CA2084659A1

CA2084659A1 - Vectors containing hiv packaging sequences, packaging defective hiv vectors, and uses thereof

Info

Publication number: CA2084659A1
Application number: CA002084659A
Authority: CA
Inventors: Joseph G. Sodroski; William A. Haseltine; Marck Poxnansky; Andrew Lever
Original assignee: Dana Farber Cancer Institute Inc
Current assignee: Dana Farber Cancer Institute Inc
Priority date: 1990-06-20
Filing date: 1991-06-18
Publication date: 1991-12-21
Also published as: EP0536234A4; EP0536234A1; JP3264281B2; WO1991019798A1; JPH05508320A

Abstract

Packaging defective and packaging proficient HIV vectors are disclosed. These vectors can be used to establish HIV packaging defective cell lines, and to package desired genes. These cell lines can be used in developing a vaccine, HIV antibodies and as part of a system for gene transfer. The packaging proficient vector can be used to target HIV target cells.

Description

W O 91/1979B PCT/US9l/04335 .

2 Q ~

VECTORS CONTAININS HIV PACKAGING SEQ~IENCES, PACKAGING_DEF~CTIVE HIV VECTORS, AND USES THEREOF
The present in~ention is directed to vectors including vectors comprising a packaging defective HIV provirus, a vector comprising a HIV packaging sequence and a gene to be transferred, the use of the packagin~ defective ~ectors ~o create HIV packaging defective cell lines, and the uses of the vectors and cell lines. Most preferably, the HIV
provirus is an HIV-l provirus.

The human immunodeficiency virus (HIV-I, also referred to as HTLV~III, LAV or HTLV-III/LAV) is the etiological agent of the acquired immune deficiency syndrome (AIDS) and relat2d disorders [Barre-Sinoussi, et al., Science 220:868-871 (1983~; Gallo et ~l, Science ~:500-503 (1984); Levy et al., Science 225:840-842 (1984); Popovic et al., Science 224:497-500 (1984); Sarngadharan et al., Science ~:506-508 (1984); Siegal et al., N. En~l. J. Med. 305:1439-1444 (1981)l. The disease is characterized by a long asy~ptomatic period followed by progressive degeneration of the im~une system and the central nervous system. Studies of the virus indicate that replication is highly regulated, and both latent and lytic infection of the CD4 positive helper subset of T-lymphocytes occur in tissue culture [Zagury et al., Science ~l:850-853 (1986)]. The expression of the virus in infected patients also appears to be regulated to enable evasion of the i~mune response. ~oleeular studies of the regulation and geno~ic organization of HIV-I show that it encqdes a number of genes [Ratner et al., Nature 313:277-284 (1985); Sanchez-Pescador et al., Science ~:484-492 ~1985);
Muesing et al., Nature 313:450-457 (1985); Wain-Hobson et al., Cell 40 9-17 (1985)].

W O 91/19798 PCT/~9t/04335 ~,~X ~9 The other primate immunodeficiency viruses, HI~-2 and simian immunodeficiency virus (SIV) also share ~any of the same struceural and regulatory genes such as ~a~, Q~, anv, tat, rev and nef [Guyader, M., et al., Nature ~26:662-669 (1987); Chakrabarti, L., et al., Nature 328:543-547 (1987), which are incorporated herein by reference].

Retroviruses are typically classified as belonging to one of three subfamilies, namely oncoviruses, spumaviruses and lentiviruses. Infection by oncoviruses is typically associated with malignane disorders. These viruses typically contain a single-stranded, plus-strand RNA genome of approximately 8,000 to lO,OOO
nucleotldes encompassing the g~g, ~1 and env, genes, as well as long terminal repeat (LTR) sequences. Oncoviruses typically contain an oncogene. It is generally believed that spumaviruses are not pathogenic ~Lvivo, although they induce foamy cytopathic changes in tissue culture. Infection by lentiviruses is generally slow and causes chronic debilitating diseases after a long latency period. ~hese viruses, in addition to the ~3g, ~l, and env genes possess a nu~ber of additional genes with regulatory functions.

The human i~munodeficiency viruses (HIV) has been classified as a lentivirus, because lt too causes slow infection and has structural properties in common with such viruses. [See Haase, A.T., Nature. ~ 130-136 (1986)].

All known retroviruses share features of the replicative cycle, including packaging of viral RNA into virions, entry into target cells, reverse transcription of viral RNA to form the DNA provirus, and stable integration of the provirus into the target cell genome [Coffin, J., J. Gen, Virol. 42:1-26 , 2~g~9 (1979)]. Replication-competent pro~iruses, at a mini~um, contain regulatory long terminal repeats (LTRs) and the ~a~, pro, pol, and env genes which encode core pro~eins, a protease, reverse transcriptase/RNAse H/integrase and envelope glycoproteins, respectively [J. Gen. Virol. 42, su~ra]. The LTRs contain c s-acting sequences iD~portant for integraeion, ~ranscription and polyadenylation.

HIV shares the ~3g, p~, pol and env genes, respectively with other retroviruses [Haseltine, W.A., Journal_of Acquired Immune Deficiencv Svndrome, 1:217-240 (1988)]. HIV also possesses additional genes modulating viral replication. The HIV-l genome encodes y~, v~r, tat, rev, v~ and nef proteins [Haseltine, W.A., Journal of Acquired Im~une Deficiency Syndrome, su~ra]. Additionally, the long terminal repeats (L~Rs) of HIV contain cis-acting sequences that are important for integration, transcription and polyadenylation.
Additional c s-acting slgnals allow regula~ion of HIV
sequences by some of the novel HIV gene products, (Haseltine, W.A., Journal of Acquired Immune Deficiency Syndrome, su~ra). Sodroski et al., Scienc~ 1549-1553 (1986); Arya et al., Science ~2~:69-73 (1985); Sodroski et al., Science 227:171-173 (1985); Sodroski et al., Nature ~ 412-417 (1986); Feinberg et al., Cell 46:807-817 (1986) Wong-Staal et al, AIDS Res. and Human Retroviruses 3: 33-39 (1987); which are all incorporated herein by reference]. Th~ region between the S' major splice donor and the ~ gene initiation coton is highly conserved in different HIV-l strains sequenced to date [Myers, G., et al, Theoretical Biolo~Y and Bi_phYsics, (1988)].

Most o~ these genes encode products that are necessary for the viral life ~ycle. For example, the tat gene encodes a 14kD protein thac is critical for HIV replication and gene expression [Rosen, C.A., et al.l Nature 319:555-559 (1986);
Sodroski, J. et al., Science 227:171-173 (1985); Arya et al, Sclence 229: supra, Sodroskl, et al., ~ 9~ 229, su~ra and Dayton, A., et al., Cell 44:941-497 (1986) which are all incorporated herein by reference]. Another gene necessary for replication is the rev gene. [Sodroski, et al., Nature 412-417 (1986), which is both incorporated herein by reference].

