference to the following detailed description and attached drawings.

Brief Description of the Drawings Figure 1 depicts three different families of vectors used to produce HIV env and which may or may not have the selectable SV-Neo cassette inserted.

Figure 2 illustrates the HIV env express' levels seen in polyacrylamide gel electrophoresis of HI env specific radioimmune precipitations of extracts human Sup T1 cells transfected with the vectors shown.

The markers are in kilodaltons, gp 160 and gp 120 mar the appropriate proteins, and 517 + tat is the positive control (HIV LTR driving env in the presence of tat).

Figure 3 depicts the protocol for testing cell killing induced in mice injected with syngeneic tumor cells expressing HIV env (the vector is pAF@env- SV-Neo).

Figure 4 graphically depicts the results the experimental protocol in Figure 3. The specific killing is seen in the top graph with BC10MEenv-39 killing vs. B/ClOME resistance to killing.

Figure 5 depicts a vector designed to express sCD4.

Figure 6 illustrates the construct Ion of the plasmids carrying the vectors TK1 (without SV-Neo) and TK3 (plus SV-Neo).

Figure 7 illustrates the construction of the plasmid carrying the vector KTVIHAX.

Figure 3 illustrates the construction of the plasmids carrying the vectors KTVIHS (without SV-Neo) and KTVIH Neo (with XV-Neo) Figure 9 illustrates construction of - plasmid carrying the vector MHMTK-Neo.

Figure 10 illustrates the construction of the plasmids carrying the tat-his (tat in sense direction) or atat (tat in antisense direction) vectors.

Figure 11 graphically depicts the preferential killing of PA317 cells infected with tathis vector (5 clones, TH1-5) compared to control PA317, upon infection with the three conditional lethal vectors shown and treatment with acyclovir (ACV).

Figure 12 depicts the construction of a virus ector carrying HIV inducible marker/reporter genes such as alkaline phosphatase (AP).

Figure 13 depicts the structure of an HIV inducible ma rker/reporter gene carried on a plasmid which can be transfected into cells.

Figure 14 graphically depicts a time curse of HIV inrection of Sup T1 cells carrying the AP marker in Figure 13 with HIV at various concentrations of ACT.

The level of HIV infection was measured by taking small aliquots of supernatant.

Figure 15 graphically depicts the results the same experiment as in Figure 14, but with ddC as the HIV inhibitor.

Figure 16 diagrammatically illustrates the number of cells surviving after phleomycin selection upon transfection of cells with a plasmid which expresses the phlemoycin resistance gene (PRG) directly from a promoter (right) and with another which expresses PRG with a coding sequence interposed between it and promoter.

Figure 17 depicts four plasmids designed to express retroviral proteins in mammalian cells. pSVgp and pRSVenv are contransfectd with a selectable marker, while pSVgp-DHFR and pRSVenv-phleo are the equivalent plasmids with the selectable marker placed downstream of the viral protein-coding regions.

Figure 18 depicts three sites of fusion of HIV env and MLV env after site-directed mutagenesis of both coding sequences to create new compatible restriction enzyme sites. The N terminal sequences are at the left in both cases. The numbering is according to nucleotide numbers. ST, SR, SE are the starts of tat, rev and while TT, TR, and TE are the corresponding terminati sites.

Figure 19 depicts the substitution of U3 in 5' LTR by a heterologous promoter@enhancet which can fused to either the Sac I, Bsst II or other site in the region.

Figure 20 illustrates a representative method for crossing transgenic mice expressing viral protein or vector R Detailed Description of the Invention I. Immunostimulation The ability to recognite and defend against foreign pathcgens is central to the unction oP t)'.e immune system. This system, through immune recognition, must be capable of distinguishing "self" from "nonself" (foreign) , which is essential to ensure that defensive mechanisms are directed toward invading entitles rather than against host tissues.The fundamental features of the immune system are the presence of highly polymorphic cell surface recognition structures (receptors) and effector mechanisms (antibodies and cytolytic cells) for the destruction of invading pathogens.

Cytolytic T lymphocytes (CTL) are induced normally by the display of processed pathogen specific peptides in conjunction with the MHC class I or class cell surface proteins. Within one embodiment of the present invention, presentation of immunogenic vIral determinants in the ccntext of appropriate MHC molecules efficiently induces optimal CTL responses withou exposing the patient to the pathogen. This vector approach to immunostimulation provides a more effective means of inducing protective and therapeutic CT responses, because the type of immunity induced by the vector more closely resembles that induced by exposure to natural infection.Based on current knowledge or several viral systems, it is unlikely that exogencusly suppiiea, nonreplicating viral antigens, such as peptldes and purified recombinant proteins, will provide sufficient stimulus to induce optimal class I-restricted CTL responses. Alternatively, vector-deliverec expression of selected viral proteins within target cells as described within the present invention provides such a stimulus.

By way of example, in the case of HIV-1 infections, patients develop antibodies specific for a variety cf- viral envelope-region determinants, some of when are capable of in vitro virus neutralization.

Nevertheless, disease progression continues and the patients eventually succumb to the disease. Low-level CTL responses against infected patients' cells (Plata et al., Nature 328:348-351, 1987) and against target cells infected with recombinant vaccinia vectors expressing HIV gag, pol, or env (Walker et al., Nature 328:345-348, 1987; Walker et al., Science 240:64-66, 1988) have been detected in scce HIV-1 seropositive patients. In addition, it has recently been shown that murine as well as human CTL can be induced by autologous stimulator cells expressing HIV gp 120 via transfection (Langlade-Demoyan et al., J. Immunol, 141:1949, 1988).

Improved CTL induction could be therapeutically advantageous to injected patients and provide effective preventive therapy to individuals under noninfectious conditions. HIV infection itself may not be producing adequate CTL response because other elements associated with HIV infection may prevent proper immune stimulation. In addition, it may be that stimulation of T-cells by infected cells is an interaction that leads to infection of the stimulated -cells.

Example 1 describes procedures for constructing plasmids capable of generating retroviral vectors in packaging cells, which then lead to expression of HIV viral antigens.

EXAMPLE : Vectors Expressing HIV Antigens A. Env Expression Vector (See Figure 1): A 2.7 kb Kpn-Xho @ DNA fragment was isolated from the HIV proviral clone BH10-R3 (for sequence, see Ratner et al., Nature 313:277, 1985) and a =400 bp Sal Kpn I DNA fragment from IIIexE7deltaenv (a Bal 31 deletion to nt. 5496) was ligated into the Sal I sits the plasmid sk@. From this clone, a 3.1 kb env DNA fragment (Xho I-Cla I) which also encodes rev, essential for env evpressicn, was purified and ligated into a retroviral vector called pAFVXM (see Kriegler et al., Cell 38: :483, 1984) . This vector was modified in that the Bgl II site was changed by linker insertion to a Xho T site to facilitate cloning of the HIV env cooing DNA fragment.

A dominant selectable marker gene corise of a SV4O early promoter driving expression of neomycin phosphotransferase gene was inserted into the vector the Cla I site to facilitate isolation of infected and transfected cell lines.

The Xho I site upstream from the ENV gene in the vector provides a convenient site to insert additional promoters into the vector construct as the RSV promoter, SV40 early or late promoter, the CMV immediate early (IE) promoter, human beta-bactin promoter, and Moloney murine MLV SL3-3 promoter.

One such promoter, the CMV Immediate Early gene promoter, a 673 bp DNA fragment Hinc II to Eag results in a tenfold increase in ENV expression 1.

human. T-cell line called Sup TI when compared to the parental construct pAF ENVr SV2 Neo.

B. Cac Sxtresson vector: A 2.5 kb Sac I-Eco RV DNA fragment was isolated from pBH10-R3 (see Ratner et al., op. cit.) an ligated into the Sac I-Sal I site of pUC3l. pUC31 derived from pUC19 with additional Xho I, Bgl Bsst II and Nco I sites inserted between the Eco R1 an Kpn I sites of the poly linker. However, this constr@ contained the major splice donor (SD) site from HIV an thus could be problematic in virus generation. The SD site was removed by subcloning a 70 bp Rsa I-Cla I fragment with a 2.1 kb Cla I-Bam H1 DNA fragment into the Hinc II-Bam H1 site of SK+. The Bam H1 site was converted Into a CIa I site by linker insertion.This construct was designated SK@ gag protease SD deita.

The 2.5 kb Xho I-Cla I DNA fragment from S.

gag protease SD delta was inserted into the Xho I/Cla - sites of the vector pAFVXM just as described above.

These plasmids, when placed in a suitable packaging cell, expressed a retroviral vector construct which contains a packaging signal. The packaging signal directed packaging of the vector construct Into a capsid and envelope along with all further proteins required for viable retroviral particles. The capsld, envelope, and other proteins are preferably produced from one or more plasmlds containing suitable genomes placed in the packaging cell. Such genomes may be proviral constructs, which in a simple case may merely have the packaging signal deleted. As a result, only the vector will be packaged.Suitable packaging or packaging cell lines, and the gencme necessary for accomplishing such packaging, are described in Miller et al. (Mol. Cell.

Bio. 6:2895, 1986), which is Incorporated herein by reference. As described by Miller et al., I is preferable that further changes be made to the provide construct other than simple delection of the packaging signal in order to reduce the chances of recombination events occurring within the packaging cell line, which may result in production o= viral partIcles which are not replication defective.

It will be understood tat Example 1 is mere illustrative of a procedure for generating an envelope glycoprotein (gp1 or otner viral antigen.

is also pcssible to provide a proviral vector construct which expresses a modified HIV envelope gp on the target cells which will likewise stimulate an immune response, but with less T-cell cytopathic effects. Envelope glycoproteins can be suitably modified using techniques well known in the art, for instance through use of the disclosure of articles such as Kowalski et al. (Science 237:1351, 1987), which is herein incorporated reference. Thus, a proviral construct may be constructed by the above technique which generates retroviral constructs expressing such a suitabl modified gp.This construct is then placed in a packaging cell as described above. The result recombinant retroviruses produced from the packagi cell lines may be used in vitro and in vivo to stimulate an immune response through the Infection of susceptIble target cells. It will be appreciated that other proteins expressed from the HIV genome, such as gag, pol, vif, nef, etc., may also elicit beneficial cellular responses in HIV-infected individuals. Provlral vectors such as those described below are designed to express such proteins so as to encourage a clinically beneficial immune response. It may be necessary for certain vectors to include rev coding sequences as well as a rev responsive element (Rosen et al., Proc. Natl. cad. Sci.

