WO1997020052A1 - Recombinant hiv and modified packaging cells and method for treating acquired immune deficiency syndrome - Google Patents

Abstract

A recombinant human immunodeficiency virus (rHIV) and recombinant mammalian cell-line (packaging systems) and a method for treating infection of cells by human immunodeficiency virus HIV. rHIV, such as rHIV-1, comprises a gene construction which includes a foreign gene. The expression of this gene is activated in human cells in the presence of wild-type HIV. This gene product can cause cell death in the presence of an appropriate drug, e.g. Acyclovir. This gene product is typically a viral thymidine kinase. rHIV is so constructed that is unable to replicate by itself due to the absence of a regulatory gene that is necessary for its replication, such as tat or rev or both. The recombinant mammalian cell-line packaging system comprises in its genome a recombinant gene construction which typically includes a functional regulatory gene from HIV which is missing from rHIV, such as the tat or rev genes or both. The method for treating infection of cells by HIV comprises administering a composition comprising rHIV, which is produced in vitro from a recombinant mammalian cell-line, and treating with a nucleoside analog, such as Acyclovir or Gancyclovir.

Description

RECOMBINANT HIV AND MODIFIED PACKAGING CELLS AND METHOD FOR TREATING ACQUIRED IMMUNE DEFICIENCY

SYNDROME

FIELD OF THE INVENTION

The present invention relates generally to genetic engineering and also to gene therapy wherein a genetically engineered gene is included into a vector for the purposes of in vivo targeted therapy.

BACKGROUND OF THE INVENTION

The availability of genetic engineering, particularly in the form of recombinant DNA technology has increased the possibility of successful gene therapy. A particular desired scenario for gene therapy involved in vivo targeted therapy which is specific for the disease to be treated. Typically, a gene, which expresses a product that is useful in the treatment of the disease, is inserted into a vector, such as a virus, which is then administered to the patient suffering from the disease. There are numerous proposals in the literature for such treatments and vectors .

It will be appreciated that in recent times, considerable effort and resources have been devoted to treating one particular disease in humans which is usually fatal. This disease, acquired immune deficiency syndrome (AIDS) is caused by a particular retrovirus known as human immunodeficiency virus (HIV) which includes the virus known as HIV-1. Numerous treatments for this disease have been proposed and tested and some are currently in use. For example, AIDS is commonly treated by administering the drug AZT to AIDS patients. Moreover, πbozyme and anti-sense technology are currently being developed as possible treatments for humans suffering from infection by HIV. Many of these new technologies are discussed in volume 260 of SCIENCE, 28 May 1993 issue. As illustrated on page 1257 of this issue of SCIENCE, numerous therapeutics have been identified for various steps in the stages of the replication of HIV. The replication of HIV is attached by these proposed therapeutics m order to treat the disease. However, it has not been possible to cure the infected individual because of the ability of HIV to remain integrated into the cellular genome and also due to the frequency of changes that appears m the viral genome m every replication cycle.

Often, these various treatments are not permanent and are not economical, and they are often not specific enough. The lack of specificity of the treatments tends to cause complications arising from the effect of the therapeutic drug on umnfected human cells in the body of the patient being treated.

The present invention seeks to provide a novel treatment for AIDS as well as a genetically engineered HIV and a genetically engineered cell line for producing the genetically engineered HIV. SUMMARY OF THE INVENTION

The invention provides a recombinant human immunodeficiency virus (r HIV) and a recombinant (modified) T-cell line having a functional regulatory gene from HIV, and a method for treating the acquired immune deficiency syndrome in a human caused by HIV.

The modified human immuno deficiency virus (rHIV) includes in its genome a modification (e.g. a deletion) of one of the HIV regulatory genes and the inclusion of a foreign gene. In one embodiment, the foreign gene is a viral kinase enzyme such as a viral thymidine kinase (tk) from the Herpes simplex virus (HSV-1) . Typically, the rHIV is unable to express at lease one functional regulatory gene product of the genome of HIV because the genome of rHIV has been modified to either remove or incapacitate the gene which encodes this functional regulatory gene product. In one embodiment, this functional regulatory gene product may be either the tat protein or the rev protein of HIV.

The present invention also provides a modified mammalian cell line, such as a modified T-cell line, having in its genome a recombinant gene construction including a gene from the genome of HIV (usually the wild-type HIV-1) . This foreign gene will typically encode a functional regulatory gene product of HIV. Typically, this functional regulatory gene product of HIV is the same functional regulatory gene product which the rHIV cannot produce or express. In this fashion, the modified T-cell line will support replication of rHIV while a normal T-cell line (or a normal in vivo T-cell) will not support replication of rHIV. In one embodiment, the modified T-cell line includes the tat gene from wild-type HIV (e.g. HIV-1) and expresses the gene product of this gene so that is becomes possible to replicate rHIV (tat-) in this cell line. This in vitro rHIV production is referred to as a packaging system.

The invention also provides a method for treating AIDS in a human by administering a composition which includes rHIV followed by administering a nucleoside analog. In a typical embodiment, the nucleoside analog is Acyclovir or Gancyclovir. In a typical implementation of this method, the rHIV is harvested from the modified T-cell line. The harvested rHIV will then be administered to the patient suffering from AIDS. rHIV will infect all HIV-infectable cells. In one embodiment, the tk gene included in rHIV will be actively expressed only in the presence of wild-type HIV. Treatment with Acyclovir completes this modality.

It will be appreciated that in the preferred embodiment rHIV (typically, rHIV-1) is incapable of replication on its own in a normal T-cell line or a normal T-cell. That is, it requires the modified T- cell line or T-cell having the regulatory gene which is depleted from rHIV-1. It will also be appreciated that rHIV produced according to the invention is highly infectious and has exactly the same host range as wild- type HIV-1. rHIV-1 is capable of super infecting human T-cells previously infected with wild-type HIV-1. Although rHIV-1 is incapable of replicating by itself in a normal cell, it will infect a previously HIV-1 infected cell as well as an HIV-1 infectable cell, but rHIV-1 will not replicate in cells that lack wild-type HIV-1.

In the case of double infection of a cell with HIV-1 and rHIV-1, rHIV-1 is stimulated to replicate and viral thymidine kinase is produced. This enzyme will phosphorylate a nucleoside analog, such as Acyclovir or Gancyclovir, which is a cytotoxic substance that will kill the HIV-1 infected cell. It will be appreciated that normal cellular thymidine kinase phosphorylates the nucleoside analog very minimally with no consequences. Thus, administering these nucleoside analogs is safe and approved (as these analogs have been successfully used to treat HSV-1 infections in humans) ; that is these drugs have been well tested due to the fact that they have been prescribed for the treatment of herpes viruses. If HIV-1 infects cells previously infected with rHIV-1, viral thymidine kinase is produced and upon treatment with Acyclovir or Gancyclovir or other nucleoside analogs, these dully infected cells would be killed. The problem of developing resistance to these compositions such as Acyclovir or Gancyclovir does not arise because (1) the time for developing a modified tk gene is not available, and (2) fresh inoculation of rHIV can always be administered to circumvent the problem.

It will be appreciated that repeated treatment may be needed to eradicate the HIV infection from infected individuals and prevent future reinfection from within. This direct in vivo gene therapy not only avoids the costs and complications of ex-vivo approaches but has the potential to become a simple out-patient office procedure. The treated patient can go on to other remedial therapies for the correction of other deficits .

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be described while referring to the following figures which are presented for purposes of illustration:

Figure 1 shows a general method of producing a modified mammalian cell or cell line according to the present invention.

Figures 2a, 2b, 2c and 2d show various maps which illustrate the construction of the plasmid that is used to produce a modified mammalian cell or cell line according to the present invention.

Figure 3 shows a general method for producing a recombinant HIV and relevant control constructs according to the present invention.

Figure 4 shows a particular plasmid which is a gene construct containing proviral HIV-1.