In some oncoviruses, cis-acting sequences located between the 5' LTR and the ~a~ gene initiation codon have been located which are necessary for the efficient packaging of the viral RNA into virions ~Bender, M.A., et al, J. Vlrol 61:1639-1646 (1987), Katz, R.A., et al, J Virol 59:163-167 (1986), Mann, R., et al, Cell 33:153-159 (1983), Pugatsch, T., et al, VirQl~gy 1~:505-511 (1983), Watanabe, S., et al, Proc Natl. Acad S~E~ 79:5986-5990 (1983) Eglitis, M.A., et al, ~ Techniqy~_~:608-614 (1988~ which are incorporated herein by reference]. In addition to these sequences, sequences overlapping the g3g gene were found to contribute to the efficiency of viral RNA encapsidation by Moloney murine leukemia virus [Adam, M.A., et al, J. Virol.
~:3802-3806 (1988); Bender, M., et al., J._Virol.
61:1639-1646 (1987)]. Certain retroviruses have been used to introduce genetic information stably in~o the genome of t~rget cells in eukaryotic cells in ~ and n _i~
[Cornetta, K., Pro~r~ce i~ N~-Cleic Acids Research and_ Molecular Biolo~y 36:311-322 ~1989); Gilboa, E., Biotechniques_4:504-512 (1986); Joyner, A., Nature 305:556-558 (lg83); Nann, R., et al., Cell 33:153-lS9 ~1983)]. Vectors containing the desired gene and packaging sequences were incorporated by packaging signal-deleted W O 91/19798 PcTtUS9l/o433s viruses generating virions capable of entry into certain cells. The signals needed for packaging of lent:iviruses RNA, (such as HIV ~NA) into virion particles have not: been identified.

Although a great deal of research has been expended on understanding HIV-l, the life cycle of this retrovir~ is not co~pletely understood.

In addition, a great deal of research has been directed to developing a vaccine to the virus, but there have been no reports of success to date. This is, in part, due to the lack of conservation in the antigenically active parts o~ the virus and in part because the functionally important regions of viral proteins and/or inactivated viral particles are poorly immunogenic.

Many methods proposed for treating HIV infected individuals would adversely affect uninfected cells as well as HIV infected cells.

Accordingly, it would be extremely useful to have a provirus that produced HIV proteins but which was not lethal because ~he viral RNA could not be packaged into virions.
Using this packaging-defective provirus vector, it would be possible to create packaging defective cell lines that could be used to investigate the packaging mechanism of the virus and to develop strategi~s to interfere with this packaging mechanism. Significantly, the virions produced by such packaging negative proviruses could be used for vaccines and as a system for eficiently introducing a desired gene into a mammalian cell.

2 ~

It would also be usaful to have a vector thae could be selectively tar~eeed to HIV target cells and could thus introduce a desired product into such cells.

Summarv of the In~ention ~ e have now disco~ered a vector co~prising a sufficient number of nucleotides corresponding to an HIV ~enome to express functional HIV gene products (HIV nucleotides), but which does not contain a sufficient number of nucleotides corresponding to nucleotides of the HIV ~enome between the 5' major splice donor and the g~g gene initiation codon to efficiently package the viral RNA into virions (HIV packaging sequence). Preferably, this HIV packaging sequence corresponds to the region between the 5' ma;or splice donor and the ga~ gene initiation codon (nucleotides 301-319).
More preferably, this sequence corresponds to a segment just downstream of the S' ma;or splice donor, and about 14 bases upstream of the g3g initiation coton. In one embodiment lt is a 19 base segment having the sequence AAAAATTTTGACTAGCCGA.

This vector can be used to transform a preselected cell line to result in an HIV packaging defective cell line.
Preferably, one would transform a cell line using at least two vectors, which collectively contain the HIV nucleotides necessary to express HIV g~g, ~1, and e~y products, but wherein each vector by itself does not contain the HIV
nucleotides necessary to express all three products. In addition, each vector does not have a sufficient number of n~cleotides corresponding to nucleotides of the HIV genome between the 5' major splice donor and the g~ gene ~o efficiently package HIV RNA. More preferably, each vector would not contain a sequence corresponding to an LTR sequence W O 91/19798 PCT/US91/0433~
2 ~ '3 downstream of the nucleotides corresponding to t:he HIV
genes. Preferably, each vector would contain a different marker gene. The transformed cell line would e~cpress HIV
virions b~t would not be able to package HIV RNA into these virions. Thus, these virions could be used to generate antibodies, as a vaccine or as a method of transferring a desired gene product to a different cell line capablP of infection by HIV.

A second vector contains a preselected gene, a sufficient number of nucleotides corresponding to an HIV packaging sequence to package HIV RNA (HIV packaging sequence), and is flanked OD each side with a sequence corresponding to a suEficient number of HIV LTR nucleotides to be packaged by the HIV packaging sequence (HIV LTR sequences), wherein the HIV packaging sequence and HIV LTR sequences correspond to the same HIV genome. This vector can be used with the pa~kaging defective vectors to transfer the desired preselected gene. Alternatively, the vector can be administered to an HIV infected cell and be packaged by the HIV virions being produced. The HIV infected cell can be in an individual. Combinations where the HIV packaging defective vector and HIV virus as a helper virus are used together are also described. The packaging sequences are located in a region from the 5' major splice donor to a site within the 5' most part of the ~g gene.

Brieg_~scr_ption of the Drawin~s Figure l is a schematic of the HIV-l genome from the 5' LTR to the ~ initiation codon showing the 5' major splice donor (SD~ and the site of the deletion in a vector representing one embodiment of this invention, pHXB Pl.

~C~

Figure 2a-e represents schematics of vectors representing various embodiments according to this invention. Figure 2a is a packaging defective vector, HXB~Pl. Figure! 2b is a packaging defective vector, HXB~Pl~en~. Fi~ure 2c is a packaging defective vector, pSVIIIenv 3-2. Figure 2d is a packaging proficient vector, HVBtSL3-Neo). Fi~lre 2e is a packaging proficient vector, HVB(SL3-~eo)~

Figure 3 is a schematic of o~e preferred embodiment showing two packaging deficient ~ectors, ~XB~Pl~env and pSVIIIenv 3-2.

Figure 4 is a chart showing p24 Levels in Culture of Infeceed Jurkat Cells by vectors representing various embodiments of this invention.

Figure 5a Ls an autoradiogram of the immunoprecipitation of 35S-labelled viral protein from COS-l cells transfected with pHXB~Pl DNA with AIDS patient serum.

Figure 5b is an electron micrograph of COS-l cells transfected with pEXBAPl showing virion particles of normal HIV-l morphology.