85:2071, 1988).

The following example demonstrates the ability of this type of treatment to elicit CTL responses in mice.

EXAMPLE 2 Immune Response to Retroviral Vector-Encoded antigens A murine tumor cell line (B/C1OME) (H-2d) was infected with the pAF envrSV40 Neo vector construct coding for HIV env. One cloned HIV-env expressing cell line (B/C10ME-29) was then utilized to stimulate HIVenv-specific CTL in syngeneic (i.e., MHC identical) Balbjc (H-2d) mice. Mice were immunized by Intraperitoneal injection with B/Cl0ME-29 cells (4 x 10 cells) and boosted on day 7-14. Responder spleen cei suspensions were prepared from these Immunized mice and the cells cultured in vitro for 4 days in the presence of either B/C10ME-29 or B/C1OME mitomycin-C-treated cells at a stimulator: responder cell ratio of 1:50 (Figure 3). The effector cells were harvested from these cultures, counted, and mixed with radiolabeled (51Cr) target cells (i.e., B/C10MEenv-29 or B/C10ME) at various effector:target (E:T) cell ratios in a standard 4-5 hr 51C~-release assay.Following Incubation, the microtiter plates were cent-ifuged, 100 ul of culture Supernate was removed, and the amount of radiolabe released from lysed cells quantltated in a Backman garda spectrometer. Target cell lysis was calculated as: % Target Lysis = Exp CPM - SR CPM/MR CPM - SR CPM x 100, where experimental counts per minute (Exp CPM) represents effectors plus targets; spontaneous release (SR) CPM represents targets alone; and maximum release (MR) CPM represents targets in the presence of 1 M HCt.

The results (Figure 4) illustrate that CTL effectors were induced which specifically lysed HIV-envexpressing target cells (B/C10MEenv-29) significantly more efficiently than B/C10ME targets. Primed spleen cells restimulated in vitro with non-HIV-en-expressing control cells (B/C1OME) did not show significant CT activity on either B/C10MEenv-29 or B/C10Me targets, particularly at lower E:T cell ratios. Spleen cells obtained from naive nonimmunited 3alb/c mice which were restimulated in vitro with B/C10MEenv-29 did not generate CTL, thus suggesting the importance of the in vivo priming and boosting event. This experiment has been repeated and similar results obtained.

In another experiment, effector cells obtained from Balb/c mice Immunized, boosted and restimulated in vitro with a different H-2d HIV-env-expressing tumor cell clone (L33-41) infected with the same pAF env SV40 Neo (HITJ-env) vector construct were capable of lysing B/C10MEenv-29 target cells. This provides additional support that the CTL generated in these mice are specifically recognizing an expressed form of HIV-env rather than simply a unique tumor cell antigen on these cells.This result also suggests that the vectordelivered antigen is presented in a similar manner by the to tumor cell lines.

Implementation of this immunostimulant application in humans requires that (1) the gene coding for the antigen of interest be delivered to cells, (2) the antigen be expressed in appropriate cells, and (3) :Aj.'C restriction requirements, i.e., class I class II antigen interaction, are satisfied.

Preparations of vector are made by growing the producer cells in normal medium, washing the cells with PBS plus human serum albumin (HSA) at 10 mg/ml, then growing the cells for 8-16 hours in PBS plus HSA. Titers obtained are typically 104 to 106/ml depending on the vector, packaging line or particular producer line clone. The vector supernatants are filtered to remove cells and are concentrated up to 100-fold by filtration through 100,000 or 300,000 pass Amicon filters (Wolff et al., Proc. Natl. Acad. Sci. 34:3344, 1987). This lets globular proteins of 100,000 or 300,000 pass but retains 99t of the viral vector as infectious particles.The stocks can be frozen for storage since they lose about 50% of the infectious units on freezing and thawing.

The most direct delivery involves administration of the appropriate gene-carrying vector into the individual and reliance upon the ability of the vector to efficiently target to the appropriate cells, which can then initiat stimulation of the immune response. The dose would be 105 - 106 infectious units/kg body weight. However, n more practical approach may involve the extracorporea treatment of patient peripheral blood lymphocytes (PBL), fibroblasts or other cells obtained from each individual with the vector.PBL could be maintained in culture hrough the use of mitogens (phytohemagglutinin) or lymphokines (e.g., IL-2) . This type of approach allows for directed vector infection, monitoring of expression and expansion of the antigen presenting cell population prior to injection, and return of vector-expressing cells to the respective partient. Other types of cells would also be explanted, vector introduced, and the tells returned to the patient. Only a moderate number of infected cells (105-106/kg body weight) is necessary to elicit strong immune responses.

A different fcrm of administration is the implantation of producer lines making retroviral vec particles. These may be immunologically urmatohec' classical producer cell lines or the patients own cells, which have been explanted, treated and returned (see Alternative Viral Vector Packaging Techniques, below).

Both types of implants (105 - 106/kg body weight) would have a limited life span in the patient, but would lead to the retroviral vector infecting large numbers (107 - 1010) of cells in their vicinity in the body.

In any case, the success of the treatment zar, be assayed by removing a small amount of blood an measuring the CTL response using as targets the individual's own cells infected with vector leading to env expression.

When It is desired to stimulate an 'AHC class I or class II restricted Immune response to pathogens, including pathogenic viruses other than HIV, suitable forms of envelope or other antigens associated with such retroviruses which will stImulate an immune response can be ascertained by one skilled in the art. In general, suitable forms of antigens associated with pathogenic agents can readily be selected which will stimulate an immune response to those pathogenic agents.

An alternative approach to creating a desired immune response is to deliver an antigen-specific T-cell receptor gene to an appropriate cell, such as a T-cell.

Another possible recipient is NN cells where the cytotoxic immune response is not C limited.

specific immunoglobulin gene could similarly be use"1 if delivered ts a 3-cell.

Blocking Agents Manv infectious diseases, cancers, autoimmune diseases, and other diseases involve the interaction of viral particles with cells, cells with cells, or cells with factors. In viral infections, viruses commonly enter cells via receptors on the surface of susceptible cells. In cancers, cells may respond inappropriately or not at all to signals from other cells or factors.

autoimmune disease, thers is inappropriate recognition of "self" markers. Within the present invention, such interactions may be blocked by producing, in vivo, an analogue to either of the partners in an interaction.

This blocking action may occur intracellularly, on the cell menbrane, or extracellularly. The blocking action of a viral or, in particular, a retroviral vector carrying a gene for a blocking agent, can be mediated either from inside a susceptible cell or by secreting a version of the blocking protein to locally block the pathogenic interaction.

In the case of HIV, the to agents c- interaction are the gp 120/gp 41 envelope protein and the C2 receptor molecule. Thus, an appropriate blocker would be a vector construct expressing either an HIV env analogue that blocks HI=1 entry without causIng pathogenic effects, or a CD4 receptor analogue. The CD4 analogue would be secreted and would function to protect neighboring cells, while the gp 120/gp 41 is secreted or produced only intracellularly so as to protect only the vector-containing cell.It may be advantageous to add human immunoglobulin heavy chains or other components to CD4 in order to enhance stability. Deliver of a retroviral vector encoding such a hybrid-soluble CD4 to a host results in a continuous supply of a stable hybrid molecule.

vector particles leading to expression of H env ma also be ccnstructed as described above. It will be evident to one skilled in the art which porticns are capable of blocking virus adsorption without overt pathogenic side effects (Willey et al., J. Virol.

6@:109, 1988; Fisher et al., Science 233:655, 1986) The fl Io'4ing example describes the construction of a CD4 vector from which infectious vector particles were made.

EXAMPLE 3 sCD4 Vector 1. A 1.7 kb Eco R1 - Hind III DNA fragment from pMV7.T4 (Maddon et al., Cell 47:333, 1986) was blunt-end ligated to the Hinc II site of SK+.

2. A universal translation termination sequence containing an Xba I site was inserted into the Nhe I site of the CD4 fragment.

3. The 1.7 kb Xho I-Cla I fragment was excised and clones into the Xho I - Cla I site pXFVXM. These vector plasmids can be used to generate infectious vector particles, as described in Example 1.

Suc infectious blocking vectors, when into human T-cell lines in culture can inhibit the spread of HIV infections . Preparation, concentrati and storage of infectious retroviral vector preparations is as for the immunostimulant. Route of administration would also be the same, it doses about 10-fold higher.

Another route which may be used is the aspiration of bone marrow, infection with retroviral vector and return of this infected marrow (Gruber et al., Science 230:1057, 1985) to the patient. SInce the marrow replication will amplify the vector expression throug cell replication, doses in the range of immunostimulant can be used (105 - 106/kg body weight,'.

In any case, the efficacy of the treatment can be assayed by measuring the usual indicators of disease progression, including antibody level, viral antigen production, infectious HIV levels, or levels of ncnspecific infections.

III. Expression of Palliatives Techniques similar to those described above can be used to produce recombinant retroviruses with vector constructs whic.: direct the expression of an agent (or "aplliative") which is capable of inhibiting a function of a pathogenic agent or gene. Within the present inventIon, "capable of inhibiting a funct@ means that the palliative either directly inhibits the function or indirectly does so, for example, by converting an agent present in the cells from one which would not normally inhibit a function of the pathogenic agent to one which does.Examples of such functions for viral diseases include adsorption, replicatIon, gene expression, assembly, and exit of the virus infected cells. Examples of such functions for cancerous diseases include cell replication, susceptibility to external signals (e.g., contact inhibition) , and lack of production of anti-oncogene proteins.