Figure 5 shows a design strategy for a particular embodiment of the present invention for inactivating a particular regulatory gene of HIV-1.

Figure 6 shows a design strategy for creating a gene construct for rHIV-1 for a particular embodiment of the present invention. Figure 7 shows a particular gene construct for a particular embodiment of the present invention; this gene construct contains a foreign gene which is to be inserted into HIV-1 m order to create rHIV-1.

Figure 8 shows a general method for treating HIV infection with a recombinant HIV, as well as a method for producing the recombinant HIV.

Figure 9 illustrates the nucleoside analog Acyclovir .

Chart 1 shows a test for the replication of progeny virus from a T cell line transfected with various gene constructs.

Chart 2 shows results from the infection of mononuclear peripheral blood cells from four normal donors by the progeny virus produced by the modified T- cell lines of the present invention.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

While the present invention will be described by referring to specific examples of gene constructs, nucleoside analogs, foreign genes, and particular cell lines, as well as other details, it will be appreciated that this description is not by construed m a manner to limit the scope and spirit of the present invention. Moreover, numerous specific details which are well understood by those m art are described briefly so that the present invention is not unnecessarily obscured. The modified mammalian cell line, such as a modified human T-cell line having in its genome a regulatory gene from wild-type HIV, is created, in one embodiment, according to the general method shown in figure 1. In a typical embodiment, the modified cell line will be a human T-cell which is transfected with a plasmid containing an HIV-1 regulatory gene, such as the tat gene, thereby producing a modified T-cell line having the tat gene in its genome. According to the general method shown in figure 1, the HIV-1 tat/rev mRNA, labeled 101, is reverse transcribed in order to produce tat/rev cDNA, which is cut with restriction enzymes Sail and BamHI to isolate the two coding exons of HIV-1 tat cDNA, and this product is labelled as 102 in Figure 1. This tat cDNA is then inserted into a vector 103, typically downstream from a promoter region 104 to produce a resulting plasmid 105 as shown in figure 1. This plasmid is then used to transfect human T or B cells (e.g. the Jurkat T cell line) to produce a modified T-cell or a modified B-cell, such as T-cell 107 or B-cell 108. It will be appreciated that other regulatory genes of HIV, such as rev may also be inserted into a plasmid along with the tat gene and this plasmid may then be used to transfect T-cells or B-cells to thereby produce a modified T-cell or B-cell. These cells are typically immortalized cell lines capable of indefinite replication and thus are useful as packaging systems to prepare large numbers of the rHIV-1 described below.

A specific example of this general method shown in Figure 1 will now be provided in conjunction with Figures 2a, 2b, 2c and 2d. Figure 2a shows a map on the proviral DNA of the genes encoding for the HIV-1 proteins. The two coding exons of the tat gene are indicated by arrows on Figure 2a. Figure 2b shows the splicing pattern for the tat/rev mRNA; the Sail and BamHI splicing sites corresponding to the tat cDNA (0.3 kb long) fragment 102 are indicated on the outline of its mRNA shown in Figure 2b. It will be appreciated that the gaps in the mRNA of Figure 2b are joined after conventional mRNA post-transcriptional processing. The resulting mRNA tat/rev is shown as fragment 101 in Figure 1, and this fragment is reverse transcribed as indicated above and then the cDNA is digested with Sail and BamHI to produce the cDNA tat fragment 102, which is 356 base pairs (bp) long and is isolated using conventional techniques.

The expression vector 103, shown in Figure 1 and Figure 2c, contains the origin of replication and the early region of the human papovavirus BK (allowing its amplification in human cells) , a "cassette" of SV40 early prometer 104, splicing and polyadenylation sequences that permit expression of a cDNA, and plasmid sequences from pBR322. The expression vector 103 also includes the bacterial gene for the aminoglycoside phosphotransferase (Neo) under the control of the SV40 early region promoter and polyadenylation sequences, which confers resistance to the aminoglycoside antibiotic G418 when expressed in euharyotic cells.

The plasmid 105, shown in Figure 2d, contains the cDNA of HIV-1 tat gene and was derived from plasmid 103, shown in Figure 2c, in the following manner. The Sall-BamHI fragment 102 of 356 bp (which contains the two coding exons of the HIV-1 tat cDNA) was filled in using the Klenow enzyme (large fragment of DNA polymerase I) and inserted by conventional blunt end ligation into the unique Xhol site of vector 103 which was previously filled m with the Klenow enzyme. This blunt end ligation of the filled in fragment 902 into the vector 103 places the tat cDNA 102 fragment between the SV40 early promoter 104 and the SV40 splicing and polyadenylation signals (shown as SV40 s.p.s. in Figure 2d) .

The Jurkat T-cell is derived from a human T-cell lymphoma and is used in an embodiment where T cells are transfected with the plasmid 105. This transfection produces a modified mammalian T-cell lone having tat+ (that is, having the tat gene which expresses functional tat protein) . Plasmid 105 was introduced into these Jurkat T cells by electroporation using conventional electroporators (e.g. from Invitrogen Corporation) . In one embodiment, a sample of 10⁷ cells was suspended m 1ml of the media RPMI 1640 (which is chilled on ice to roughly about 0'C) . Plasmid DNA (m the form of Plasmid 105) was added at concentrations of lOOng to 1 icrogram per sample. Electroporation m a 0.4cm wide cuvette was carried out at 280mV and 960μF. Transfected cells were selected using the antibiotic geneticm (G418) . Viable cells were recovered and expanded into cell lines which stably produce the HIV-1 tat protein.

The modified human immunodeficiency virus (e.g. rHIV-1) is produced according to the general method shown in figure 3. This method begins with a proviral HIV-1 DNA, labeled 201, which is inserted, using conventional recombinant DNA technology, into a plasmid to create plasmid 203. It will be appreciated that this proviral HIV-1 DNA in one embodiment was constructed from two wild-type HIV ana accordingly has the necessary regulatory genes which are essential for its replication. This is not only a replication competent virus, it is also highly infectious. Accordingly, the tat gene will be present m plasmid 203. The plasmid 203 may then be replicated in culture (e.g. m a bacterial culture) to produce a large number of such plasmids using conventional cultuπng technology. Some of these plasmids may then be harvested for the knock out or removal operation shown figure 3 which creates plasmid 205 from plasmid 203, and similarly some of these plasmids 203 may be harvested for an insertion operation where m the tk gene 207 is inserted into the plasmid 203 to produce the plasmid 210. In an analogous fashion, the plasmid 205 is used to insert the tk gene 207 to produce the plasmid 209. A specific example is given below for incapacitating the tat gene order to produce the plasmid 205 from the plasmid 203. It will be appreciated by those the art that the tat gene or other essential regulatory genes may be removed or knocked out in order to render the gene incapable of expressing a functional regulatory gene product. For example, the rev gene bay be knocked out in addition to the removal or knock out of the tat gene.

After producing plasmids 210 and 209 from plasmids 203 and 205 respectively, conventional transfection operations are performed in order to introduce the respective plasmid into normal T-cell lines or modified T-cell lines. Alternatively, transfection operations may be performed on normal B-cell lines or modified B- cell lines. Currently, transfection of modified T-cell lines containing the tat gene of HIV-1 (wild-type) is preferred. Given this preference for transfection into T-cell or T-cell lines, this transfection procedure may be carried out as described above. Typically, the electroporation technique is used to cause transfection in order to efficiently introduce the DNA construct into the packaging cells. However, alternative methods of transfection may also be performed. In one embodiment a DNA construct, such as plasmid 209, may be transfected into T-cells (e.g. modified Jurkat tat+ cells) by first cleaving the construct into two pieces (e.g. including a cleavage at the Sail site) and then sequentially introducing (by for example electroporation) one piece into the T-cells and then the other piece into the same T-cells. Then, the T- cells may assemble the pieces to produce a competent cell packaging system (e.g. T-cells 211) which will produce the rHIV of the present invention.