Figure 6 is an autoradiogram of immunoprecipitation of labelled viral proteins from Jurkat T cell lysates or supernatants exposed to supernatants froM COS-l cells that were transfected or mock transfected.

Figure 7 is an RNA dot blot test.

Figure 8 is an autoradiogram showing the Sourthern blo~
of toeal DNA of G4l8-resistant Jurkat cells.

_tailed Description of the Inventi_n We have now discovered that it is possible to make HIV
packaging defeceive vectors and cell lines. We have fou~d that the region between the 5' major splice donor and the g~g gene initiation codon in HIV viruses contains ;sequences necessary for packaging of HI~ ~NA into virions. One can prepare a vector co~prising a packaging defectiYe HIV
provirus wherein the vector contains a nucleotide sequence which corresponds to a sufficient number of nucleotides from an HIV genome to express desired HIV products, but does not correspond to a sufficient number of nucleotides corresponding to the region between the 5' major splice donor and the g~g gene initiation codon to efficiently package HIV
RNA (the HlV packaging sequence).

These sequences preferably correspond to the genome of HIV-l, HIV-2 and simian immunodeficiency virus (SIV). [See Ratner, et al, Nature 313, su~_, Sanchez-Pescados et al, : Science 227, supra, Muesing, et al, ~ature 313, supra, Uain-Hobson et al, Cell 40, ~E~, Guyader, ~. et al, Nature 326, su~ra (1987); Chakrabarti et al, ~ature 328, supra :; (1987~ and Hirsch, V., et al, Gell 49:307-319 (1987) which are all incorporated herein by reference].

The term corresponding means that conservative additions, deletions and substitutions are permitted.

Preferably, the vector does not contain the HIV packaging sequence corresponding to the segment immediately downstrea~
of the 5' major splice donor and just upstream of the g~g gene initiation codon. Typically, the vector could contain nucleotides ranging from about 14 bases to 2 bases upstream of the g~g initiation codon (for ex~mple either the 14 '2~

upstream bases or 5 upstream bases) and still be packaging deficient. In one embodiment the vector does not contain a nucleotide sequence beginning about 9 bases downstream of the 5' ma;or splice donor and continuing to about 14 bases upstream of the ~ag initiation codon. The nu~ber of bases that need to be left out can vary greatly, for example, the 19 base pair deletion AAAAATTTTGACTAGCGGA deletion in HIV-l (nucleotides 301-319) is sufficient to resule in loss of packaging ability (See Figure 1)~ However, even smaller deletions in this region should also result in loss of packaging efficiencies. Indeed, it is expected that a deletion as small as about 5 base pairs in this region should remove packaging ability. Thus the size of a particular deletion can readi.ly be determined based upon the present disclosure by the person of ordinary skill in the art.

The vector should contain an HIV nucleotide se~ment containing a su~ficient nu~ber of nucleotides corresponding to nucleotides of the HIV genome to express functional ~IIV
gene products, but as aforesald, should not contain a sufficient number of nucleotides corresponding to the region between the 5' major splice donor and ehe g~g gene initiation codon to permit ef~icient packaging of the viral RNA into virions.- In using these vectors to establish HIV packaging defective cell lines it is preferred that such cell lines do not produce any infectious HIV. Although a cell line transformed by these packaging deficient vectors would have low infectivity because the cells are packaging defective, some RNA can still be packaged into the virion. Accordingly, it is preferable that the HIV nucleotide segmen~ does not correspond to the entire ~IV genome so that if some of the viral RNA is packa~ed into the virion, what is packaged will not be a replication competent virus~

W O 91/19798 PCT/US9i/04335 -Ll-Preferably, one would want to have at least two different vectors, each containing a different portion of the HIV
genome and also not containing the sequence necessary for viral packaging. Then by co~transfecti.ng a cell wich each vector the cell would still be able to express all the HIY
structural proteins and produce virions. In one preferred embodiment the vector would not contain sequences corresponding to an HIV LTR but would contain sequences corresponding to a promoter region and/or another genome's polyadenylation sequences. Selection of particular promoters and polyadenylation sequences can readily be determined based upon the particular host cell. Preferably, the LTR which the s~quences do not correspond to is the 3' LTR. For example, see Figure 3.

In one preferred embodiment one vector would include sequences permitting expression o HIV proteins upstream of env and the second vector would permit expression of the remaining proteins. For example, one vector would contain an HIV nucleotide segment corresponding to a sufficient number of nucLeotides upstream of the ~g initiation codon to the env gene sequence to express the 5'-mose gene products. The other vector would contain an HIV nucleotide segment correspond~ng to a sufficient number of nucleotldes downstream of the ~a~ gene sequence and including a functional env gene sequence. Such vectors can be chemically synthesized from the reported sequences of the HIV genomes or derived from the many available HIV proviruses, by taking ad~antage of the kno~n restriction endonuclease sites in these viruses by the skilled artisan based upon the prcsent disclosure ~Figure 3). Preferably, one would also add a different marker ~en2 to each vector, i.e., co-transfect a preselected cell line with these differen~ vectors and by W O 91/19798 PCT~US91/04335 looking for a cell containing both markers, one would have a cell line that has been co-transfected with the two vectorS
Such a cell would be able to produce all of the HIV
proteins. However, although virions would be produced, the RNA corresponding to the entire viral sequences would not be packaged in these virions. One can use more than two vectors if desired, e.g. a g~g:~l vector, an ~E~ v~ctor and a vif/v~u vector.

For example, one could have a vector comprising nucleotides corresponding to a sufficient number of nucleotides of an HI~ LTR at the 5' end to result in a functional LTR, (preferably it corresponds to the 5' LTR~, nucleoeides corresponding to the ~ gene and the env gene downstream of the LTR, and at the 3' end, where the sequences do not correspond to another LTR, the sequences correspond to polyadenylation sequences, such as polyadenylation sequences corresponding to the SV40 virus (e.g., pSVIIIenv 3-2 in Figure 3). A second vector would contain other HIV or SIV
genes and contain a deletion in the packaging sequence and a deletion for the env gene. Thi5 vector would also noe have a 3' LTR, but would have a polyadenylation sequence. For example, one could have a vector which would not contain a sufficient number of nucleotides corresponding to HIV
packaging sequence to package HIV RNA, but would contain a nucleotide segment corresponding to a sufficient number of nucleotides corresponding to a sufficient number of nucleotides of the HIV g~g and E~ genes to express functional ~a~ and ~1 products. Preferably, this vector would also contain a sufficient nu~ber of nucleotides corre~ponding to a functional tat gene. The vec~or would not coneain a sufficient number of nucleotides to encode a functional en~ protein. More preferably, the vec~or would W O 91/19798 PCT/~S91/0433', 2 ~ c.D

also contain nucleotides corresponding to other HIV
regulatory genes to express functional gene proclucts, such as vpr, v~u, vif, etc. Other combinations of vectors can also be prepared. For example, a vector that does not contain a sufficient number of nucleotides to correspond to a functlonal g~g gene, but would have a sufficient number of nucleotides to correspond tD functional P~l and ~a~ genes, other vectoss include one that does not contain a sufficient number of nucleotides to encode a func~ional r,ol protein, but would have a sufficient number of nucleotides to encode a different functional HIV protein, etc.