(i) Inhibitor Palliatives In or.e aspect of the present invention, the vector construct directs the expression of antisense RNA (or complementary RNA) to RNA of a pathogenic virus, such as HIV (or a pathogenic gene, such as an oncogene,, to thereby inhibit its re 1 cation or pathogenesis.

Such express Ion may either Se essentially continuous or in response to the presence in the cell of another agent associated with the pathogenic condition (an "identifying agent") . Alternatively, the expression be tissue-specific due either to targeting of vecte entry or to tissue-specific control sequences in the vector.

In one embodiment, retroviral viruses which express RNA complementary to key pathogenic gene transcripts (for example, S viral 1 gene product cr an activated cellular oncogene) on be used to inh@ translation of that transcript into protein, such as the inhibition of translation of the HI tat protein. Since expression of this protein is essential for viral raplication. cells containing the vector would be resistant to HIV replication.

In a second embodiment, where the pathoge agent is a single-stranded virus having a package signal, RNA complementary to the viral packaging singal (e.g., an HIV packaging signal when the palliative is directed against HIV) is expressed, so that the assocIatIon of these molecules with the viral signal will, in the case of retroviruses, inhibi locp formation or tRNA primer binding require ~r proper encapsldation or replication of the retroviral RNA genome.

In a third embodiment, a retroviral vector be introduced which expresses a protein that interferes with the pathogenic state. In the case of HIV, one example is a mutant tat protein which lacks the ability to transactivate expression from the HIV LTR and interferes (in a transdominant manner) with the normal functIoning of tat protein. Such a mutant as been identified for HTLV II tat protein ("XII Leu5" mutant) see Wachsman et al., ScIence 235:674, i987). A mutant transrepressor tat should inhibit replication much as has been shown for an analogous mutant repressor HSV-1 $Friedmann et al., Nature 335:452, 1988).

Such a transcriptional repressor protein ma': be selected for in tissue culture using any vira specific transcriptional promoter whose expression is stimulated by a virus-specific transactivating protein (as described above). In the specific case of HIV, a cell line expressing HI tat protein and the HS.:TK gene driven by the HIV promoter will die in the presence of ACV. However, if a series of mutated tat genes are introduced to the system, a mutant with the appropriate pr (i . .e., represses transcript Ion from the promoter in the presence of wild-type tat) will grow ant be selected for. The mutant gene can then be reisolated from these cells.A cell line containing multiple copies of the conditionally lethal vector/tat system may be used to assure that surviving cell clones are not caused by endogenous mutations in these genes. A battery of randomly mutagenited tat genes are then introduced into these cells using a "rescuable" retroviral vector (i.e., one that expresses the mutant tat protein and contains a bacterial origin replication and drug resistance marker for growth and selection in becteria). This allows a large number random mutations to be evaluated and permits faciie subsequent molecular cloning of the desired mutant line.This procedure may be used to identify utilize mutations in a variety of viral transcriptional activator/viral promoter systems for potential antivi therapies.

In a fourth embodiment, the HSVTK gene product is used to more effectively metabolize potentially antiviral nucleoside analogues, such as AZT or ddC. The HSVTK gene is expressed under the control of 3 constitutive macrophage or T-cell-specific promoter introduced into these cell types. AZT (and other nucleoside antivirals) must be metabolized by cellular mechanisms to the nucleotide triphosphate form in order to specifically inhibit retroviral reverse transcriptase and thus HIV replication (Furmam et al., Proc. Natl.

Acad. Sci. USA 83:8333-8337, 1986). Constitutive expression of HSVTK (a nucleotide and nuclectide phosphate kinase with very broad substrate specificity) results in more effective metabolism of these drugs to their biologically active mucleotide triphosphate form.

AZT or ddC therapy will thereby be more effective, allowing lower doses, less generalized toxicity, and higher potency against productive infection. Additional nuclecside analogues whose nucleotide triphosphate forms show selectivity for retroviral reverse transcriptase but, as a result of the substrate specificit of cellular nucleoside and nucleotide kinases are not phosphorylated, will be made more efficacious. A description of this method is et forth in Example 4.

EXAMPLE 4 Vectors Desianed to Potentiate the Antiviral Effect of AZT and Analogues I. All of the following retroviral vectcrs are based on the "N2" vector (see Keller et al., Nature 318:149-154, 1985). Consequently, 5' and 3' Eco R1 LTR fragments (2.8 and 1.0 kb, respectively) were initially subcloned into plasmids containing polylinkers (into SKto give pN2R5(+/=); into pUC31 to give p31N2R5[+/-] and p31N2R3[+/-] to facilitate vector construction. In one case, a 1.2 kb CIa I/Eco R1 5' LTR fragment was subcloned into the same sites of an SK+ vector to give pN2C. In another case, the 5' LTR containing a 6 bp deletion of the splice donor sequence was subcloned as 1.8 bp Eco R1 fragment into pUC31 (p31N25delta[+]).The coding region and transcriptional termination signals of HSV-l thymidine kinase gene was isolated as a 1.3 k@ Bgl II/Pvu II fragment from plasmid 322TK (3.5 kb Bam H1 fragment of HSVTK cloned into Bam H1 of pBR322) and cloned into Bgl II/Sma I-digested pUC31 (pUCTK). For constructs which require deletion of the terminato@ signals, pUCTK was digested with Sma. I. and Bam H1. The remaining coding sequences and sticky-end Bam H1 overhang were reconstituted with a double-stranded oligonuclectide made from the following ollgomers: 5' GG AGA TGG GGG AGG CTA ACT GAG 3' and 5' GAT CCT CAG TTA GCC TCC CCC ATC TCT C 3' forming the construct pTK delta k.

For diagnostic purposes, the oligos were designed to destroy the Sma I site while keeping its Ava I site without changing the translated protein.

The d.6 kb HIV promoter sequences were cloned as a Dra I/Hind III fragment from pCV-l (see Arya et al., Science 229:69-73, 1985) into Hinc II/Hind IIIcut SK@ (SKHL).

A. Construction of TK-1 and TK-3 retroviral vect@@s (see Figure 6).

1. The 5 kb Xho I/Hind III 5' LTR and plasmid sequences were isolated from p31N2R5(+).

2. HSVTK coding sequences lacking transcriptional termination sequences were isolated as 1.2 kb Xho I/Ban H1 fragment from pTKdeltaA.

3. 3' LTR sequences were isolated as a 1.0 kb Bam H1/Hind III fragment from pN2R3(-) 4. The fragments from steps 1-3 were mixed, ligated, transformed into bacteria, and individual clones identified by restriction enzyme analysis (TK-1).

5. TK-3 was constructed by linearizing T with Baa H1, filling in the 5' overhang and blunt-end ligating a 5'-filled Cla I fragment containing bacterial lac UV5 promoter, S':40 early promoter, Tn5 Neor gene. Kanamycin-resistant clones were isolate and individual clones were screened for the prop@ orientation by restriction entyme analysis.

These constructs were used to generate infectious recombinant vector particles in conjunction with a packaging cell line, such as PA317, as described above.

Administration of these retroviral vectors human T-cell and macrophage/monocyte cell lines increase their resistance to HIV in the presence of and ddc compared to the same zells without retroviral vector treatment.

Preparation, concentration and storage of the retroviral vector preparations would be as described above. Treatment wculd be as previously described but ex cor-ore treatment of patients' cells would aim =cr uninfected potentially susceptible T-cells or monocytes.

One preferred method of targeting the susceptible cell is with vectors which carry HIV env or hybrid env (see Section VIII Cell Line Specific Retroviruses, below) to direct absorption of vector particles to CD4- cells.

Normal adults have about 5 x 109 T cells in their total blccd and about the same number of monocytes.

A preferred method of administration would be leukophoresis, where about 20% of an individual's PBLs can be removed at any one time and manipulated in vitro.

Thus, about 2 x 109 cells would be treated and replaced.

Since the current maximum titres are around 106/ml, thIs would require 2 to 20 liters of starting viral supernatant. Repeat treatments would be performed.

Alternatively, bone marrow would be treated and allowed to amplify the effect as described above. Treatment with AZT would be at lower than normal levels to avoid toxic side effects, but still efficiently inhibit spread of HIV. The course of the treatment would be followed as described for the blocker.

A fifth embodiment for producing inhibitor palliatives Involves the expression of defective inter=ering viral structural proteins, which innirit viral assembly. Vectors would code for defective gag, pol, er.v or other viral particle proteins or peptides, and these would inhibit in a dominant fashion the assembly of viral particles. This occurs because the interaction of normal subunits of the viral particle is disturbed by interaction with the defective subunits.

A sixth such embodiment involves te express Ion of inhibiting peptides specific for vIra protease. Viral protease cleaves the viral gag and gag/pol proteins into a number of smaller peptides.

Failure of this cleavage in all cases leads to complete inhibition of production of infectious retroviral particles. The HI'J protease is known to be an aspartyl protease, and these are known to be inhibited by peptides made from anino acids from protein or analogues. Vectors to inhibit HIV will express one or multiple fused copies of such peptide inhibitors.

A seventh embodiment Involves the deliver of supressor genes which, when deleted or not expressed In a cell type, lead to tumorigenesis n that cell type Reintroduction of the deleted gene by means of a viral vector leads to regress Ion of the tumor phenotype in these cells. Examples of such cancers are retinoblastoma and Wilts Tumor. Since malignancy can be considered to be an inhibition of cellular terminal differentiation compared with cell growth, the retroviral delivery and expression of gene products which lead to differentiation of a tumor should also, general, lead to regression.

In an eighth embodiment, the retrovira construct provides a therapeutic effect by insertin itself into a virus, oncogene, or pathogenic gene, thereby inhibiting a function required for pathogenesIs.

This embodiment requires the direction of retrov' integration to a specific site In the genome homologous recombination, integrase modification, other methods (described below).