In the case of the plasmid 210 which contains a complete copy of the HIV-1 genome as well as a complete copy of the tk gene (from, for example, HSV-1) , this plasmid is transfected into a normal (tat-) T-cell or T-cell line, thereby producing a T-cell 212 having an integrated proviral HIV-1 genome which includes the tk gene from HSV-1. Transfection of a modified (tat+) T- cell or modified T-cell line which contains the tat gene from HIV-1 (Wild-type) with plasmid 210 produces a T-cell 214 which contains a integrated proviral copy of the HIV-1 genome with the tk gene as well as a further copy of the tat gene in the genome of the T-cell. It will be appreciated that these modified T-cells are produced according to the method described in conjunction with figure 1 above. It will be appreciated that T-cells 212 and T-cells 214 are used as controls in the experiments described below which establish the efficacy of the methods for producing the proper and desired gene constructs and the desired rHIV-1 and modified T-cells/T-cell lines.

Plasmid 209 is also used to transfect both normal T-cells or T-cell lines and modified T-cells or modified T-cell lines. Plasmid 209 contains a full copy of the HIV-1 genome with the exception that a regulatory gene has been disabled or totally removed (e.g. tat-), and it also includes a copy of the tk gene (full functional copy capable of expressing viral thymidine kinase) . In the embodiment shown in figure 3 the functional regulatory gene of HIV which has been disabled is the tat gene, and thus the plasmid 209 is labeled as "tat-" to indicate the absence of this functional regulatory gene of HIV-1. After producing many copies of the plasmid 209 and harvesting these plasmids from their bacterial host, one sample of plasmids 209 is used to transfect a normal (tat-) T- cell or T-cell line, and another sample of plasmid 209 is used to transfect a modified (tat+) T-cell or T-cell line, thereby producing T-cell 213 or T-cell 211 respectively. The transfection procedures are similar to those described above. The T-cell 213 will be incapable of producing complete HIV-1 viral particles due to the absence of the tat gene on both the genome of the T-cell and the proviral copy of HIV-1 in the genome of the T-cell. These T-cells, such as T-cell 213 may be used as a control in the experiments shown below to establish the efficacy and functionality of rHIV-1 as well as the modified T-cell line. The T-cell 211 will be able to produce multiple viral products, in this case the modified or recombinant human immunodeficiency virus referred to as rHIV-1 which lacks the functional regulatory gene tat and consequently is unable to express the gene product of this gene but does include a copy of the HSV-1 thymidine kinase gene. This viral product may then be used to treat AIDS m the method described below. The T-cell 211 is capable of producing this recombinant virus by virtue of having a separate copy of the tat gene which s expressed by the transcription and translation system of the modified T-cell. That tat gene product expressed in this T-cell 211 allows the proviral HIV-1 having the tk gene (tk+) but not having a functional copy of the regulatory gene tat (tat-) to replicate itself to produce multiple copies of rHIV-1 (tat-, tk+) . Thus, T cell 211 may be considered a packaging system for producing this recombinant virus.

It will be appreciated that numerous gene constructs such as the plasmids 203, 205, 209, and 210 may be constructed m various ways to achieve the results described herein. Thus, the specific examples given below for these particular gene constructs are merely one implementation of the present invention. In a particular embodiment of the present invention, plasmid 203 is pNL 43, shown m figure 4. In this same embodiment, plasmid 205 is plasmid pNL43dBM, shown partially in figure 5, which shows a design strategy for the mactivation of the HIV-1 tat gene. The tk gene insert 207 shown figure 3 is produced m this same embodiment according to the design procedure shown m figure 7. The plasmid 209 m this same embodiment, which is the plasmid of interest since it produces rHIV-1 m the modified T-cell line, is the plasmid pNL43dBMtk and is produced according to the design procedure shown m Figure 6. The control plasmid 210 in this embodiment is known as plasmid pNL43tk.

After producing multiple copies of the recombinant HIV, which m the particular embodiment described above is rHIV-1 (tat-, tk+) , the treatment of humans infected with HIV-1 may then be performed by administering the recombinant virus and a nucleoside analog to the same patient. This will be described by referring to figure 8 which also provides an overview of the invention. While figure 8 presents one example of the method according to the present invention, it will be appreciated that certain of the steps (e.g. 801, 803, 805, and 807) may be performed a sequence which is different from that shown m figure 8. The method begins in step 801 wherein a T-cell line (or a B-cell line) is transfected with a plasmid which contains the functional HIV regulatory gene (e.g. tat gene) m order to produce a modified T-cell line which will be the packaging system used to produce the recombinant HIV of the present invention. In step 803, a proviral HIV construct is created without the functional HIV regulatory gene (e.g. tat gene) which has been inserted into the modified T-cell in step 801. Then in step 805, a foreign gene such as the HSV-1 thymidine kinase gene is inserted into the proviral HIV construct created step 803. This creates, for example, rHIV-1 (tat-, tk+) . Then in step 807, this recombinant DNA [(for example, rHIV-1 (tat-, tk+) DNA] is then inserted into the modified T-cell line packaging system which was created m step 801. This produces in step 809 many copies of the recombinant virus [(for example, rHIV (tat-, tk+) ] . Then m step 811, this recombinant virus is harvested and then injected into a human patient suffering from infection by wild-type HIV-1. Alternatively, a sample of the patient's blood may be taken and cultured with the recombinant virus (to infect T cells in the sample) and then the sample is injected back into the patient. Then, in step 813, a nucleoside analog such as Acyclovir is administered orally or Gancyclovir is injected. The standard therapeutic dosages of this nucleoside analog may typically be used several times over a period of time. While administering Acyclovir, blood samples from the patient may be taken to determine the progress of the treatment (by measuring the quantity of HIV present and/or the number of infected cells) . As shown by the dashed lines in figure 8, further injections of the recombinant virus into the patient may be performed followed by further administrations of the nucleoside analog. Again, the progress of the treatment may be monitored by taking samples of the patient's blood and measuring the quantity of HIV present and/or the number of cells infected by HIV.

A particular implementation of the present invention will now be described by showing particular gene constructs which were used to create an embodiment of the recombinant human immunodeficiency virus of the present invention. Figure 2a shows a gene map of the proviral form of the HIV-1 genome. As is known, this genome is also similar to the HIV-2 genome and therefore the invention may be used to treat infections by HIV-2. As is known, the tat and rev regulatory genes of HIV-1 are mapped to two non contiguous regions of the genome, and the messenger RNA for tat is a spliced copy of each segment. The inactivation strategy according to the present invention for inactivating tat seeks to disrupt the reading frame of the tat exon.

Figure 4 shows a particular plasmid, the plasmid pNL43 which contains two full copies of HIV from two different isolates of HIV (wild-type) . These isolates are referred to as NY5 (5¹ ) and LAV (3' ) and were cloned directly from genomic DNA. See, generally, Adachi , A., et al . , Journal of Virology, vol. 59, at pages 284-291 (1986) . This plasmid pNL43 is used as the basis for the creation of the plasmid pNL43dBM, and the plasmid pNL43dBMtk.

Figure 5 shows a design strategy, according to one embodiment, for the HIV-1 (tat-) gene construct, which has been labelled pNL43dBM. The tat gene is inactivated, according to this strategy, by a 16 base pair deletion between Banll and Mval restriction sites in the HIV-1 proviral DNA. This deletion renders the tat protein functionally inactive. As shown in Figure 5, the plasmid pNL43dBM (generally represented as plasmid 205 in Figure 3) is derived from the plasmid pNL43 (which is generally represented as plasmid 203 in Figure 3) . A portion of the HIV-1 DNA in plasmid 203 about 70 nucleotides downstream from the Sail restriction site ( in the tat gene) and 25 nucleotides downstream from the ATG codon was cut with Banll and Mval restriction endonucleases . The fragments were then treated with SI nuclease to digest the single- stranded portions of the fragments in order to blunt end them. The fragments were then bound to each other by the T4 DNA ligase enzyme. Deletion of the 16 base pair stretch was confirmed by sequencing. Also, see the paper by Shibata, et al . concerning research on tat mutants; Shibata, R., et al . , Archives of Virology, V. 114, pages 243-250 (1990) .