As used herein, the term a sufficient number of nucleotides permits additions, deletions and substitutions as long as the claimed functional ability is not lost. For example, if one is referring to the functional ability of packaging HIV RNA then the resultant vector must have a sequence that can package HIV RNA.

HIV-l can be pseudotyped with the envelope glycoproteins of other viruses. [(Lusso, P., et al., ~Ç~nÇ~ 247:848-851 tl99G)]. Consequently, one can prepare a vector containing a sufficient number of nucleotides to correspond to a functional env gene from a different retrovirus. Preferably, the 5' LTR of this vector would be o~ the same genome as the ~_ gene. Such a vector could be used instead of an env packaging deficient vector eo create virions. By such a change, the resultant vector system can be used in a wider host range.

Virtually any cell line can be used. Preferably, one would use a m,~mmalian eell line, for example, CV-l, Hela, Raji, RD, SU480 or CHO cell lines.

2 ~ 9 In order to increase production of the viral cellular products, one could use a different promoter than the 5' LTR, i.e., replace the 5' LTR with a promoter that will preferentially express genes under its control in a particular cell line. For exa~ple, the CMV promoter will preferentially express genes in CV-l or Hela cells. The particular promoter used can be readily deter~ined by ~he person of ordinary skill in the art, based upon the particular host cell line to be used.

In srder to increase the level-of viral cellular products one can also add enhancer sequences to the vector to get enhancement of the HIV LTR and/or promoter. Particular enhancer sequences can readily be determined by the person of ordinary skill in the art depending upon ehe host cell line.

One can also add vectors that express viral enhancer proteins, such as those of herpes virus, hepatitis B vir~s, which act on ~IV LTRs to enhance the level of virus product, or cellular transaceivator proteins. Cellular transactivation proteins include NF ~-B, W light responsive factors and other T cell activation factors well known to the person of ordinary skill in the art.

By using a series of vectors that together would contain the co~plete HIV genome, one can create cell lines that produce a virion that is ident~cal to the HIV virion, except that the virion does not contain the HIV RNA. The virions can readily be obtained from these cells. For example, the cells would be cultured and supernatant collected. Depending upon the desired use the supernatant containing the virions can be used or these virions can be separated from the supernatant by standard techniques. Typically, this would W O 91/19798 PCr/US91/04335 2 ~ 6 ~ .~

include gradiant centrifugation, filtering, etc.

These attenuated virions would be extremely useful in preparing a vaccine. The virions can be used to generate an antigenic response to the HIV virions and because ehese virions are identical to the actual HIV virionsV except that the interior of these virions do not contain the viral ~NA, the vaccine created should be particularly usef~

These virions can also be used to raise antibodies to the virion that can then be used for a variety of purposes, e.g.
scxeening for the virion, developing target system for the virions, etc.

Additionally, these HIV packaging deficient cell lines can be extremely useful as a means oi` introducing a desLrecl gene, for example, a heterologous gene into ma~malian cells.

These virions could be used as an extremely efficient way to package desired genetic sequences into target cells infectable by HIV. Thi5 would be done by preparing a vector containing a nucleotide seg~ent containing a sùfficient number of nucleotides corresponding to the packaging nucleotides of the HIV virus (HIV packaging region), a predetermined gene, and flanking the packaging sequence and the predetermined gene with sequences corresponding to a sufficient number of sequences from within and near the LTRs for packaging, reverse transcription, integration and gene expression. The packaging region used would preferably correspond to at least the region between the 5' ~ajor splice donor and just upstream of the g3g initiation codon, more preferably the region between the 5' major splice donor and the Bal I site (2202 in HIV-l) in the g3g gene.

2a~4~

For example, a sufficient number of HIV-l sequences to be packaged, reverse transcribed, integrated and expressed in the target cells would inlclude the U3, R and U5 sequences of the LTRs, the packa~ing sequences, and some ~equences flanking the LTRs (required for reverse transcription). Fro~
the 5' LTR, the R and U5 regions would be included which, in HIV-l, extend from ~l to 183. The sequences flanking the 5' LTR necessary for reversa transcripticn and packaging would extend from 183 to about 335. Although not wishing to be bound by theory, applicants believe the inclusion of additional sequences from the ~ gene in the vector (up to the Bal I site, nucleotide 2202) should enhance packaging eficiency. The regions from the 3' LTR and the i~mediate flanking sequences to be included extend from about 8645 to about 9213 (U3 and R regions). Analagous regions would be included in a vector based upon HIV-2 or SIV.

When this vector is used to trans1ect one of the HIV
packaging deficien~ cells, it is the nucleotide sequence from this vector ~hat will be packaged in the ~irions. These ~HIV
packaged" genes could then be targeted to cells infectable by ~IV. This method cf transformation is expected to be much more efficien~ than current ~ethods. Further, by appropriate choice of genes, one could also monitor the method oE HIV
infection.

For example, the ~ector could contain a sufficient number of nucleotides corresponding to both 5' and 3' LTRs of HIV-l, HIV-2 or SIV to be expressed, re~erse transcribed and integrated, a sufficient nu~ber of nucleotides corresponding to an HIV packaging sequence to be packaged, for example a seg~ent between the 5' major splice donor and just upstream of the g~g initiation codon (e.g., nucleotide 381). The 2 ~ 8 ~

vector would also contain a sufficient number of nucleotides of the gene which is desired to be transferred to produce a functional gene (e.g , gene segment). The gene can be any gene desired, for example, the gene for neo~ycin phosphotransferase (NeoR). More preferably, the "genP"
would express a product that adversely affects HIV
replication or integration such as a 3~ -dominant inhibitor, anti-sense RNAs, catalytic RNAs or soluble CD4 derivative. With such a ~ene, the vector of the presene invention can be used to ~arget HIV target cells. One would preferably include a promoter for the desired gene, although the LTR sequence, itself, can serve as a promoter. Virtually any promoter can be used. Preferably, one would use a promoter that would facilitate expression of the gene in the host cell to which the gene is to be transfered. Preferred promoters include viral promoters, such as SL-3, murine retroviral LTR, etc. Enhancers sequ~nces are also preferably used ~n the vector. One would also preferably include polyadenylation sequenc~s for the gene. One can use any polyadenylation sequence, for example, the sequences corresponding to SV-40 polyadenylation sequences. The desired gene can be inserted in the present vector, in either the sense or anti-sense orientation with respect to the LTRs. The vector can contain more than one gene or pseudogene sequence, permitting the expression of multiple genes of interest.