(i) Conditional Toxic Palliatives Another approach for inhibiting a pathogen it agent is to express a palliative which is toxic for the cell expressing the pathogenic condition. In this case, expression of the palliative rom the proviral vector should be limited by the presence of some intracellul signal identifying the pathogenic state in ormer to avoid destruction or nonpathogenic cells. This co' type specificity may also be conferred at the level of infection by targeting recombinant retrovirus carr the vector to cells having or being susceptible to pathogenic condition.

ifl one embodiment of this method, recombinant retrovirus (preferably, but not necessarily, but not necessarily, a recombinant NLV retrovirus) carries a vector const containing a cytotoxic gene (such as ricin) expressec from an event-specific promoter, such as a cell cycledependent promoter (e.g., human cellular thymidine kinase or transferrin receptor promoters). which will be transcriptlonally active only in rapidly proliferating cells, such as tors. In this manner, rapt replicating cells, which contain factors capable activating transcription from these promoters, are preferentially destroyed by the cytotoxic agent produces by the viral construct.

In a second embodiment, the gene producing the cytotoxic agent is under control of a tissue-specific promoter, where the tissue specificity corresponds to the origin of the tumor. Since the viral vector preferentially integrates into the genome of replicating cells (for example, normal liver cells are not replicating, while those of a hepatocarcinoma are) these to levels of specificity (viral integration/replication and t ssue-specific transcriptional regulation) lead to preferential killing of tumor cells.Additionally, event-specific and tissuespecific promoter elements may be artificially combined such that the cytotoxic gene product is expressed only in cell types satisfying both criteria (e.g., in the example above, combined promoter elements are functional only in rapidly dividing liver cells). Transcriptional control elements may also be amplified to increase the stringency of cell-type specificity.

These transcriptional promoter/enhancer elements need not necessarily be present as an internal promoter (lying between the viral LTRs) but may oe added to or replace the transcriptional control elements in the viral LTRs which are themselves transcriptiona promoters, such that condition-specific transcriptional expression will occur directly from the modified viral LTR.In this case, either the condition for maxima express ion will need to be mimicked in retroviral packaging cell lines (e.g., by altering growth conditions, supplying necessary transregulators of express Ion or using the appropriate cell line as a Farent for a packaging line), or the LTR modification is limited to the 3' LTR '3 region, to obtaIn maximal recombinant viral titters. In the are case, after one @ound of infection/integration the 3' LTR U3 is now also the 5' LTR U3 giving the desired tissue specific expression.

In a third embodiment, the proviral vector construct is similarly activated but expresses a protein which is not itself cytotoxlc, and which processes within the target cells a compound or a drug with little or no cytotoxicity into one which is cytotoxic (a "conditionally lethal" gene product) . Specifically, the proviral vector construct carries the herpes simplex virus thymidlne kinase ("HSVTK") gene downstream and under the transcriptional control of an HIV promoter (which is known to be transcriptionally silent except when activated by HIV at protein).Expression of the tat gene product in human cells infected with HIV and carrying the proviral vector construct causes increased production of HSVTK. The cells (either in vitro cr in vivo) are then exposed to 3 drug such as acyclovir or its analogues (FIAU, FIAC, @HPG). These drugs are known to be phosphorylated by HSVTK (but not by cellular thymidine kinase) to their corresponding active nucleotide triphosphate forms (see, for example, Schaeffer et al., Mature @@@:583, 1978). Acyclovir and FIAU triphosphates inhibit cellular polymerases in general, leading to the specific destruction of cells expressIng HSVTK in transgenic mice (see Borrelli et al., Proc. Natl. Acad. Sci.USA 85:7572, 1988).

Those cells containing the recombinant vector and expressing H tat protein are selectively killed by the presence of a specific dose of these drugs.

addition, an extra level of specificity is achieved by including in the vector the HIV rev protein, responsive CRS/CAR sequences. In the presence of the CRs sequence gene expression is suppressed, except in the presence of the CAR sequences and the rev protein. Example provides an illustration of this technique.

EXAMPLE 5 Vector to Conditionally Potentiate the Toxic Action of ACV or its Analoaues Construction of Vectors A. Construction of pKTVIHAX (see Fiaure 7).

1. A 9.2 kb Asu II/Xho I fragment was isolated from vector pN2 DNA.

2. A 0.6 kb Xho I/Bam H1 promoter fragment was isolated from plasmid pSKHL.

3. A 0.3 kb Bg lII/Acc I and a 1.5 Acc I/Acc I fragment were purified from pUCTK.

4. The fragments from 1, 2, and 3 were ligated, transformed into bacteria, and appropriate Amp@ clones of the given structure identified by restriction enzyme analysis.

B. Construction of vKTVIH-5 and pKTVIH5 Net retroviral vectors (see Figure 85.

1. 4.5 kb 5' LTR and vector was isolated as an Xho I/Bam H1 fragment from vector p31N25delta(+).

2. The 1.0 kb 3' LTR was isolated as Apa I/Bam Hi fragment from pN2R3(+) fragment.

3. The 0.6 kb HIV promoter element was isolated from pSKHL as an Apa I/Eco RI fragment.

4. The HSVTK coding sequence ant transcriptional termination sequences were Isolated 1.3 kp Eco Rl/Sal I fragment from pUCTK.

5. The fragments from 1-4 were combined, ligated, transformed into bacteria, and clones of the given structure were identified by restriction enzyme analysis (pKTVIH-5).

6. pKTVIH5 Neo was constructed 5 linearizing pKTVIH5 with Cla I; mixing with a 1.3 t;~ CIa I fragment containing the bacterial lac UV5 promoter, SV40 early promoter, and Tn5 Neor marker, 5. A 2. kb Bgl II/Sal I fragment containing the HSVTK coding region upstream of the SV40 ear promoter driving Neo taken from pTK-3 (see Figure 3).

6. 3-5 were mixed, ligated, used to transform bacteria, and appropriate clones were identified by restriction enzyme analysis.

D. Construction of tat and anti-tat expression vectors (see Figure 10).

These vectors are used as pseudo-HIV to testactivate tat-dependent HSVTK vectors.

1. The Hisr expression vector p8amHis was linearized with Bam H1 and treated with calf intestinal phosphatase.

2. The Sac I site of pCV-l was mutagenized to a Ban H1 site and the 350 bp Bam H1 coding sequence 0= ,. tat was isolated, 3. The fragments purified in steps 1 and were mixed, ligated, used to transform bacteria, ant clones with tat in both orientations were identified by restriction enzyme analysis.

These constructs were used to generate infectious recombinant vector particles in conjunction with a packaging cell line such as PA317, as described above.

The biological properties of these retroviral vectors are described hereinafter. The HIV tat gene ("tathis" vector -- see Figure 10) was transfected into mouse PA317 cells. Five individual histldinol-resistant subclones were obtained (TH 1-5) which express HIV tat.

These cells are thus an experimental model for TIV In=ection. The vectors KTVIHAX, KTVIH5NEO, and MHMTKNEO, ere subsequently introduced by infection into these tat-expressing cell lines as well as their parent cell line lacking tat. Cell viability was then determined in various concentrations of the HSVTK- s~ec .ic cytotoxic drug, acyclovir. The data are reported here as LD50 (the drug concentration at which ligating, transforming bacteria and selecting for kanamycin resistance. Clones with the insert in the indicated orientation were identified by restriction analysis.

C. Construction of MHMTK Neo retroviral vector (see Figure 9).

1. Construction of intermediate plasmid MHM-1: a) plasmid pN2CR5 was linearized by partial digestion with Fok I, the 5' overhang filled in with deoxynucleotide triphosphates using Klenow DNA polymerase, and Hind III linkers inserted. After transformation into bacteria, a clone with a Hind III linker inserted in the MLV LTR Fok I site was identified by restricton enzyme analysis (pN2CR5FH).

b) pN2CR5FH waws linearized with Nhe I, the 5' overhang filled in with Klenow polvmerase digested with Hind III, and the 4.3 kb fragmen with promoterless ML'? sequences Isolatet.

c) 0.5 kg Eco RV/Hind III HIV promote sequences were isolated from pSKHL.

d) b and z were mixed ligated, used to transform bacteria, and the structure of MHM-1 was confirmed by restriction enzyme analysis.

2. A 0.7 kb Eco RV. Bal I fragment isolate from MHM-1 was subcloned into the Eco RV site of plasmi 1308 (a modified IBI30 plasmid containing additiona Bgl II, Bst II, Neo I and de I sites in te polylinker). After transformation into bacteria, clones with the appropriate orientation were identified b restriction enzyme analysis (pMHMB).

3. pMHMB was digested with Apa I and Xho I and gel purified.

4. MHM-1 was digested with Apa I/Bam H1 and the 1.8 kb MHMLTR/leader sequence gel purified.

50% toxicity is observed). The parental cell lin containing the vector but lacking tat (non-HIV-infecte model) showed no detectable toxicIty by ACi at the concentrations tested (see Figure 11). These cells thus require 100 uM AC'? or greater for cytotoxicity. This is true also for these cells lacing the vectors. Thus the vectors alone, AC7 alone, or even the vector +ACV (solid boxes) is not cytotoxic. However, cell lines which express HIV tat (the experimental representation of an HIV infection) are effectively killed by ACI. This is true to varying degrees for all three vectors tested.

These data indicate that MIV-infected cells will be killed in the presence of ACV and "potentiator" vectors.

Similarly, HIS In=ectlon of human T-cell lines +/- FIAU shower preferential inhibition (through cell killing) or HIV infection. Cultures were first treated with vector, then challenged with low multiplicity HIV infection for 4 days. Viral supernatants were titred using the HIV, as described in Section IV.