The tk gene from HSV-1 is obtained from the PvuII- Pvull fragment 207 shown m figure 7 which may be obtained from the plasmid 701 shown in figure 7. In particular one embodiment, a portion of the HSV-1 genome, containing the tk gene (m the BamHII-BamHII fragment 703) , was cloned into a plasmid vector, thus obtaining the plasmid 701. See generally the article by McKnight, S.L. and Grace, E.R. , Nucleic Acid Research, Vol. 8, starting at page 5981 (1980) . The PvuII-PvuII fragment 207 shown m figure 7 is a fragment containing the complete tk gene that can be expressed under an HIV-1 promoter. This tk fragment 207 is inserted into the HIV proviral genome at the Sail site as shown in figure 6. Figure 6 illustrates a schematic representation of the construction of plasmids 209 and 210 into which the tk fragment is inserted. This insertion is produced by partial digestion by Sail followed by a fill-m reaction to produce blunt ends and the insertion and ligation of the PvuII-PvuII fragment 207. In particular, to make Plasmids 209 and 210, plasmids 203 and 205 respectively were cut with Sail (which is located just after the splice acceptor site for the tat/rev mRNA) . The resulting single strand portions of DNA were filled-m with Klenow DNA polymerase producing blunt ends . The PvuII-PvuII fragment 207 of the HSV-1 subclone containing the tk gene (about 2 kilobase pairs in length) was inserted between the blunt ends of opened plasmids 203 and 205 by conventional blunt end ligation. Charts 1 and 2 illustrate various experimental results which demonstrate the in vitro effectiveness of the treatment method of the present invention and establish and verify the proper construction and functional operation of rHIV-1 (tat-, tk+) and the proper construction and operation of the modified T- cell lines. Chart 1 shows the results of assaying for the presence of p24 (an HIV-1 structural protein) , assaying for RT (reverse transcπptase) , observing cytopathic effect (CPE) and cell lysis in the presence of Acyclovir. In each case for the Jurkat tat cells (a modified cell-line of human T-cells with tat+) , these cells were transfected with one of the three plasmids

(pNL43, pNL43tk, or pNL43dBMtk as respectively indicated m rows 3, 4, & 5 of Chart 1) and then the cultures of the respective transfected cells were assayed for p24, RT, CPE and cell lysis (after treatment with 10 microMolar of Acyclovir) . It would be expected that the modified T-cells transfected with pNL43BdMtk would produce rHIV 1 (tat-, tk+) and that these cells would experience cell death by lysis from treatments of Acyclovir (due to internal expression of the viral tk gene product) . The results shown chart 1 confirm this expectation as only 13% of the Jurkat

(tat+) cells transfected with pNL43dBMtk were positive for HIV-1 p24 after only one treatment. 100% of the control Jurkat (tat+) cells untreated with Acyclovir but transfected with pNL43dBMtk were positive for HIV-1

(not shown in Chart 1) . Accordingly some RT and CPE were also detected and observed. Moreover, the same

(13%) number survived after cell lysis indicating that cell death by lysis did occur the presence of Acyclovir. These are the same 13% cells that were present m the IFA test. The potency of pNL43 as an HIV infective carrier is demonstrated by these assays. Control T cells lines (tat-) (i.e. not transformed to include the tat gent) transfected with tat-, tk- (e.g. pNL43dBM) or tat-, tk+ (e.g. pNL43dBMtk) did not synthesize virus-specifIC antigens and reverse transcriptase was not detected during an extended period of cultunng of these control T cell lines. However, PCR (polymerase chain reaction) analysis did indicate the presence of HIV-1 specific sequences in these control cells, thereby verifying an effective transfection with the plasmids. As shown m Chart 1, the modified Jurkat (tat+) cells supported the production of infectious virus efficiently, including those cells transfected with pNL43, pNL43tk and pNL43dBMtk.

CHART 1 Test of pNL43 transfected Jurkat (tat+) cells for the replication of the progency virus and response to Acyclovir (10μM)

Primary Transfectant Jurkat (tat+)

Specimen IFA Cell Lysis Supernatant p24 (%+) RT CPE after Acvclovir

PBMC

Negative

Jurkat pNL43 45 pNL43tk 43 pNL43dBMtk 13

HIV (LAV) 47

Chart 2 demonstrates the infectiousness of the various forms of recombinant virus [including both of the control viruses produced by transfection of T-cellε with pNL43 and pNL43tk and the desired recombinant virus rHIV-1 (tat-, tk+) produced by transfection of T- cells with pNL43dBMtk] . Note that these recombinant viruses were used for the infection of normal (not infected by HIV) fresh peripheral blood mononuclear cells (PBMC) , which are known to be targets of wild- type HIV-1; these cells were also exposed to a wild- type HIV-1 (the HIV-1 LAV isolate) to verify their expected infectability. The PBMC exposed to rHIV-1

(tat-, tk+) were not killed by this virus while those PBMC cells exposed to the control recombinant viruses

(shown m the columns pNL43 and pNL43tk) were killed as expected. In the case of the column labeled pNL43dBM, the supernatant from transfected T (tat+) cells

(transfected with pNL43dBM) was applied to the culture containing the PBMC cells; it will be appreciated that it would be expected, based on the teachings of this invention, that no virus would be produced m this case, and no infection or cell lysis would occur. This expectation was also confirmed.

CHART 2

Infection of PBMC from 4 normal donors by the progency virus produced by the pNL43 series of virus produced from Jurkat tat+ cells. Specimen HIV- 1 Umnfected number pNL43 pNL43dBM pNL43tk pNL43dBMtk ( LAV) Control

1 CPE/ cel no CPE/ cel no CPE/cel Good 1 lysi s respons 1 lys is respons 1 lysis e e

2 CPE/cel no CPE/cel no CPE/cel Good 1 lysis respons 1 lysis respons 1 lysis e e

3 CPE/cel no CPE/cel no CPE/cel Good 1 lysis respons 1 lysis respons 1 lysis e e

4 CPE/cel no CPE/cel no CPE/cel Good 1 lysis respons 1 lysis respons 1 lysis e e

For the columns labelled pNL43 and HIV-1 (LAV) , CPE were observed in the infected cells within 3 to 5 days and all cells in these cultures died by 12-15 days. For the column labelled pNL43tk, CPE were observed by the 10th day in culture, although many of these cells remained healthy, and these cells were killed by applying 10 microMolar of Acyclovir.

This evidence demonstrates that the insertion of the tk gene is effective in producing cell death in the presence of a nucleoside analog such as Acyclovir or Gancyclovir and yet does not effect normal HIV specificity and infectivity. This also demonstrates that rHIV (tat-, tk+) is effective in infecting and killing T-cells specifically in the presence of the nucleoside analog. This also demonstrates that inactivation of the tat gene will prevent viral replication. In addition to the uses described herein for the recombinant virus (e.g. rHIV-1) , this virus is useful for research and development into the molecular biology of HIV, and it can be used in animal studies for potentiating immune responses. This virus can also be used to test the response of an animal to the virus in a specially developed murine system which is transplanted with human lymphoid organs. These are mice with sever combined immunodeficiency (SCID-hu) . Moreover, this virus can be used in regulated gene therapy strategies by adding a therapeutic gene to the rHIV, and injecting this modified rHIV into an animal and regulating over expression by injecting Acyclovir and injecting tat protein to cause expression of the therapeutic gene. The rHIV of the present invention may also be used in research and experimentation in the field of immunosuppression; for example, rHIV may be used to produce immunosuppresion to enhance organ transplantation or may be used to treat autoimmune disorders. In one case, rHIV may be introduced into an organ transplant patient and then the tat gene product may be injected intravenously while Acyclovir is introduced in the patient; in this manner, the infected CD4 lymphocytes may be selectively killed to suppress the immune system.