~ lis vector can preferably be used with the packaging-defective vectors described above. In such a situation, ona preferably uses HIV LTRs in the vector corresponding to the genome of the package-deficient vector to facilitate packaging e~ficiency. However, in addition to use with a packaging-deficient virus, this vector can also be W V 91/19798 PCT/USgl/04335 2 ~ 18- `
used with helper virus for gene transfer. For ex~mple, when one wants to deliver a gene to treat an individual infected with AIDS, one could insert this vector into tha~ indi~idual and it would be incorporated into HIV virions being produced in that indi~idual. This would facilitate the delivery of the desired gene to the appropriate target cell.
Accordingly, one could use this to deli~er ~ s-dominant inhibitors, anti-sense R~As, catalytic RNAs, or soluble CD4 derivatives which are also aimed at inhibitin~ HIV-l functions critical for viral replication. One could also deliver this material by using the packaging-defective vectors described herein or such packagin~ defecti~e vectors in combination with the HIV virus in an infected indiYidual.

Additionally, these HIV packagirlg defective cell lines can be used to study various stages of the HIV life cycle, both in~ivo and ~ E~ systems by a system because the cells will express HIV cellular proteins, but will not package the RNA.

The present invention is further illustrated by the following examples. These examples are provided to aid in the understanding of the i~vention and are not to be construed as li~itation thereof.

The region between the HIV-l 5' LTR and the ~a~ gene is shown in Figure l which shows the 5' major splice donor (SD) and site of deletion in a vec~or described below, pHXB~Pl. A l9 base-pair deleeion in this region was crea~ed in an infectious HIV-l pro~;ral clone contained on the plasmid pHXBc2 of Fisher, A.G., et al, Nature_316:
262-265 (1985). This plasmid also contains an SV40 origin of replication to allow efficient gene expression in COS-l 2 ~ 5 9 cells. The mu~ation was produced by the site-directed mutagenesis as described in Kunkel, T.A., et al, ~gçhes___n~
Enzymolo~Y 154, 367-382 (1987), and the sequence confirmed by DNA saquencing (Sanger, F., ee al, Proc. Natl. Acad. Sci _ 74:5463-5467 (1977). The mutated plasmid was designated PHXB~Pl.

An additional out-of-frame deletion between the Bgl II
sltes (nucleotides 6620 to 7199) was created in the env gene. See Figure 2b. Then, polyadenylation signals from SV40 were substituted ~or the 3' end of the pHXB~Pl provirus beginning at the BamHI site (nucleotide 8053) to prepare HXB~Pl~env. The pSVIIIenv3-2 plasmid encodes the HIV-l env and env genes, with polyadenylation signals derived from SV40. In the pSVIIIenv 3-2 plasmid, the rev and ~nv genes of HIV-l are under the control of the HIV-l LTR.
~e construction of this vector has heen previously d~scribed. (See Sodroski, et al., ~S~E~ ~21:412-417 (I986) and Sodroski, et al., Nature 322:470-474 (1986), which are incorporated herein by reference. The maps of the relevant portions of the plasmids used are shown in Fi~ure 2. For the HXBQPl and HXB~Pl~env vectors, the positions of the HIV-l ~enes are shown along with the location of a nineteen base pair deletion previously described (Al9 bp). And for HXB~Plaenv and pSVIIIenv 3-2, the }~y responsive element (RRE), and the SV40 polyadenylation signal (SV40 polyA) are shown.

COS-l cells were maintained in Dulbecco's modified Eagle's medium DMEM (Hazelton Biologics, Lenexa) supplemented with 10~ fetal calf serum (Gibco, Long Island, NY) and antibiotics. Jurkas cells were maintained in culture in RP~I
1640 with 10~ fetal calf serum and antibioeics. Jurkat cells W O 91~19798 PCT/US91/04335 were preventatively treated for mycoplasma with B.M. Cyclin I
and II and human serum two weeks prior Lo infection. The HXBc2 (~g+, pro+, e~l+, ~_+, ~~, xæ~~, tat~, rev+, e~v+, ~ ) provirus was used in all plasmids was used in all plasmid and vector con~;tructs.

To evaluate the effect of Che ~utation on viral protein expression and virion production, COS-l cells were transfected with the pHXBc2 and pHXB~Pl plasmids by the DEAE-dextran procedure [Lopata et al, Nucl. Acids_Res.
12:5707-5717 (1984); Queen and Baltimors, Cell 33:741-748 (1983); (Sodroski, J., et al, Science_231:1549-1553 (1986) which are incorporated herein by reference]. COS-l cell lysates and supernatants radiolabelled with 35S-cysteine (Sodroski, J., et al, Science 231, su~ra) at 48 hours a~ter transfection were precipitated with 19501 AIDS patient serum. The overall level of viral p.rotein detected in cell lysates was comparable for the vectoxs containing the wild-type HXBc2 and the HIV packaging defective HXB~Pl.
See Fi~ure 5A, which shows immunoprecipitation of 35S-labelled viral proteins fro~ COS-l cell lysates (lanes 1-3) or supernatants (lanes 4-6) with 1950L patient serum, after transfection with no DNA (lanes 1 and 4), lO~g pHXBc2 (lanes 2 and 5) or 10~ pHXB~Pl ~lanes 3 and 6). The overall level of viral protein detected in cell lysates was comparable for cells transfected by either HXBc2 or HXB~Pl. The level of viral proteins precipitated from the supernatants of COS-l cells was sli~htly less with HXB~Pl than with HXBc2. The amount of reverse transcriptase (RT) activity measured in the supernatants of COS-l cell transfected by pHXB~Pl was 60~ of that measured in cells transfected with the HXBc2 vector ~data not shown). COS-l cells transfected with pHXBAPl were fixed 2 ~

48 hours following transfection and examined by ~lectron microscopy. Viral particles, including budding i^orms, of normal HIV-l morphology were obse~ved~ Figure 5B is an electron micrographs of COS-l cells transfected with PHXB~Pl showing virus particles of normal HIV-l morphology.

To evaluate the effect of the HXB~Pl mutation on HIV-l replication, supernatants from COS-l cells transfected with pHXBc2 and pHXB~Pl were filtered ~0.2~) and RT
measured. Supernatants containing equal amounts of RT
activity of mutant and wild-type viruses were added to Jurkat human T lymphocytes. The Jurka~ cultures along with a mock-infected culture were maintained with medium changes every three days. At intervals aliquots of Jurkat cells were labelled and assessed for expression of HIV-l proteins by im~unopreclpitation with 19501 AIDS patient serum. Figure 6 shows immunoprecipitation of labelled viral proteins from Jurkat T cell lysates tlanss 1-3, 7-9 and 13-15) or supernatants (lanes 4-~, 10-12 and 16-18) exposed to supernatants from COS-l cells that were mock transfected ~lanes 1, 4, 7, 10, 13 and 16), pHXBc2 (lanes 2, 5, 8, 11, 14 and 17), or transfected with pHXB~Pl (lanes 3, 6, 9, 12, 15 and 18). The Jurkat cells were examined at day 7 (lanes 1-6), day 14 (lanes 7-12~ and day 21 (13-18) following infection. Jur~at cultures exposed to HXB~Pl exhibited marked delays in and lower levels of viral protein production relative to those exposed to pHXBc2, the wild-type virus.
Virus replication in human T ly~phocytes transfected by X~B~Pl is thus seen eo be significantly attenuated compared with cells tra~sfected by HXBc2.