In the case of HIV-infected cells, expressIon of the conditionally lethal HSVTK gene was made eve.

more HIV specific by including cis-acting elements ir.

the transcript ("CRS/CAR") , which require an additcnai HIV gene product, rev, or optimal activity (Rcsen et al., Proc. Natl. cad. Sci. USA 35:2071, 1988).

generally, cis elements present in mRNAs have been shown in some cases to regulate mRJiA stability or translatability. Sequence of this type (i.e., posttranscriptional regulation of gene expression) may be used for event- or tissue-specific regulation of vector gene expression. In addition, multimerization of these sequences (k.e., rev-responsive "CRS/CAR" of tatresponsive "TAR" elements for HIV) could result in even greater specificity. It should be noted that this kind of conditional activation of an inactive precursor an active product in cells may also be achieved using other viral vectors with a shorter term effect, e.g., adenov'rus vectors.

Production, concentration and Storage of vector preparations is as previously described.

Administration is by direct in vivo administration as before or by ex corpore treatment of PBL and/or bone marrow. Doses will be at approximately the same levels as for Example 4. Targeting of viral vector infection will not be through the CD4 receptor, but may be accomplIshed through making vector particles with hybrid 4LVenv-CD4 "envelope" proteins (see Section VII) to target gp 120 expressing cells (i.e., those infected with HIV). This inversion of the normal virus-receptor interaction in order to target virally infected cells can be used wIth all types of viruses.

In a similar manner to the preceding embodiment, the retroviral vector construct can carry a gene for phosphorylation, phosphoribosylation, ribosylation, or other metabolism of a purine- or pyrimiaine-based drug. This gene may have no equivalent in mammalian cells and might come from organisms such as a virus, bacterium, fungus, or protozoan. An example of this would be the E. coli guanine phosphoribosyl transferase gene product, which is lethal in the presence of thioxanthine (see Besnard et al., Mol. Cell.

3iol. 7:4139-4141, 1987). Conditionally lethal gene products of this type have potential application to man presently known purine- or pyrimidine-based ant cancer drugs, which often require intracellular ribosylation or phosphorylation in order to become effective cytotoxic agents. The conditionally lethal gene product could also metabolize a nontoxic drug, which is not a urine or pyrimidine analogue, to a cytoxic form.

Mammalian viruses in general tend to have "immediate early" genes which are necessary for subsequent transcriptional activation from other viral promoter elements. Gene products of this nature are excellent candidates for intracellular signals (or "identifying agents") of viral infection. Thus, conditionally lethal genes from transcriptional promoter elements responsive to these viral "immediate early" gene products could specifically kill cells infected with any particular virus.Additionally, since the human a and ss interferon promoter elements are transcriptionally activated in response to infection by a wide variety of nonrelated viruses, the introducton of vectors expressing a conditionally lethal gene product like HSVTK, for example, from these virairesponsive elements (VRE) could result in the destruction of cells infected with a variety of different viruses.

In a fourth embodiment, the recombinant retrovirus carries a gene specifying a product whion is not in itself toxic, but when processed by a protease specific to a viral or other pathogen, is converted into a toxic form.

In a fifth embodiment, the retrovira construct may express a "reporting product" on the surface of the target cells in response to the presence of an identifying agent in the cells (such as HIV tat protein). This surface protein can be recognized by a cytotoxic agent, such as antibodies for the reporting protein or by cytotoxic T-cells. In a similar manner, such a system can be used as a detection system (see below) to simply identify those cells having a particular gene which expresses an identifying protein, such as the HIV tat gene.

Similarly, in a sixth embodiment, a surface protein could be expressed which would itself be therapeutically beneficial. In the specific case of HIV, expression of the human CD4 protein in HIV-inected cells may be beneficial in two ways: 1. Binding of CD4 to HIV env intracellularlv could inhibit the formation of viable viral particles much as soluble CD4 has been shown to do for free virus, but without the problen of systematic clearance ant possible immunogenicity, since the protein will remain membrane bound and is structurally identical to endogenous CD4 (to which the patient should be immunologically tolerant).

2. Since the CD4/HIV env complex has been implicated as a cause of cell death, additional expression of CD4 (in the presence of excess HIV-env present in HIV-infected cells) leads to more rapid cell death and thus inhibits viral dissemination. This may be particularly applicable to monocytes and macrophages, which act as a reservoir for virus production as a result of their relative refractility to HIV-inducer cytotoxiclty (which, in turn, is apparently due to the relative lac of CD4 on their cell surfaces).

(iii) immune Down-Reaulation Specific down-regulation of inappropriate or unwanted immune response, such as in chronic hepatitis or in transplants of heterologous tissue such as bone marrow, can be engineered using anti-MHC class I genes, such as immune-suppressive viral genes. Group C adenoviruses Ad2 and Ad5 possess a 19 kd glycoprotein (gp 19) encoded in the E3 region of the virus. This gp 19 molecule binds to class I MHC molecules in the endoplasmic reticulum of cells and prevents terminal glycosylatlon and translation of class I MHC to the cell surface. For example, prior to bone marrow transplantation, donor bone marrow cells may be infected with gp 19-encoding vector constructs which upon express Ion of the gp 19 inhibit the surface expression of IHC class I transplantation antigens. These donor cells may be transplanted with low risk of graft rejection and may require a minimal immunosuppresslve regimen for the transplant patient. This may allow an acceptable dcncr-recipient chimeric state to exist with fewer complications.Similar treatments may be used to treat the range of so-called autoimmune diseases, including lupus erythromiatis multiple sclerosis, rheumatoid arthritis or chronic hepatitis 3 infection.

IV. Expression of Markers The above-described technique of expressing a palliative in a cell, in response to some identifying agent, can also be modified to enable detection of a particular gene in a cell which expresses an identifying protein (for example, a gene carried by a particular virus) , and hence enable detection of cells carrying that virus. In addition, this technique enables the detection of viruses (such as HIV) In a clinical sample of cells carrying an identifying protein associated wit the virus.

This modification can be accomplished providing a genome coding for a product, the presence of which can be readily identified (the "marker product") and carrying a promoter, which responds to the presence of the identifying protein in indicator cells, by switching express Ion of the reporting product between expressing and nonexpressing states.For example, HI,t, when it infects suitable indicator cells, makes tat and ev. The indicator cells tan thus be provided with a genome (such as bv infection witn an appropriate recombinant retrovirus) which codes for a marker gene, such as the alkaline phosphatase gene, ss-galactosidase gene or the luciferase gene, and a promoter, such as the HIV promoter, which controls expression of the marker gene.When the indicator cells are exposed to a clinical sample to be tested, and the sample contains HIV, the indicator cells become infected with HI'., resulting in tat and/or rev expression (an identifying protein) therein.The HIV expression controls in the indicator cells would then respond to tat and'or rev proteins by switching expression of genes encoding S- galactosidase, luciferase, or alkaline phosphatase (marker products) from normally "off" to "on." In the case of ss-galactosidase or alkaline phosphatase, exposing the cells to substrate analogues results in a color or fluorescence change if the sample is positive for HIV. in the case of luciferase, exposing the sample to luciferin will result in luminescence if the sample is positive for HIV. For intracellular enzymes such as ss-galactosidase, the viral titre can be measured directly by countIng colored or fluorescent cells, or making cell extracts and performing a suitable assay.

For secreted enzymes, such as an engineered form of alkaline phosphatase, small samples of culture supernatant are assayed for activity, allowing continuous monitoring of a single culture over tIme.

Thus, different forms of this marker system can be set for different purposes. These include counting active virus or sensitively and simply measuring viral spread in a culture and the Inhibition of this spread by various rugs.

Further specificity can be incorporated i@ the preceding system by testing for the presence of the virus either with or without neutralizing antibodies to that virus. For example, in one portion of the clinics sample being tested, neutralizing antibodies to HIV may te present; whereas In another portion there would be neutralizing antibodies. If the tests were negative the System where there were antibodies and positive where there were no antibodies, this would assist confirming the presence of HIV.

Within an analogous system for an in vitr@ assay, the presence of a particular gene, such as a viral gene, may be determined in a cell sample. In tis case, the cells of the sample are infected with a suitable retrovlral vector which carries the reporter ger.e linked to the expression controls of the virus nterest. The reporter gene, after entering the sample cells, will express its reporting product (such as -galactosidase or luciferase) only if the host cell expresses the appropriate viral proteins.

These assays are more rapid and sensitive, since the reporter gene can express a greater amount of reporting product than identifying agent present, which results in an amplification effect. Example 6 describes a representative technique for detecting a gene which expresses an identifying protein.

EXAMPLE 6 HIV-Specific Marker System or Assay A. Constructs Reporter constructs under the control of the HIV expression system are shown in Figure 12 (a recombinant retroviral vector) and in Figure 13 (a simple plasmid used by transfaction). The pieces of these preferred vector and plasmid reporters were derived as follows: The retroviral backbone was derived from the construct pAFVXM (Krieger et al., Cell 38:384, 1984), which had been linearized usIng Xho I and Cla I. SV2neo was obtained from the plasmid pKoneo (Hanahen, un?ubl.) by isolation of the 1.8 kb Cla I fragment.

The HIV LTR was Isolated as a 0.7 kb Hind III fragment from the plasmid pC15CAT (Arya et al., Science 229:69, 1985). Beta-gal was obtained from the plasmid pSP65 ss-gal (Cepko, pera. comm.) as a Hind III-Sma fragment. A secreted form of human placental alkaline phosphatase was produced by introduction of a universal terminator sequence after amino-acid 489 of the cell surface form of alkaline phosphatase (as described by Berger et al., Gene 66:1, 1988). The secreted alkaline phosphatase gene was isolated as a 1.8 kb Hind III to Kpn I fragment.The CRS-CAR sequences from HIV env were obtained by isolating the 2.1 kb Kpn I to Barn HI fragment from HTLVIIIB/BH10R3 (Fisher et al., Science 233:655, 1986). This fragment was inserted into pCC31 linearized by Bam HI, and Kpn I pUC31 is PUC19 (Yanisch-Perron et al., Gene 33:103, 1985) with extra Xho I, B-gl II, Bssh II and Nco I sites between the Eco R1 and Kpn I sites of pUC19. The Ban H1 site of the resulting construct was converted to a Nco I site to allow resection of the CRS-CAR sequences by Nco I digestion. The SV40 t intron was obtained from pSVOL (de Wet et al., Mol. Cell.Biol. 7:725, 1987) as a 0.8 kb Nco I to Ban H1 fragment.