Various of the plasmids and cell lines described above have been deposited with the American Type Culture Collection (ATCC) . In particular, samples of the modified Jurkat (tat+) T cell line have been deposited with the ATCC, 12301 Parklawn Drive, Rockville, MD, U.S.A. on October 3, 1995 under ATCC accession No. CRL 11987. Samples of the unmodified (control) Jurkat T cell (untransfected with plasmid 105) have been deposited with the ATCC, 12301 Parklawn Drive, Rockville, MD. USA on October 3, 1995 under ATCC Accession No. CRL 11988. Samples of the plasmids pNL43, in samples contained within Escherichia coli bacteria, have been deposited with the ATCC, 12301 Parklawn Drive, Rockville, MD, USA on October 3, 1995 under ATCC Accession No. 69928. Samples of the plasmid pNL43dBM, contained within bacteria Escherichia coli, have been deposited with the ATCC, 12301 Parklawn Drive, Rockville, MD, USA on October 3, 1995 under ATCC Accession No. 69927. Samples of the plasmid pNL43dBMtk, contained within bacteria Escherichia coli, have been deposited with the ATCC, 12301 Parklawn Drive, Rockville, MD, USA on October 3, 1995 under ATCC Accession No. 69929.

Applicant s or i gent's file International application No rcfereac,; number 02094. P001D

INDICATIONS RELATING TO Λ DCI'OSITCD MICROORGANISM

Λ. The indications made below relate to the microorganism referred in tn the description on page 23 . line 22 (Jurkat tat +) ιι. inεNTiπcΛTiO or ΠLΓOSIT rυrlli r deposits .ire identified on .in additional shcel K

Name of dc_|røsιtιry institution

American Type Culture Collection (ATCC)

Address of depositary institution {rncluJmς postal oJ and country)

ATCC

12301 Parklawn Drive Rockville, Maryland 20852 United States of America

Date ol deposit Λccei On Number

03 October 1995 (03.10.95) ATCC NO. CRL 11987

C. ADDITIONA INDICATIONS {lease Muni_* rf oi pplicah ) Tins inforππiion is continued on an additional sheet [ 1

A Jurkat (tat +) T cell line (designated as 65AJT) ; a modified form of a human

T cell line produced as described in the description at pages 12-14 . This cell line is useful for packaging and/or producing a recombinant HIV as described in the description at pages 17-18 .

Applicant hereby desires to avail itself of the benefit of the expert option under Rule 28(4) EPC.

D. DESICNΛ^'I HO STΛ'IT.S KOK WHICH INDICΛ'l IONS ΛUK MΛUK (./'<-.^■ rnJicuuonj arenctfor all u._Sn iι States)

All designated states.

i:. SU'ΛRΛTK r^'UKNISIUNC O ^* INDICATIONS ( aveh nl f n„, a_rPl,cal.U)

ITic indications listed below will be submit led to the International IJure.iu later {sp_*.crf, th r.n_*.ral natwc n{ tlu: i dication! e g , 'Λcc xiσn Su**rher of Depasrl ')

Applicant s or agent's Tile lntcrr..ιiιυnΛl.ιpplι jtιθnNo reference number 02094. P001D

INDICATIONS RELATING lOΛ DEPOSITED MICROORGANISM

(PCT Rule Ijύis)

Λ. The indications, made below relate lo the microorganism referred to in the description on page 23 . line 33 (Jurkat tat-) ii. IDENTIΠCΛTIO or DEPOSIT rurther deposits .ire identified on .in .iddition.il sheet X

Name of dcjxisitary institution American Type Culture Collection (ATCC)

Address of depositary institution (tπeludin postal code and country)

ATCC

12301 Parklawn Drive Rockville, Maryland 20852 United States of America

D.itc ol^" deposit ΛtctiMfin Number

03 October 1995 (03.10.95) ATCC NO. CRL 11988

C. ADDITIONAL INDICATIONS ( as- filan. if not apphcahte) Tins informniion is continued on an additional sheet j |

A Jurkat T cell line (designated as 65BJ); a form of a human T cell line which is available and is useful for producing the Jurkat (tat-r) T cell line as described in pages 12-14 of the description.

D. DESIGNATED STΛTT.S OIt WHICH INDICΛ^'I IONS AUK MADE {\f tlu: indications arc not for all signated States)

All designated states.

C. SEPARATE KURNISIIINC Ol^" INDICATIONS ( -mcb r,!, ι/.« applicahl,)

The in ications listed below will be submitted to the liucrn.ntion.il IJurcau later (specify ilie general nature of ^'tire in i ations e $ . 'Accession umber of Deposit')

Tor receiving Office use only (^"or In.enuiion.il Bureau use only

\ ζ This sheet was received with the intcrn.inon.il application I ^"Hns sheel wn^ receiv d by ihe Irucrn.ilion.il βurc.iu on

Authorized officer Authorized officer rt' Ml

Form PCΪ7KO/I 4 (July 1992) Applicant s or s-: n[ s tile I rw.vr. iiυm. pplication No rcterenci numoer 02094. P001D

INDICATION'S RELΛ^'lliNC TO A DlZl'OSITCϋ MICKOORCΛMSM

(PCT Rule I3i )

Torni PCTΛ<0/l3-i (Juiv 1992) Applicant * or agent s [i ) Iπtern tiυn si replicatio i">0 reference number 02094. P001D

INDICATIONS RELATING TO Λ DEI'OSI^'ITD MICISOOKCΛNISM

Λ. The indications made below relate to the microorganism retcrrcd to in the description on page _^_ . line g (pNL43dBM)

11. IDENTIFICATION OI^' OLIOS IT urther deposits arc identified on an additional sheet k;

Name of de_|iosιtar) institution

American Type Culture Collection (ATCC)

Address of depositar institution (including postal eode and country)

ATCC

12301 Parklawn Drive Rockville, Maryland 20852 United States of America

Date ol deposit Λι. .iMθπ um er 03 October 1995 (03.10.95) ATCC NO. 69927

C. Λ ODITIONΛL IN 1)1 C,\^'l IONS {leu*-e blank rf not applicable) T ns inforrn-iiion is continued on 3t\ additional sheet [ ]

Bacteria with pNL43dBM plasmid (designated as Escherichia coli strain DH5) . These bacteria are produced as described in the description at pages 14 and 21 These bacteria are useful for producing a modified version of the ρNL43dBM plasmid, such as pNL43dBMtk.

Applicant hereby desires to avail itself of the benefit of the expert option under Rule 28(4) EPC.

0. DLSIC Λ^'I^'l.D S^'lΛ ITS KOK WHICH \ \)lCΛ^'V OSS ,\Ul. ^\.\i)i: {, [ I, ee indr atwns are not for all desrgnated Slates)

All designated states.

I^".. SKI'ΛllΛ^'l i: KURNISIIINC Ol^" INDICATIONS {Luse blank, f not applicable)

^'Die indications listed be I osv w. ill be submitted to tlie I nieπn i loin! liureau ler {specify the gen al atu nftiu. indication., e . of Deposit^')

I or Iπi πi llt ml Bur au use only

I ^'iliis, stiCLt ΛΓU received by ihe Inicrmiioml fiurc.iu on

Λυlliori/cd olfi er

INDICATIONS RCLΛTIN'C TO A DCΓOSITCD MICISOORCΛNIS_IM

A The indications made belo" relate to the microorganism referred lo in the description on p ς 2 .line 12 (pNL43dBMtk)

'is. cNTiπcΛ'uoN or DLΓOSIT jrtin ( deposits are identified on an additional sheet [ I

Name of deposit ιr) institution

American Type Culture Collection (ATCC)

Ad ress ofdeposttary institution (ineludrng postal code and s uuntry)

ATCC

12301 Parklawn Drive Rockville, Maryland 20582 United States of America

ϋiicol deposit Λ n.isιθιι Number

03 October 1995 (03.10.95) ATCC NO. 69929

C. A H HIT IONA INΠICA I ION {teuie blank if not applicable) ^"111 is intorπiation is continued on an additional sheet [ j

Bacteria with pNL43dBMtk plasmid (designated as Escherichia coli strain DH5). These bacteria are produced as described in the description at pages 14-15/21-22 These bacteria are useful for producing the plasmid _PNL43dBMtk which may then be used to create a modified HIV ("rHIV").