Supernatants from the above cultures were .9 0~2~-filtered and equivalent a~ounts of reverse transcriptase activity pelleted by centrifugation at 12000xg for one hour at 20C. Viral pellets were. lysed by NP40 in the presence of vanadyl ribonucleotides and dilutions of virus dot-blotted ontD nitrocellulose filters. Some samples were treated ~ith sodium hydroxide (SM at 60C for 15 minutes) prior to dot-blotting. Fil~ers were hybridized with a DNA probe consisting of HIV-l ~a~ and env gene sequences, washed and autoradiographed as previously described in Maniatis, T., et al, Molecular Cloning, Cold Spring Harbor Laboratory, (1982). For the ~ild-type HXBc2 virus, a signal specific for RNA could be detected after blotting lO00 reverse transcriptase units of filtered supernatant (not shown). For the HXBQPl, even 5 X 104 reverse transcriptase units of supernatant gave no detectable signal. Figure 7 is an RNA dot blot without (colu~n l) and with (column 2) sodium hytroxide treatment following blotting of filtered supernata~ts from the Jurkat cultures. The supernatants contained a reverse transcriptase activi~y of 5 x 104 cpm of HXBaPl (row A), 5 x 104 cpm of HXBc2 (row B), or 1 x 105 cpm of HXBc2 (row C). These results indicate that the efficiency of pac~aging virus-specific RNA
into virions for cells transfected with a packaging defective viral ~ector according to the present inven~ion is less than 2~ of the wild-type virus.

The results indicate that the region.between the 5' LTR
and g~g gene of HIV-l is important for packaging viral RNA
into virions. A mutation in this region exhibits minimal effects on the ability of the provirus to produce proteins and virion particles following transfection, but markedly decreases the level of virion RNA and attenuates virus replication in a human CD4-positive lymphocyte line. HIV-l ,.. : . : .

fi ~ ~

replicates in cultured GD4-positive cells via cell-free transmission and cell-to-cell transmission, the latter involving the contact of infected and uninfected cells [Fisher, A.G., et al, ~ :262-265 (1985), Sodroski, J., et al, Science 231:~549-1553 (1986), Strebel, ~., et al, :728-730 (1987~].

~ ectors based on HIV-l packaging sequences were constructed.

The pHVB(SL3-Neo)sense (pHVB ($L3-Neo)) and pHVB
(SL3-Neo)anti-sense (PHVB (SL3-~eo)) plasmids contain the coding sequence of the neo gene under the control of the SL3-3 murine leukemia virus LTR, with polyadenylation signals derived from SV40. The pHVB(SL3-~eo)sense and pHVB(SL3-Neo) anti-sense plasmids contain complete 5' and 3' ~IV-l LTRs and flanking viral sequences nucleotides 183-381 near the 5' Ll~
and nucleotides 8504-8661 adjacent to the 3' LTR. A unique BEm HI site was insert~d at the boundaries of the ma~or deletion in the HXBc2 provirus ~nucleotides 382 to 8593), and the SL3 ~TR-~eo ~ranscription unit was cloned into this site in either the sense (pHVBtSL3-Neo)) or antisense (pHVB(SL3-Neo)) orientation with respect to the }IIV^l LTRs.
All of the plasmids contained SV40 origins of replication.
See Figure 2. These vectors include the 19 base pair sequence shown by deletion to be important for packaging viral RNA. The vectors cannot encode any of the HIV-l gene products. The vectors contain an insert in which ~he SL3-3 murine retroviral LTR, which functions as an efficient promoter in T ly~phocytes, promotes the expression of the neomycin phosphotransferase (NeoR) gene. The polyadenylation signals for the NeQ transcript are provided by sequences from SV40. Numbers above the plasmid in Figure W O 91/19798 PCT/U~91/04335 2 indicate tha nucleotide of the HXBc2 sequence that form the boundaries of provirus/insert. The SL3-3 murine leukemia virus LT~ is indicated SL3. The position of the major 5' splice donor (SD) and ag gene initiation codon (g~gATG) are shown in the figure.

50~ confluent COS-l cells were transfected with plasmid DNA for the systems HXB~Pl + HVB (SL3-Neo)sense;
HXB~Pl + HVB(SL3-Neo)anti-sense, HVB(SL3-Neo)sense ~pH~3~Pl~env ~pSVIIIenv 3-2; and HVB(SL3-Neo)anti-sense+ pHXB~Pl~env + pSVIIIenv 3-2 using 10 ~g/ml of each plasmid by the calcium phosphate method [Chen, C., et al., Mol. Cell. Biol. 7:2745-2752 (1987)] to generate recombinant viruses. DMEM containing FCS
and antibiotics was placed in COS-l cell cultures 12 hours after transfection. COS-l sup~rnatants were harvested and filtered through a 0.2 ~m filter (Millipore) 72 hours post transfection. The p24 level in the COS-l supernatants was determined by a p24 radioimmunoassay (Dupont).

Jurkat cells to be infected with virus were seeded into 6-well culture plates at a 2.5 X 105 cells per well in 2.5 ml of complete medium. Ten-fold serial dilutions of transfected COS-l supernatants were then applied to each well and the virus allowed to absorb to the Jurkat cells at 37C
for 4 hours. After this the Jurkat cells were pelleted and resuspended ln complete medium. Twenty-four hours later the medium was replaced by complete medium containing G418 (G~bco, NY) at an actlve concentration of O.8 mg/ml and the cells dispensed into 24-well culture plates at 1.0 X 104 cells per well. Culture medium was changed every 4 days.
Positive wells containing clusters of viable G418-resistant cells were identified and counted in cultures 18 days W ~ gl/19798 PCT/US91/04335 2~34~!3 post-infection. Levels of p24 in infected Jurkae cell culture medium were determined at 5, 10, and 20 days post-infection by radioi~unoassay. The level of syncytiu~
formation by infected Jurkat cells was also determined in culeures 5 to 20 days post-infection.

To clone the G418-resistant Jurkat cells, the cells were washed in phosphate-buffered saline and diluted to a concentration of 0.5 viable cells per 100 ~1 medium.
Then 100 ~1 of the cell suspension was dispensed into each well of a 96-well culture plate. ~ells containing single cells were identified by phase contract microscopy and individual cells were expanded to 107 cells in complete medium containing O.8 mg/ml G418.