B. Indicator Cells and Retroviral Vectors Human T-cell (H-9, CEM and Sup TI) and monocyte (U-937) cell lines were obtained from ATCC, ant maintained in RPM1 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin.

The nonretroviral vectors were introduced into cell lines by electroporation using a Bio-Rad Gene Pulser. The cell lines were selected in G-418 (1 mg/ml, for 2-3 weeks to obtain stable G-418R cell lines, ten dilution cloned to obtain clonal cell lines.

The pAF vectors were transfected into te PA317 packaging cell line as a calcium phosphate precipitate (Wigler et al., Cell 16:777, i979). The virus-producing PA317 cells were co-cultivated wi human monocyte eell lines for 24 hours in the presence of polybrene, after which the suspension cells were removed and selected in G-418 and subcloned as above.

C. Assav Stable cell lines were infected with HIV (HTLV III@) and the cells (ss-gal) or media (alkaline phosphatase) assayed on a daily basis for 6 days post-infection.

ss-Galactosidase Assav In infected cells could be assayed by either: (i) In situ histochemical staining as described by MacGregor et al. Somatic Cell and Mol. Genetics 13:253, 1987) ; or (ii) by using cell extracts in a solution enzymatic assay with ONPG as a substrate (Norton and Coffin, Mol. Cel. Biol. 5:281, 1985).

Soluble Alkaline Phosphatase Assav Medium was removed from infected cells, microfuged for 10 seconds, and then heated to 68 C for 10 minutes to destroy endogenous phosphatases. The medium was then microfuged for 2 minutes and an aliquot (10-50 l removed for assay. 100 l of buffer (1 M diethanolamine, pH 9.8; 0.5 Mm MgCl2; 10 L-homoarginine) was added and then 20 l of 120 M p-nitrophenylphosphate (in buffers) was added. The A 05 of the reaction mixture was monitored using an automatic plate reader.

Figures 14 and 15 depict typical results of a tine course of infection of Sup Tl cells using the alkaline phosphatase assay in the presence of varying concentrations of antiviral drugs. The "+" and "-" on day 6 indicate the presence or absence of syncytia.

The present invention provides a number of other techniques (described below) which can be used with the retroviral vector systems employed above, so as to enhance their performance, Alternatively, these techniques may be used it other gene-delivery systems.

V. Packaging Cell Selection This aspect of the present invention is based, in part, upon the discovery of the major causes of low recombinant virus titres from packaging cells: and of techniques to correct those causes. Basically, at least five factors may be postulated as causes for 1 low recombinant virus titres: 1. the limited availability of viral packaging proteins; 2. the limited availability of retroviral vector RNA genomes; 3. the limited availability of cell membrane for budding of the recombinant retroviruses; the limited intrinsic packaging efficiency of the retroviral vector genome; and 5. the density of the receptor specific for the envelope of a given retrovirus.

As noted above, the limited availability of viral packaging proteins is the initial limiting factor in recombinant retrovirus production from packaging cells. When the level of packaging protein in the packaging cells is increased, titre increases to about 5 infectious units/milliliter, following which increasing packaging protein level has no further effect on titres. However, titres can be further augmented by also increasing the level of retroviral vector genome available for packaging. Thus, as described herein, it is advantageous to select producer cells that manufacture the maximum levels of packaging proteins ant retroviral vector genomes.It has been discovered that the methods of identifying, and thus selecting, packaging cells and producer cells, described earlier under the sectlon entitled "Background of the InventIon," tend to lead to selection of many producer cells which produce low titres for the reasons descrIbed below.

The present invention takes advantage of the previously disadvantageous fact that the protein expression level of a gene downstream from the 5' LTR or other promoter, and spaced therefrom by an intervening gene, is substantially less than if the intervening gene were absent. In the present invention, the selectabie gene is placed downstream from a gene of the packaging genome or the gene of interest carried by the vector construct, but is still transcribed under the control or the viral ' LTR or other promoter without any splice donor or splIce acceptor sites. This accomplishes t.:c things.First, since the packaging genes or genes or interest are now upstream with no intervening gene between themselves and the promoter, their corresponoing proteins (packaging protein or protein of Interest) will be expressed at a higher level (5- to 20-fold) than the selectabie protein. Second, the selectable protein will be expressed on average at a lower level, with the distribution of level of expression shifting toward lower levels. In the case of the phleor protein, this shift in distribution is illustrated by the broken curve indicated in Figure 16. However, the selection level for resistance to phleomycin remains the same, so that only the top-end expressing cells survive.The levels of the packaging protein or of the protein of interest will still be proportional, only in this case, a higher level of selectable protein corresponds to a much higher level of packaging protein or protein of interest.

Preferably, the foregoing procedure is performed using a plasmid carrying one of the provirai gag/pol or env packaging genes, along with a first selectable gene. These cells are then screened for the cells producing the highest levels of protein by reaction with an antibody against env (or possIbly gag/pol), a second fluorescent antibody, and then sorted on a fluorescence-actIvated cell sorter (FACS).

Alternatively, other tests for protein level may be used. Subsequently, the procedure and screening are repeated using those selected cells, and the other of the gag/pol or env packaging @g genes. In this step, a second selectable gene (different from the first) would be required downstream from the packaging gene and the cells producing the large amount of the second viral protein selected. The procedure and screening are then repeated using the surviving cells, with a plasmid carrying the proviral vector construct bearing the gene of interest and a third selectable gene, different from the first or second selectable gene.As a result or this procedure, cells producing high titres of the desired recombinant retrovirus will be selected, and these can be cultured as required to supply recombinant retrovirus. In addition, gag and pol can oe independently introduced and selected for.

Example 7 describes the construction of gag/pol and env plasmids designed to use these procedures.

EXAMPLE 7 Plasmids Designed to Make High Levels of Packaging Proteins (Figure 7) 1. The 2.7 kb Xba I fragment from pPAM (Miller et al., Mol. Cell. Biol. 5:431, 1985), which contains the amphotropic env segment, was cloned in pU18 at the Xba I site, then removed with Hind III ar.d Sma I. This fragment was cloned into the vector pRSV neo (Gorman et al., Mol. Cell. Biol. 2:1044, 1982, Southern at al., J. Mol. Appl., Genet. 1:327, 1982) cut with Hind IiI and Pvu II, to give pRS'J env. A 0.7 kb Bam H1 to BstE II fragment from the plasmid pUT507 (Mulsant et al., Somat. Cell. Mol.Genet. 14:243, 1988) with the BstE II end filed in carries the phleo resistance coding Sequence. The 4.2 kb Ban H1 to Xho fragment, the contiguous 1.6 kb Sho I to Xba I (Xba illed in) from RSVenv, and the phleo fragment were ligated to give pRSVenv-phleo.

2. A fragment from the Pst I site at nucleotide 563 of MLY (RNA Tumor Viruses, Vol. II, 1985, Cold Spring Harbor) to the Sc. I site at 5870 was derived from pMLV-K (Miller et al., 1985, op. cit.) and cloned in the Pst I to Bam H1 (Bam H1 filled-in) fragment from p4aA8 (Jolly et al., Proc. Natl. Acad.

Sci. USA 80:477, 1983) that has the SV40 promoter, the pBR322 ampicillin resistance and origin of replication and the SV40 poly A site. This gives pSVgp. pSVgpDHFR was made using the following fragments: the 3. 6 kb Hind III to Sal I fragment from pSVgp containing the SV4O promoter plus MLY gag and some pol sequences; the 2.1 kb Sal I to Sca I fragment from pMLV-K with the rest of the pol gene, the 3.2 ko Xba I (Xba I filled-in) to Pst I fragment from pF400 with the DHFR gene plus poly A site, pBR322 origin and half the ampicillin resistance gene; the 0.7 kb Pst I to Hind III fragment from pBR322 with the other half of the ampicillin resistance gene.

This gives pSVgp-DHFR. All these constructs are shown in Figure 7. These plasmids can be transfected into 3T3 cells or other cells and high levels of gag, pol or env obtained.

An additional method for accomplishing selection is to use a gene selection in one round and its antisense in a subsequent round. For example, gag/pol may be introduced into an HPRT-deficient cell with the HPRT gene and selected for the presence of this gene using that media which requires HPRT for the salvage of purines. In the net rotund, the antlsense to HPRT could be delivered downstream to env and the cel selected in 6 thioguanine or the HPRT-deficient phenotype.Large amounts of antlsense HPRT would be required in order to inactivate the HPRT gene transcripts, assuming no reversion occurred.

Envelcce Substitutions The ability to express gagpol and env function separately allows for manipulation of these functions independently. A cell line tat expresses ample amounts of gagpol can be used, for example, to address questions of titre with regard to env. One factor resulting in low titres is the density of appropriate receptor molecules on the target cell or tissue. Given that env expression is from a separate unit, a variety or envelope genes (requiring different receptor proteins), such as xenotropic, polytropic, or amphotropic envs from a variety of sources, can be tested for highest titres on a specific target tissue.Furthermore, envelopes from nonmurine retrovirus sources can be used for pseudotyping a vector. In addition, hybrid envelopes (as described below) can be used in this system as well, to tailor the tropism (and effectively increase titres) of a retroviral vector. Conversely, a cell line that expresses ample amounts of a given envelope gene can be employed to address questions of titre with regard to gag and pol.

VI. Alternative Viral Vector Packaaina Techniques Two additional alternative systems can he use to produce recombinant retroviruses carrying the vector construct. Each of these systems takes advantage of the fact that the insect virus, baculovirus, and the mammalian viruses, vaccinia and adenovirus, have been adapted recently to make large amounts of any given protein for which the gene has been cloned. For example, see Smith et al. (Mol. Cell. Biol. 3:12, 1983); Piccini et al. (Meth. Enzymology, 153:545, 1987); and Mansour et al. (Proc. Natl. Acad. Sci. USA 82:1359, 1985).