Genomic DNA was prepared from clones, digested with S~cI
and Southern blotted as previously described by Southern, E.N, J. Mol. Biol. ~.503-517 (1975). Southern blots were hybridized to a 3.3 Xb fragment containing the SL3 LTR, neo gene and SV40 polyadenylation sequences that had been labelled by random priming with oligonucleotides. Souehern blots were washed under conditions of high stringency.

The pHXB~Pl plasmid and the HIV-l veceors were cotransfected into COS-l cells as described above. Table 1 shcws that ~g p24 protein of ~IV-l was detectable in the supernatant of these transfected ceLls on the third day after transfection. The filtered COS-l supernatants were serially diluted and incubated with Jur~at lymphocytes, which were selected for G418-resistance. The number of G418-resistant Jurkat cells generated (Table 1) ranged from 102 to 105 per milliliter of COS-l supernatant, with the HVB(SL3-Neo)anti-sense vector yielding higher ti~ers than the ~a~

HVB(SL3-Neo)sense vector. No G418-resistan~ Jurkat cells were generaced following incubation wi.th supernatant derived from COS-l cells transfected with no ~NA, the vectors alone, or the pHX3~Pl plasmid alone.

The EXB~Pl provirus is not completely replication defectl~e. Thus, the production of ~iral p24 antigen and the formation of syncytia were examined eo dstermine the amount of infectious virus in the G418-resistant Jurkat cells.
Figure 4 shows that HIV-l p24 antigen was detectable in the supernatants of the Jurkat cultures. Syncytium formation was ~isible and increased with time in these cultures, indicating the expression of the HIV-l envelope glycoproteins ln the target cells. The induction of significant cytophathic effect in these cultures made cloning of the G418-resistant Jurkat cells difficult, and further suggested the presence of replication-competent viruses in the target cells. See also Table 2.

WO 91tl9798 PCT/US91~04335 2 ~

Taolel - Viral p24 Antigen Expression And Reco~Binane V~ral_Titers In Superna~an~s of Transfecte _COS-l Cellsl G418-~esistant titers Transfected DNA _ ~24~a~ml)a _ cfu~ml)a_ =
None ~ o pHYB(SL3-Neo)sense 0 0 pHVB(SL3-Neo~anti-sense 0 0 pHXB~Pl 0.6 0 pHXB~Pl + 2 pHVB(SL3-Neo)sense 6.1 5 x 10 pHXB~Pl + 4 pHVB(SL3-Neo)anti-sense 22.0 1 x 10 __ .
None o o pSVIIIenv3-2 0 0 pHXB~Pl~y 5.6 0 pHXB~PlQenv +
pSVIII~y3-2 + 5 pHVB(SL3-Neo))sense 2.S 1 x 10 pHXB~Pl~en~ +
pSVIIIenv3-2 +
pHVB(SL3-Neo)anti-sense 1.1 1 x 10 a Values shown are the mean results of three experiments.

. .

~ 28-Two other plas~ids described above encoding ehe HIV-1 proteins were utilized instead of the HXB~P1 provirus.
The pHXB~Pl~en~ plas~id is identical to the pBXB~Pl plas~id e~cept that the provi:rus in the former contains a deletion in the env gene and contains a polyadenylation signal rom SV40 in place of thle 3' LTR. The pSVIIIenv3-2 plasmid encodes both rev and env g~nes of HIV-l under the control of the HIV-1 LTR, with polyadenyla~ion signals derived from SV40. Uhen these two plasmids were cotransfected into COS-l cells along with the pHVB(SL3-Neo) sense or pHVB(SL3-Neo) anti-sense plasmids by the methods described above, p24 ra~ antigen could be detected in the supernatants three days after transfection (Table 1).
Incubation of these supernatants with Jurkat ly~phocytes and selection of the JurXat cells with G418 indicated that the level of recombinane virus ranged fon~ 104 to 106 colony-forming units per millilliter of filtered supernatant. In these experiments, no significant differences were noted between experiments, and no significant difference was noted between the HVB~SL3-Neo)sense and HVB(SL3-Neo)anti-sense vectors in ~hree sepPrate experiments.

These G418-resis~ant Jurkat cells were cloned and used for isolation of DNA. The genomic DNA was digested with SacI, which cuts the vectors once in each of the BIV-l LTRs to produce a 3.3 Kb fragment. A fragment which contains sequences derived fro~ the SL3-3 LTR, the coding sequences of the NeoR gene, and SV40 polyadenylation signals was used as a probe. Clones derived fro~ both the HVB(SL3-Neo)sense and HVB(SL3-Neo)anti-sense vectors demons~rated a single hybridizing band at 3.3 Rb that was not seen in control Jurkat cells. See Figure 3. This demonstrates that the W O 91/19798 PCT/US91/0433~

HVBtSL3-Neo)sense and HVB(SL3-Neo)anti-sense sequences had been transferred without rearrangement or recombination into the G418-resistant Jurkat cells.

p24 g~g protein was measured in cell supernaLtants and syncytia were scored up to 40 days ollowing the! initial infection. In all of the clones examined, no syncytia were observed and p24 antigen was undetectcible. See Table 2.
This indicates that no replication-competent virus was present in these G418-resistant Jurkat cells.
Tabla 2 Sync~tium Formation in G41.8-resistant Jurkat Culturesa-Days After Infection DNA Transfected into COS-l Cells 5- 10 20 _ . 40 pi~B~Pl +
pHVB(SL3-Neo)sense - + +++ ND
pHXB~Pl +
pHVB~SL3-Neo)anti-sense ~ ND
PH~B~Pl~env +
pSVIIIenv3-2 ~
pHVB(SL3-Neo)sense pHXB~Pl~env pSVIIIenv3-2 ~
pHVB(SL3-Neo)anti-sense a Syncytia were scored according to the following criteria:
-, no syncytia observed; +, 1-5 syncytia/hpf; ++, 5-10 syncytia/hpf; +++, ~reater than 10 syncytia/hpE (hpf-high power field).
The results suggest that the packaging-defective HXB~Pl provirus can provlde trans-acting viral functions required for the transfer of a HIV-l vector to Jurkat lymphocytes. The HIV-l LTRs and the i~ediate flanking .

2 ~

sequsnces, including the sequences defined to be ;.mportant for packaging viral ~NA, appear to be sufficient t:o allow packaging, reverse transcription and integration. By providing ehe trans-acting func~ions on two separate plasmids, each lacking a 3' LTR, the transfer of vector sequences occurs in the apparent absence of replication-co~petent virus. The titers of recombinant virus in this helper virus-free context appear to be improved relative to those observed in the presence of replication-competent virus, probably because of the induction of significant cytopathic effects by the latter.
The higher titer observed for the HVB(SL3-Neo)anti-sense vector relative to the HVB(SL3-Neo)sense vector in the presence of replication-competent virus may in part relate to a suppresive effect of anti-sense read-through transcripts from the SL3 promoter on helper virus replication.

The HIV-l vector described herein provides a simple, efficient means of introducing individual genes of interest into potential HIV-l target cells. In lighe of recent observations demonstrating that HIV-l can be pseudotyped with the envelope glycoproteins of other viruses, increasing the host range of these vectors is feasible. Given the ability of the wild-type HIV-l genome to encode multiple gene products, these vectors are readily adaptable for the expression of multiple genes of interest in the target cells.

It is evident that those skilled in the art, given the benefit of the foregoing disclosure, may make numerous modifications thereof and departures from the specific embodiments described herein, without departing from the inventive concepts and the present invention is eo be limited solely by the scope and spirit of the appended claims.

Claims

We claim:

1. A vector containing:
(a) a sufficient number of nucleotides corresponding to an HIV genome to encode functional HIV protein, referred to as the HIV segment, but not containing a sufficient number of nucleotides corresponding to nucleotides of the HIV genome between the 5' major splice donor and the gag gene to effectively package HIV RNA, referred to as the HIV packaging segment;
(b) a promoter upstream of the HIV segment; and (c) a sufficient number of nucleotides downstream of the HIV segment corresponding to a polyadenylation sequence but not containing a sufficient number of sequences corresponding to a functional LTR.

2. The vector of claim 1, wherein the vector does not contain a sufficient number of nucleotides corresponding to all the HIV genes to encode functional protein by all the HIV
genes.

3. The vector of claim 2, wherein the vector does not contain a sufficient number of nucleotides corresponding to an env gene to encode functional env protein.

4. The vector of claim 2, wherein the vector does not contain a sufficient number of nucleotides corresponding to a gag gene to encode functional gag protein.

5. The vector of claim 1, wherein the vector does not contain a sufficient number of nucleotides corresponding to HIV structural genes to encode functional proteins by all the structural genes.

6. The vector of claim 1, wherein the promoter is an HIV
LTR.

7. The vector of claim 1, wherein the HIV packaging segment is a nucleotide sequence downstream of the 5' major splice donor to about 5 bases upstream of the gag gene initiation codon.

8. The vector of claim 1, wherein the HIV genome is selected from the group consisting of HIV-1, HIV-2, and SIV.

9. The vector of claim 1, wherein the HIV genome is the HIV-1 genome.

10. The vector of claim 1, wherein the HIV genome is the HIV-2 genome.

11. The vector of claim 2, wherein the HIV segment contains a sufficient number of nucleotides corresponding to a gag gene and a pol gene to produce functional gag and pol proteins.

12. The vector of claim 2, wherein the HIV segment contains a sufficient number of nucleotides corresponding to an env gene to produce functional env protein.

13. The vector of claim 11, wherein the promoter is an HIV
LTR.

14. The vector of claim 12, wherein the promoter is an HIV
LTR.

15. The vector of claim 1, wherein the polyadenylation sequence is the SV40 polyadenylation sequence.

16. The vector of claim 11, wherein the polyadenylation sequence is the SV40 polyadenylation sequence.

17. The vector of claim 12, wherein the polyadenylation sequence is the SV40 polyadenylation sequence.

18. The vector of claim 17, wherein the HIV segment contains a sufficient number of nucleotides corresponding to a rev gene to produce functional rev protein.

19. A vector comprising:
(a) a preselected gene and a promoter for the preselected gene;
(b) a sufficient number of nucleotides corresponding to an HIV packaging sequence to package HIV RNA, referred to as the HIV packaging sequence;
wherein the vector is flanked on each side with a sequence corresponding to a sufficient number of nucleotides corresponding to HIV LTRs and flanking sequences to be expressed, reverse transcribed and integrated, referred to as HIV LTR sequences, wherein the HIV packaging sequences and the HIV LTR sequences correspond to the same HIV genome.

20. The vector of claim 19, wherein one of the HIV LTR
sequences acts as the promoter for the preselected gene.

21. The vector of claim 19, wherein there is a separate promoter sequence in addition to the HIV LTR sequences as the promoter for the preselected gene.

22. The vector of claim 19, wherein adjacent to the preselected gene are nucleotide sequences corresponding to polyadenylation sequences.

23. The vector of claim 19, which contains a second preselected gene.

24. The vector of claim 19, wherein the HIV packaging sequence corresponds to the nucleotides from the 5' major splice donor to a site in the 5' most part of the gag gene.

25. The vector of claim 24, wherein the site in the 5' most part of the gag gene is between about HIV-1 nucleotides 338 to 385.

26. The vector of claim 25, wherein the site is between about HIV-1 nucleotides 350 to about 381.

27. The vector of claim 19, wherein the HIV packaging sequences are in a segment corresponding to a sufficient number of nucleotides from the 5' major splice donor to about Bal I site at the 3' end of the gag gene.

28. The vector of claim 19, wherein the promoter is a viral promoter.

29. The vector of claim 28, wherein the viral promoter is the SL3 promoter.

30. The vector of claim 19, wherein the preselected gene encodes a trans-dominant inhibitor, an anti-sense RNA, a catalytic RNA or a soluble CD4 derivative.

31. A method of transferring genes to mammalian cells, which comprises:

(a) transfecting the cell of an HIV infected individual with the vector of claim 19;
(b) waiting for the cell in step (a) to be transformed and produce virions;
(c) having virions produced from the cell of step (b) contact other cells resulting in transfer of the desired gene.

32. The method of claim 31, wherein the cell of an HIV
infected individual is transfected in vivo.

33. The method of claim 31, wherein the cell of an HIV
infected individual is transfected n vitro.

34. A method of transferring genes to mammalian cells, which comprises:
(a) transfecting a cell with the vector of claim 19;
(b) cotransfecting the cell with at least two vectors, which collectively contain a sufficient number of nucleotides corresponding to an HIV genome to express functional HIV gag, pol, env and regulatory products but wherein each vector by itself does not contain a sufficient number of nucleotides to express all the functional HIV gag, pol, env and regulatory products, wherein the vectors do not have a sufficient number of nucleotides corresponding to nucleotides of the HIV genome between the 5' major splice donor and the gag gene to effectively package HIV RNA, and wherein each of the vectors at their 3' end do not contain a sufficient number of nucleotides corresponding to HIV LTR to be packaged by HIV
packaging sequences;
(c) culturing the cells transformed in step (b) to produce virions (d) having virions produced in step (c) contact mammalian cells.

35. The method of claim 34, wherein the mammalian cell is propagated in tissue culture.

36. The method of claim 34, wherein the mammalian cell is located in an animal host.