These viral vectors can be used to produce proteins in tissue culture cells by insertion of appropriate genes into the viral vector and, hence, could be adapted to make retroviral vector partIcles.

Adenovirus vectors are derived from nuclear replicating viruses and can be defective. Genes can be inserted into vectors and used to express proteins in mammalian cells either by in vitro construction (Ballay et al., EMBO J. 4:3861, 1985) or by recombination in cells (Thummel et al., J. Mol. Attl. Genetics 1:435, 1982).

One preferred method is to construct plasmlds using the adenovirus Major Late Promoter (MLP) driving: (1) gag/pol, (2) env, (3) a modified viral vector construct. A modified viral vector construct is possible because the U3 region of the 5' LTR, which contains the viral vector promoter, can be replaced by other promoter sequences (see, for example, Hartman, Nucl. Acids Res. 16:9345, 1988) . This portion will be replaced after one round of reverse transcriptase by the U3 from the 3' LTR.

These plasmids can then be used to make adenovirus genomes in vitro (Ballay et al., op. cit.), and these transfected in 293 cells (a human cell line making adenovirus E1A protein), for which the adenoviral vectors are defective, to yield pure stocks of gag, pol, env and retroviral vector carried separately in defective adenovirus vectcrs. Since the titres of such vectors are typically 107-10/ml, these stocks can be used to infect tissue culture cells simultaneously at high multiplicity. The cells will then be programmed to produce retroviral proteins ar.d retroviral vector genomes at high levels. Since the adenovirus vectors are defective, no large amounts of direct cell lysls will occur and retroviral vectors can be harvested from the cell supernatants.

In an alternative system (which is more truly extracellular), the following components are used: 1. gag/pol and env proteins made in the baculovirus system in a similar manner as described in Smith et al. (supra) (or in other protein production systems, such as yeast or E. coli) 2. viral vector R?'TA made in the known T7 or SP6 or other in vitro RNA-generating system (see, for example, Flamant and Sorge, j. Virol. 62::1827, 1988); 3. tRNA made as In (2) or purified rom yeast or mammalian tissue culture cells; liposomes (with embedded env protein) and 5. cell extract or purified necessary components (when identified) (typically from mouse cells) to provide env processing, and any or other necessary cell-derived functions.

Within this procedure (1), (2) and (3) are mixed, and then env protein, cell extract and pre- lipcsome mix (lipid in a suitable solvent) added.

may, however, be necessary to earlier embed the env protein in the liposomes prior to adding the resulting liposome-embedded env to the mixture of (1), (2), ant (3). The mix is treated (e.g., by sonication, temperature manipulation, or rotary dialysis) to allow encapsidation of the nascent viral particles with lipid plus embedded env protein in a manner similar to that for liposome encapsidation of pharmaceuticals, as described in Gould-Fogerite et al., Anal. Biochem.

145:15, 1985). This procedure allows the production of high titres of replication incompetent recombinant retroviruses without contamination with pathogenic retroviruses or replication-competent retroviruses.

VII. Cell. Line-Specific Retroviruses - "Hybrid Envelope" The host cell range specificit of a retrovirus is determined in part by the env gene products. For example, Coffin, J. (RNA Tumor Viruses 2:25-27, Cold Spring Harbor, 1985) notes that the extracellular component of the proteins from marine leukemia virus (ML'J) and Rous Sarcoma virus (RSV) are responsible for specific receptor binding. The cytoplasmic domain of envelope proteins, on the other hand, are understood to play a role in virion formation.

While pseudotyping (i.e., the encapsidation of viral RNA from one species by viral proteins of another species) does occur at a low frequency, the envelope protein has some specificity for virion formation of a given retrovirus. The present invention recognizes that by creating a hybrid env gene product (i.e., specifically, an env protein having cytoplasmic regions and exogenous binding regions which are not in the same protein molecule in nature) the host range specificity may be changed independently from the cytoplasmic functIon.

Thus, recombinant retroviruses can be produced which will specifically bind to preselected target cells.

In order to make a hybrid protein in which the receptor binding component and the cytoplasmic component are from different retroviruses, a preferred location for recombination is within the membrane-spanning region of the cytoplasmic component. Example 8 describes the construction of a hybrid env gene which expresses a protein with the CD binding portion of the HIV envelope protein coupled to the cytoplasmic domain of the MLV envelope protein.

EXAMPLE 3 Hybrid HIV-MLV Envelopes A hybrid envelope gene is prepared using In vitro mutagenesis (Kunkal, Proc. Natl. Acad. Sci. US 82:488-492, 1985) to introduce a new restriction site at an appropriate point of junction. Alternatively, if the to envelope sequences are on the same plasmid, they can be joined directly at any desired point using in vitro mutagenesis. The end result in either case is a hybrla' gene containing the 5' end of the MIV gp 160 and the 3' end of ML3 p15E.The hybrid protein expressed by the resulting recombinant gene is illustrated in Figure 13 and contains the HIV gp120 (CD4 receptor binding protein) , the extracellular portion of HIV gp =1 (the gp 120 binding and fsigenie regions), and the cytoplasmic portion of ML'? p15E, with the joInt occurring at any of several points within the host membrane. A hybrid gene made by fusion at the new Eco R1 site shown in Figure 16 Is expressed under control of the CMVIE gene in human Sup T1 cells and leads to syncytia, as is the case with the authentic HIV env gene.Thus the hybrid is correctly transported to the cell surface and displayed there.

While Example S illustrates one hybrid protein produced from to different retroviruses, the possibilities are not limited to retroviruses or even simply other viruses. For e:.a.*,pie, the beta-recer cr portion of human interleukin-2 may be combined with the envelope protein of MLV. In this case, a recombination would preferably be located in the gp 70 portion of the MLV env gene, leaving an intact p15E protein.

Furthermore, the foregoing technique may be used to create a recombinant retrovirus with an envelope protein which recognizes antibody Fc segments. Monoclonal antibodies which recognize only preselected target cells only could then be bound to such a recombinant retrovirus exhibiting such envelope proteins so that the retrovirus would bind to and infect only those preselected target cells.

VIII. Site-Specific Integration Targeting a retroviral vector to a predetermined locus on a chromosome increases the benefits of gene-delivery systems. A measure of safes is gained by direct integration to a "safe" spot cn a chromosome, i.e., one that is proven to have nc deleterIous effects from the insertion of a vector.

Another potential benefit is the ability to direct a gene to an "open" region of a chromosome, where its expression would be maximized. Two techniques for integrating retrovIruses at specific sites are described below.

(i) Homologous Recombination One technique for integrating an exogenous gene of a vector construct of a recombinant retrovirus into a specific site in a target cell's DNA employs homologous recombination. Plasmids containing sequences of DNA of greater than about 300 bp that are homologous to gnomic sequences have been shown to interact (either by replacement or insertion) with those genomic sequences at a rate that is greater than 10 -fold over a specific interaction in the absence of such homology (see Thomas and Capecchi, Cell 51:503-12, 1957; ant Doetscheman et al., Nature 330::576-78, 1957). It has been shown that an Insertion event, or alternatively, a replacement event, may be driven by the specific design of the vector.

In order to employ homologous recombination in site-specific retroviral integration, a vector construct should be modified such that (a) homologous sequences (to the target cell's genome) are incorporated into the construct at an appropriate location; and (b) the norma mechanism of integration does not interfere with the targeting occurring due to homologous sequences. A preferred approach in this regard is to add homologous sequences (greater than about 300 bp) in the 3' LTR, downstream from the U3 Iwierted repeat. In this approach, the construct is initially made with a region of homology inserted in the 3' LTR at the Nhe 1 site in U3.Reverse transcription in the host cell will result in a duplication of the region of horology in the 5' LTR within 31 bp of the end of the inverted repeat (IR).

Integration into the host genome will occur in the presence or absence of the normal integration mechanism.

The gene in the vector may be expressed, whether the LTR or from an internal promoter. This approach has the effect of placing a region of homology near a potential free end of the double-stranded retrovirus vector genome. Free ends are known to increase the frequency of homologous recombination by a factor of approximately 10. In this approach, it may be necessary to defeat the normal mechanism of integration, or to at least modify it to slow down the process, allowing tIme for homologous DNAs to line up. Whether this latter modification is required in a particular case can be readily ascertained by one skilled in the art.

(ii) Intearase Modification Another technique for integrating vector construct into specific, preselected sites of a target cell's genome involves integrase modification.

The retrovirus pol gene product is generally processed into four parts: (i) a protease which processes the viral gag and pol products; (ii) the reverse transcriptase; and (iii) RNase H, which degrades RNA of an RNA/DNA duplex; and (iv) the endonuclease or "integrase." The general integrase structure has been analyzed by Johnson et al. (Proc. Natl. Acad. Sci. USt 83:7648-7652, 1986). It has been proposed that this protein has a zinc binding finger with which 1 interacts with the host DNA before integratIng the retroviral sequences.

In other proteins, such "fingers" allow the protein to bind to DNA at particular sequences. One illustrative example is the steroid receptors. In this case, one can make the estrogen receptor, responding estrogens, have the effect of a glucocorticoid receptor, responding to glucocorticoids, simply by substituting the glucocortlcoid receptor "finger" (i.e., DNA bind""- segment) in place of the estrogen receptor finger segment in the estrogen receptor gene. In this example, the position in the genome to which the proteins are targeted has been changed.Such directing sequences can also be substItuted into the integrase gene in place the present zinc finger. For instance, the segment coding for the DNA binding region of the human estrogen receptor gene may be substituted in place of the DNA binding region of the integrase in a packaging genome.

Initially, specific integration would be tested by means of an in vitro integration system (Brown et al., Cell 29:347-356, 1987). To confirm that the specificit would be seen in vivo, this packaging genome 15 used to ake infections vector particles, and infection of a@ for expression in either the whole animal or targeted tissue through the selective use of housekeeping tissue-specific promoters to transcribe the packaging functions. The vector to be delivered is also inserted into a line of transgenic animals with a tissue-specific or housekeeping promoter.As discussed above, te vector can be driven off such a promoter substituting for the U3 region of the 5' LTR (Figure 19). This transgene could be inducible or ubiquitous in its expression. This vector, however, is not packaged.

These lines of animals are then mated to the gag/pol/env animal and subsequent progeny produce packaged vector.

The progeny, which are essentially identical, are characterized and offer an unlimited source of primary producing cells. Alternatively, primary cells making gag/pol and env and derived from transgenic animals can be infected or transfected in bulk with retrovirus vectors to make a primary cell producer line. Many different transgenic animals or insects could produce these vectors, such as mice, rats, chickens, sine, rabbits, cows, sheep, fish and flies. The vector ar,d packaging genomes would be tailored for species infection specificity and tissue-specific expression through the use of tissue-specific promoters art different envelope proteins. An example of such procedure is illustrated in Figure 20.

Although the following examples of transgenic production of primary packagIng lines are described only for mice, these procedures could be extended to other species by those skilled in the art. Given the horology to MLV sequences in mice genome, the final preferred animals would not be mice.

integration into estrogen-sensitIve and estrogen- nonsensitlve cells compared in culture.

Through use of this technique, incoming viral vectors may be directed to integrate into preselected sites on the target cell's genome, dictated by the genome-binding properties of site-specisic DNA-binding protein segments spliced into the integrase genome. It will be understood by those skilled in the art that the integration site must, in fact, be receptive to the fingers of the modified integrase. For example, most cells are sensitive to glucocorticoids and hence their chromatin has sites for glucocorticoid receptors.Thus, for most cells, a modified ed integrase having a glucocorticoid receptor finger would be suitable to integrate the proviral vector construct at thcse glucocortlcoid receptor-bindIng sites.

IX. Production of Recombinant Retroviral Vectors Transsonic Animals Two problems p prevIously described with helper line generation of retroviral vectors (a) difficulty in generating large quantities of vectors; and (b) the current need to use permanent instead of primary cells to make vectors. These problems can be overcome with producer or packaging lines that are generated in transgenic animals. These animals would carry the packaging genomes and retrovlral vector genomes. Current technology does not allow the generation of packaging cell lines and desired vector producing lines in primary cells due to their limited life span. The current technology is such that extensive characterization is necessary, which elininates the use of primary cells because of senescence. However, individual lines of transgenic animals can be generated by the methods provided herein which produce the packaging functions, such as gag, pcl or env. These lines of animals are then characterlzec EXAMPLE 9 Production of Gaa-Pol Proteins Using House:;:een Promoters for Ubiauitous Expression in Transqenic Animals An example of a well-characterized housekeeping promoter is the HPRT promoter. HPRT is a purine salvage enzyme which expresses in all tissues.

(See Patel et al., Mol. Cell Biol. 6:4 - -403, 1986 and Melton et al., Proc. Natl. Acad. Sci. 81:2147-2151, 1984). This promoter would be inserted in front of various gag/pol fragments (e.g., Bal I/Sca I; Aat II/Sca I; Pst I/Sca I of MoMLV; see RNA Tunor Viruses 2, 1985, Cold Spring Harbor Laboratory, 1985) that are cloned in Bluescript plasmids (Strategene, Inc.) using recombinant DNA techniques (see Maniatis et al., Molecular Clonina: A Laboratorv Manual, Cold Spring Harbor, 1982). The resulting plasmids are purified (Maniatis et al., op. cit.) and the relevant genetic information isolated using Geneclean (Bio 101) or electroelution (see Hogan et al.(Eds.) Manipulating the Mouse Embryo: A Laboratorv Manual, Cold Spri Harbor, 1986).

These fully characterized DNAs would be microinjected in the pronucleus of fertilized mouse ova at a concentration of 2 ug/ml. Live born mice would be screened by tail blot analyses (see Hogan et al., op.

cit.). Transgenic-positive animals would be characterized for expression levels of gag-pol proteins by immunoprecipitation of radiolabeled primary cells, such as fibroblast (see Harlow et al., eds. Antibodies: A Laboratorv Manual, Cold Spring Harbor, 1988). Animals would then be bred to homozygosity for establishment of animal lines that produce characterized levels of gag-pol.

EXAMPLE 10 Production of env Proteins/Mvbrid Envelope Proteins Using Housekeeping Promoters for Ubiauitous Expression in Transqenic Animals This example utilizes the HPRT promoter for express ion of either envelope or hybrid envelope proteins. The envelope proteins could be from any retrovirus that is capable of complementing the relevant gag-pol, in this case that of MLV. Examples are ecotropic MLV, amphotropic MLV, xenotropic MLV, polytropic MLV, or hybrid envelopes. As above, the envelope gene would be cloned behind the HPRT promoter using recombinant DNA techniques (see Maniatis et al., op. cit.). The resulting "minigene" would be isolated (see Hogan et al., op. cit.), and expression of envelope protein would be determined (Harlow et al., op. cit.).

The transgenic envelope animals would be bred to hcmozygosity to establish a well-characterized envelope animal.

EXAMPLE 11 Production of ga@-pol-env Animals Using Housekeeping Promoters for Ubicuitous expression in Transaenic Animals This would use the well-characterized gag-pcl animals, as well as the animals for the establishment o a permanent gag-pol/envelope animal line. This would involve breeding to homozygosity and the establishment of a well-characterized line. These lines would then be used to establish primary mouse embryo lines that could be used for packaging vectors in tissue culture.

Furthermore, animals containing the retroviral vector could be bred into this line.

EXAMPLE 12 Production of Tissue-Scecific ExDression of gag-ool-env or Hybrid Envelope in Transaenic Animals The example given here is to direct tissue expression of the gagpol, envelope, or hybrid envelope to specific tissues, such as T-cells. This involves the use of CD2 sequences (see Lang et al., EMBO J. 7:16751682, 1988) that give position and copy number independence. The 1.5 kb Bam H1/Hind III fragment from the CD2 gene would be inserted in front of gag-pol, envelope, or hybrid envelope fragments using recombinant DNA techniques. These genes would be inserted into fertilized mouse ova by microinjection. Transgenic animals would be characterized as before. Expression in T-cells would be established.Animals would be bred te homozygosity to establish well-characterized lines of transgenic animals. Gag-pol animals would be mated to envelope animals to establish gag-pol-env animals expressing only in T-cells. The T-cells of these animals would then be a source for T-cells capable of packaging retroviral vectors. Again, vector animals could be bred into these gag-pol-env animals to establish T-cells expressing the vector.

This technique allows the use of other tissuespecific promoters, such as milk-specific (whey) pancreatic (insulin or elastase), or neuronal (myelin basic protein) promoters. Through the use of promoters, such as milk-specific promoters, recombinant retroviruses may be isolated directly from the biological fluid of the progeny.

EXAMPLE 13 Production of Either Housekeepina or Tissue-Stecific Retroviral Vectors in Transaenic Animals The insertion of retroviruses or retroviral vectors into the germ line of transgenic animals results in little or no expression. This effect, described Sy Jaenisch (see Jahner et al., Nature 298:623-623, i932), is attributed to methylatlon of S' retroviral TR sequences. This technique would overcome the methylation effect by substituting either a housekeeping or tissue-specific promoter to express the retroviral vector/retrovirus.The U3 region of the 5' LTR, which contains the enhancer elements, is replaced with regulatory sequences from housekeeping or tissuespecific promoters (see Figure 20). The 3' LTR is fully retained, as it contains sequences necessary for polyadenylation of the viral RNA and integration. As the result of unique properties of retroviral replication, the U3 region of the 5' LTR of the integrated provirus is generated by the U3 region of the 3' LTR of the infecting virus. Hence, the ' is necessary, while the 5' U3 is dispensable. Substitution of the 5' LTR U3 sequences with promoters and insertion into the germ line of transgenic animals results in lines of animals capable of producing retroviral vector transcripts. These animals would then be mated to gag-pol-env animals to generate retroviral-produc Ing animals (see Figure 20).

From the foregoing it will be appreciated that, although specific em~odiments of the invention have been described herein for purposes of illustration, varIous modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Claims (5)

1. A method of testing a sample for the presence of a virus carrying a particular gene, said gene being capable of expressing an identifying protein in indicator cells having (1) a marker gene which is capable of expressing a marker product; and (2) control sequences which respond to the presence of the identifying protein by switching expression of the marker gene between expressing and non-expressing states; the method comprising contacting indicator cells with the sample, and testing for the presence of the marker product.

2. A method of testing for the presence of a particular gene in a cell sample, said gene being capable of expressing an identifying protein, comprising: infecting cells within the sample with a recombinant retrovirus comprising a vector construct coding for (1) a marker gene which is capable of expressing a marker product in the cell sample; and (2) control sequences which respond to the presence of the identifying protein in the cells within the sample by switching expression of the marker gene between expressing and non-expressing states; and testing for the presence of the marker product.

3. An indicator cell suitable for testing for the presence of a virus carrying a particular gene which is capable of expressing an identifying protein in the indicator cell, the indicator cell having a genome coding for (1) a marker gene which can express a marker product in the indicator cell; and (2) control sequences which respond to the presence of the identifying protein in the indicator cell by switching expression of the marker gene between expressing and nonexpressing states.

4. A hybrid env gene useful for preparing a retrovirus which can selectively carry a gene of interest to a target cell, the env gene coding for: (a) a cytoplasmic segment of a first retroviral phenotype; and (b) a binding segment exogenous to the first retroviral phenotype, the binding segment being capable of selectively binding to the target cell.

5. A method of producing recombinant retroviruses, comprising: mating a transgenic animal or insect containing a gag/pol env viral construct, with a transgenic animal or insect containing a vector construct containing a promoter; isolating the progeny of said transgenic animals or insects; isolating selected cells from the progeny; growing said cells in an appropriate medium; and isolating recombinant retroviruses from the cells.