Applicant hereby desires to avail itself of the benefit of the exper_t option under Rule 28(4) EPC

π. DIZSICNΛ'I ι;υ SIΛI I.S IOK WHICH I.N'DICΛ^'I IO.NS ΛKI: ,MΛI . (rfι,» nJitu/ioJuoi_^w/o .w_^jMJu^iJiu)

All designated states.

ι:. SI ΛIOVI 1: KUKNISΠINC OI IMMCΛ I IONS I O C blank, t n_Ut liable)

^"lϊic indications listed below vs ill be submitted to t e Inlcrmlioin I IJureiu liter {specif, the general nature ofllu. indication.! g , 'Acc_ession iSurnber of Deposit^')

Tor recei i g Olfiee use only I or Inivrπincitnl [lur<.ju u^c only

V] Tins sheet ss s rccciscd with the intern i lion il application [ Hits sh(_r_ι v. is r<_ccιvι_d by llie Inicrmiioml flurciυ on

Authorized officer Aut orized ot c r

It ^■ . form ΓCΓ,^" O/ XI3J ('JJulvl |W?) These deposits with the ATCC provide for permanent availability of the progeny of these cell lines to the public on the issuance of the U.S. patent describing and identifying the deposit or the publication or laying open to the public of any US. or foreign patent applications, which ever comes first, and for availability of the progeny of these cell lines to one determined by the U.S Commission of patents and Trademarks to be entitled thereto according to 35 USC Section 122 and the Commissioner's rules pursuant thereto. The owner of the present application has agreed that if the cell lines on deposit should die or be lost or destroyed when cultivated under suitable conditions, they will be promptly replaced on notification with a viable culture of the same cell line .

The present invention is not to be limited in scope by the microorganisms or cells deposited since the deposited embodiment is intended as a single illustration of one aspect of the invention and any microorganisms or cells or modified viruses which are functionally equivalent are within the scope of this invention. Indeed various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

Although the present invention has been described with preferred embodiments, it is to be understood that modifications and variations may be resorted to, without departing from the spirit and scope of this invention, as those skilled in the art will readily understand.

Various forms of the HIV virus may be modified, as described herein, to include the tk gene and to incapacitate the tat gene to produce an rHIV virus of the present invention. A search of the GenBank database may be performed to obtain these HIV viruses. As is well known, the GenBank includes many nucleotide sequences and is maintained by the National Center for Biotechnology Information (National Library of Medicine, National Institutes of Health) . The GenBank is available through the home page of the National Center for Biotechnology Information on the Works Wide Web (at the Uniform Resource Locator http: //www.ncbi . lm.nih.gov/ ) .

A search of the GenBank for HIV which contain proviral sequences similar to a portion of the pNL43 sequence was performed to obtain examples of forms of HIV which are expected to be compatible with the methods of the present invention such that these forms of HIV may be modified to produce a corresponding form of rHIV. Listed below in the accompanying Sequence Listing are portions of 15 examples of these forms of HIV. The following chart (CHART 3) identifies these particular examples by referring to the GenBank' s accession number. The portion of the pNL43 sequence used to search the GenBank is shown in the accompanying Sequence Listing as "M19921" (SEQ.ID No: 1) . Accession No.

M19921 L31963 K03455 U12055 K02013 M15654 104549 X01762 107983 K02083 M17449 112142 M38429 L02317 M96155

Seσuence Listing

(1) GENERAL INFORMATION:

(i) APPLICANTS: Syed Zaki Salhuddin and Nickolas Chelyapov

(ii) TITLE OF INVENTION: Recombinant HIV and Modified Packaging Cells and Method for Testing Acquired Immune Deficiency Syndrome

(iii) NUMBER OF SEQUENCES: 15

(iv) CORRESPONDENCE ADDRESS:

(A) ADRESSEE: Blakely, Sokoloff, Taylor & Zafman

(B) STREET: 12400 Wilshire Boulevard, Suite 700

(C) CITY: Los Angeles

(D) STATE: California

(E) COUNTRY: U.S.A.

(F) ZIP: 90025

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: 3.5 inch, double-sided, double- density disk.

(B) OPERATING SYSTEM: Apple Macintosh. (C) SOFTWARE: Microsoft Word Version 5.1a.

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA

(A) APPLICATION NUMBER:

(B) FILING DATE:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: James Scheller, Jr.

(B) REGISTRATION NUMBER: 31,195

(C) REFERENCE/DOCKET NUMBER: 02094. P001

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (408) 720-8598.

<B) TELEFAX: (408) 720-9397. (2) INFORMATION FOR SEQ. ID NO: 1

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 351

(B) TYPE: Nucleic acid

(C) STRANDEDNESS : Double-stranded

(D) TOPOLOGY: Unknown

(ii) MOLECULE TYPE:

(A) DESCRIPTION: GenBank Accession No: M19921; Human immunodeficiency virus type 1, NY5/BRU (LAV-1) recombinant clone pNL4-3.

(xi) Sequence Description: SEQ. ID NO: 1:

GGGATACTTG GGCAGGAGTG GAAGCCATAA TAAGAATTCT GCAACAACTG CTGTTTATCC 60

ATTTCAGAAT TGGGTGTCGA CATAGCAGAA TAGGCGTTAC TCGACAGAGG AGAGCAAGAA 120

ATGGAGCCAG TAGATCCTAG ACTAGAGCCC TGGAAGCATC CAGGAAGTCA GCCTAAAACT 180

GCTTGTACCA ATTGCTATTG TAAAAAGTGT TGCTTTCATT GCCAAGTTTG TTTCATGACA 240 AAAGCCTTAG GCATCTCCTA TGGCAGGAAG AAGCGGAGAC AGCGACGAAG AGCTCATCAG 300

AACAGTCAGA CTCATCAAGC TTCTCTATCA AAGCAGTAAG TAGTACATGT A 351

(2) INFORMATION FOR SEQ. ID NO: 2:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 351

(B) TYPE: Nucleic acid

(C) STRANDEDNESS : Double-stranded

(D) TOPOLOGY: Unknown

(ii) MOLECULE TYPE:

(A) DESCRIPTION: GenBank Accession No: L31963; Human Immunodeficiency virus type 1 (individual isolate: TH4- 7-5) gene.

[xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 2: GGGATACTTG GGCAGGAGTG GAAGCCATAA TAAGAATTCT GCAACAACTG CTGTTTATCC 60

ATTTCAGAAT TGGGTGTCGA CATAGCAGAA TAGGCGTTAC TCAACAGAGG AGAGCAAGAA 120

ATGGAGCCAG TAGATCCTAG ACTAGAGCCC TGGAAGCATC CAGGAAGTCA GCCTAAAACT 180

GCTTGTACCA CTTGCTATTG TAAAAAGTGT TGCTTTCATT GCCAAGTTTG TTTCATGACA 240

AAAGCCTTAG GCATCTCCTA TGGCAGGAAG AAGCGGAGAC AGCGACGAAG AGCTCATCAG 300

AACAGTCAGA CTCATCAAGC TTCTCTATCA AAGCAGTAAG TAGTACATGT A 351

(2) INFORMATION FOR SEQ. ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 351

(B) TYPE: Nucleic acid

(C) STRANDEDNESS: Double-stranded

(D) TOPOLOGY: Unknown (ii) MOLECULE TYPE:

(A) DESCRIPTION: GenBank Accession No: K03455; Human immunodeficiency virus type 1 (HXB2), complete genome; HIV1/HTLV-III/LAV reference genome.

(xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 3

GGGATACTTG GGCAGGAGTG GAAGCCATAA TAAGAATTCT GCAACAACTG CTGTTTATCC 60

ATTTCAGAAT TGGGTGTCGA CATAGCAGAA TAGGCGTTAC TCGACAGAGG AGAGCAAGAA 120

ATGGAGCCAG TAGATCCTAG ACTAGAGCCC TGGAAGCATC CAGGAAGTCA GCCTAAAACT 180

GCTTGTACCA ATTGCTATTG TAAAAAGTGT TGCTTTCATT GCCAAGTTTG TTTCATAACA 240

AAAGCCTTAG GCATCTCCTA TGGCAGGAAG AAGCGGAGAC AGCGACGAAG AGCTCATCAG 300

AACAGTCAGA CTCATCAAGC TTCTCTATCA AAGCAGTAAG TAGTACATGT A 351 (2) INFORMATION FOR SEQ. ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 351

(B) TYPE: Nucleic acid

(C) STRANDEDNESS: Double-stranded

(D) TOPOLOGY: Unknown

(ii) MOLECULE TYPE:

(A) DESCRIPTION: GenBank Accession No: U12055; Human immunodeficiency virus type 1 isolate LW12.3 from infected lab worker, complete genome.

;xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 4:

GGGATACTTG GGCAGGAGTG GAAGCCATAA TAAGAATTCT GCAACAACTG CTGTTTACCC 60

ATTTCAGAAT TGGGTGTCGA CATAGCAGAA TAGGCGTTAC TCGACAGAGG AGAGCAAGAA 120

ATGGAGCCAG TAGATCCTAG ACTAGAGCCT TGGAAGCATC CAGGAAGTCA GCCTAAAACT 180 GCTTGTACCA ATTGCTATTG TAAAAAGTGT TGCTTTCATT GCCAAGTTTG

TTTCATAACA 240

AAAGCCTTAG GCATCTCCTA TGGCAGGAAG AAGCGGAGAC AGCGACGAAG

ACCTCCTCAA 300

AGCAGTCAGA CTCATCAAGT TTCTCTATCA AAGCAGTAAG TAGTACATGT

A 351

(2) INFORMATION FOR SEQ. ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 351

(B) TYPE: Nucleic acid

(C) STRANDEDNESS: Double-stranded

(D) TOPOLOGY: Unknown

(ii) MOLECULE TYPE:

(A) DESCRIPTION: GenBank Accession No: K02013; Human immunodeficiency virus type 1, isolate BRU, complete genome (LAV-1 ) .

[xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 5 GGGATACTTG GGCAGGAGTG GAAGCCATAA TAAGAATTCT GCAACAACTG

CTGTTTATCC 60

ATTTCAGAAT TGGGTGTCGA CATAGCAGAA TAGGCGTTAC TCAACAGAGG

AGAGCAAGAA 120

ATGGAGCCAG TAGATCCTAG ACTAGAGCCC TGGAAGCATC CAGGAAGTCA

GCCTAAAACT 180

GCTTGTACCA CTTGCTATTG TAAAAAGTGT TGCTTTCATT GCCAAGTTTG

TTTCACAACA 240

AAAGCCTTAG GCATCTCCTA TGGCAGGAAG AAGCGGAGAC AGCGACGAAG

ACCTCCTCAA 300

GGCAGTCAGA CTCATCAAGT TTCTCTATCA AAGCAGTAAG TAGTACATGT

A 351

(2) INFORMATION FOR SEQ. ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 351

<B) TYPE: Nucleic acid

(C) STRANDEDNESS: Double-stranded [D) TOPOLOGY: Unknown

(ii) MOLECULE TYPE:

(A) DESCRIPTION: GenBank Accession No: M15654; Human immunodeficiency virus type 1, isolate BH10, genome.

[xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 6

GGGATACTTG GGCAGGAGTG GAAGCCATAA TAAGAATTCT GCAACAACTG CTGTTTATCC 60

ATTTCAGAAT TGGGTGTCGA CATAGCAGAA TAGGCGTTAC TCGACAGAGG AGAGCAAGAA 120

ATGGAGCCAG TAGATCCTAG ACTAGAGCCC TGGAAGCATC CAGGAAGTCA GCCTAAAACT 180

GCTTGTACCA ATTGCTATTG TAAAAAGTGT TGCTTTCATT GCCAAGTTTG TTTCATAACA 240

AAAGCCTTAG GCATCTCCTA TGGCAGGAAG AAGCGGAGAC AGCGACGAAG ACCTCCTCAA 300

GGCAGTCAGA CTCATCAAGT TTCTCTATCA AAGCAGTAAG TAGTACATGT A 351 (2) INFORMATION FOR SEQ. ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 351

(B) TYPE: Nucleic acid

(C) STRANDEDNESS: Double-stranded

(D) TOPOLOGY: Unknown

(ii) MOLECULE TYPE:

(A) DESCRIPTION: GenBank Accession No: 104549; Sequence 11 from patent EP 0187041

(xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 7:

GGGATACTTG GGCAGGAGTG GAAGCCATAA TAAGAATTCT GCAACAACTG

CTGTTTATCC 60

ATTTCAGAAT TGGGTGTCGA CATAGCAGAA TAGGCGTTAC TCGACAGAGG

AGAGCAAGAA 120

ATGGAGCCAG TAGATCCTAG ACTAGAGCCC TGGAAGCATC CAGGAAGTCA

GCCTAAAACT 180 GCTTGTACCA ATTGCTATTG TAAAAAGTGT TGCTTTCATT GCCAAGTTTG

TTTCATAACA 240

AAAGCCTTAG GCATCTCCTA TGGCAGGAAG AAGCGGAGAC AGCGACGAAG

ACCTCCTCAA 300

GGCAGTCAGA CTCATCAAGT TTCTCTATCA AAGCAGTAAG TAGTACATGT

A 351

(2) INFORMATION FOR SEQ. ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 351

(B) TYPE: Nucleic acid

(C) STRANDEDNESS: Double-stranded

(D) TOPOLOGY: Unknown

(ii) MOLECULE TYPE:

(A) DESCRIPTION: GenBank Accession No: X01762; Human T-cell leukemia type III (HTLV-III) proviral genome (AIDS virus for acquired immune deficiency syndrome) ;xi) SEQUENCE DESCRIPTION: SEQ. ID NO.

GGGATACTTG GGCAGGAGTG GAAGCCATAA TAAGAATTCT GCAACAACTG CTGTTTATCC 60

ATTTCAGAAT TGGGTGTCGA CATAGCAGAA TAGGCGTTAC TCGACAGAGG AGAGCAAGAA 120

ATGGAGCCAG TAGATCCTAG ACTAGAGCCC TGGAAGCATC CAGGAAGTCA GCCTAAAACT 180

GCTTGTACCA ATTGCTATTG TAAAAAGTGT TGCTTTCATT GCCAAGTTTG TTTCATAACA 240

AAAGCCTTAG GCATCTCCTA TGGCAGGAAG AAGCGGAGAC AGCGACGAAG ACCTCCTCAA 300

GGCAGTCAGA CTCATCAAGT TTCTCTATCA AAGCAGTAAG TAGTACATGT A 351

(2) INFORMATION FOR SEQ. ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 351

(B) TYPE: Nucleic acid

(C) STRANDEDNESS: Double-stranded (D) TOPOLOGY: Unknown

(ii) MOLECULE TYPE:

(A) DESCRIPTION: GenBank Accession No: 1079083; Sequence 1 from patent EP 0185444

[xi) SEQUENCE DESCRIPTION: SEQ.ID NO: 9:

GGGATACTTG GGCAGGAGTG GAAGCCATAA TAAGAATTCT GCAACAACTG CTGTTTATCC 60

ATTTCAGAAT TGGGTGTCGA CATAGCAGAA TAGGCGTTAC TCGACAGAGG AGAGCAAGAA 120

ATGGAGCCAG TAGATCCTAG ACTAGAGCCC TGGAAGCATC CAGGAAGTCA GCCTAAAACT 180

GCTTGTACCA ATTGCTATTG TAAAAAGTGT TGCTTTCATT GCCAAGTTTG TTTCATAACA 240

AAAGCCTTAG GCATCTCCTA TGGCAGGAAG AAGCGGAGAC AGCGACGAAG ACCTCCTCAA 300

GGCAGTCAGA CTCATCAAGT TTCTCTATCA AAGCAGTAAG TAGTACATGT A 351 (2) INFORMATION FOR SEQ. ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 351

(B) TYPE: Nucleic acid

(C) STRANDEDNESS: Double-stranded

(D) TOPOLOGY: Unknown

(ii) MOLECULE TYPE:

(A) DESCRIPTION: GenBank Accession No: K02083; Human immunodeficiency virus type 1, isolate PV22, complete genome (H9/HTLV-III proviral DNA) .

(xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 10:

GGGATACTTG GGCAGGAGTG GAAGCCATAA TAAGAATTCT GCAACAACTG CTGTTTATCC 60

ATTTCAGAAT TGGGTGTCGA CATAGCAGAA TAGGCGTTAC TCGACAGAGG AGAGCAAGAA 120

ATGGAGCCAG TAGATCCTAG ACTAGAGCCC TGGAAGCATC CAGGAAGTCA GCCTAAAACT 180 GCTTGTACCA ATTGCTATTG TAAAAAGTGT TGCTTTCATT GCCAAGTTTG TTTCATAACA 240

AAAGCCTTAG GCATCTCCTA TGGCAGGAAG AAGCGGAGAC AGCGACGAAG ACCTCCTCAA 300

GGCAGTCAGA CTCATCAAGT TTCTCTATCA AAGCAGTAAG TAGTACATGT A 351

(2) INFORMATION FOR SEQ. ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 351

(B) TYPE: Nucleic acid

(C) STRANDEDNESS: Double-stranded

(D) TOPOLOGY: Unknown

(ii) MOLECULE TYPE:

(A) DESCRIPTION: GenBank Accession No: M17449; HIV- 1, isolate MN [xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 11

GGGATACTTG GGCAGGAGTG GAAGCCATAA TAAGAATTCT ACAACAACTG

CTGTTTATTC 60

ATTTCAGAAT TGGGTGTCGA CATAGCAGAA TAGGCATTAT TCGACAGAGG

AGAGCAAGAA 120

ATGGAGCCAG TAGATCCTAG ACTAGAGCCC TGGAAGCATC CAGGAAGTCA

GCCTAAGACT 180

GCTTGTACCA CTTGCTATTG TAAAAAGTGT TGCTTTCATT GCCAAGTTTG

TTTCACAAAA 240

AAAGCCTTAG GCATCTCCTA TGGCAGGAAG AAGCGGAGAC AGCGACGAAG

AGCTCCTGAA 300

GACAGTCAGA CTCATCAAGT TTCTCTACCA AAGCAGTAAG TAGTACATGT

A 351

(2) INFORMATION FOR SEQ. ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 351

(B) TYPE: Nucleic acid

(C) STRANDEDNESS: Double-stranded (D) TOPOLOGY: Unknown

ii) MOLECULE TYPE:

(A) DESCRIPTION: GenBank Accession No: 112142 Sequence 1 from U.S. Patent No. 5,420,030.

[xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 12:

GGGATACTTG GGCAGGAGTG GAAGCCATAA TAAGAATTCT ACAACAACTG CTGTTTATTC 60

ATTTCAGAAT TGGGTGTCGA CATAGCAGAA TAGGCATTAT TCGACAGAGG AGAGCAAGAA 120

ATGGAGCCAG TAGATCCTAG ACTAGAGCCC TGGAAGCATC CAGGAAGTCA GCCTAAGACT 180

GCTTGTACCA CTTGCTATTG TAAAAAGTGT TGCTTTCATT GCCAAGTTTG TTTCACAAAA 240

AAAGCCTTAG GCATCTCCTA TGGCAGGAAG AAGCGGAGAC AGCGACGAAG AGCTCCTGAA 300

GACAGTCAGA CTCATCAAGT TTCTCTACCA AAGCAGTAAG TAGTACATGT A 351 (2) INFORMATION FOR SEQ. ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 351

(B) TYPE: Nucleic acid

(C) STRANDEDNESS: Double-stranded

(D) TOPOLOGY: Unknown

(ii) MOLECULE TYPE:

(A) DESCRIPTION: GenBank Accession No: M38429; Human immunodeficiency virus type 1, isolate JRCSF, complete genome.

(xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 13:

GGGATACTTG GGCAGGAGTG GAAGCCATAA TAAGAATACT GCAACAGCTG CTGTTTATTC 60

ATTTCAGAAT TGGGTGTCGA CATAGCAGAA TAGGCATTAC TCGACAGAGG AGAGCAAGAA 120

ATGGAGCCAG TAGATCCTAG CCTAGAGCCC TGGAAGCATC CAGGAAGTCA GCCTAAGACT 180 GCTTGTACCA ATTGCTATTG TAAAAAGTGT TGCCTTCATT GCCAAGTTTG TTTCACAACA 240

AAAGGCTTAG GCATCTCCTA TGGCAGGAAG AAGCGGAGAC AGCGACGAAG ACCTCCTCAA 300

GACAGTCAGA CTCATCAAGT TTCTCTACCA AAGCAGTAAG TAGTGCATGT A 351

(2) INFORMATION FOR SEQ. ID NO: 14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 351

(B) TYPE: Nucleic acid

(C) STRANDEDNESS: Double-stranded

(D) TOPOLOGY: Unknown

(ii) MOLECULE TYPE:

(A) DESCRIPTION: GenBank Accession No: L02317; Human immunodeficiency virus type 1 [xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 14

GGGATACTTG GGCAGGAGTG GAAGCCCTAG TAAGAACTCT GCAACAACTG CTGTTTACTC 60

TTTTCAGAAT TGGGTGTCGA CATAGCAGAA TAGGCATTAC TCAACGAAGA AGAGCAAGAA 120

ATGGAGCCAG TAGATCCTAG ACTAGAGCCC TGGAAGCATC CAGGAAGCCA GCCTAAAACT 180

GCTTGTACCA AATGCTATTG TAAAAAGTGT TGCTTACATT GCCAAGTTTG TTTCATGACA 240

AAAGGCTTAG GCATCTCCTA TGGCAGGAAG AAGCGGAGAC AGCGACGAAG AGCTCCTCAA 300

GACAGTCAGA CTCATCAAGC TTCTCTATCA AAGCAGTAAG TAGTGCATGT A 351

(2) INFORMATION FOR SEQ. ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 351

(B) TYPE: Nucleic acid

(C) STRANDEDNESS: Double-stranded (D) TOPOLOGY: Unknown

(ii) MOLECULE TYPE:

(A) DESCRIPTION: GenBank Accession No: M96155; Human immunodeficiency virus type 1 proviral DNA encoding the vif, vpr, vpu, env, tat, rev, and nef genes, complete cds, and 3 ' LTR.

(xi) SEQUENCE DESCRIPTION: SEQ.ID NO: 15:

GGGATACTTG GACAGGAGTG GAAGCCTTAA TAAGAATTCT GCAACAACTG CTGTTTATTC 60

ATTTCAGAAT TGGGTGTCGA CATAGCAGAA TAGGCATTAT TCAACACAGG AGAACAAGAA 120

ATGGAGCCAG TAAATCCTAG CCTAGAGCCC TGGAAGCATC CAGGAAGTCA GCCTAAAACT 180

GCTTGTACCA ATTGCTATTG CAAAAAATGT TGCTTTCATT GCCAAGCTTG TTTCATAACA 240

AAAGGCTTAG GCATCTCCTA TGGCAGGAAG AAGCGGAGAC AGCGACGAAG ACCTCCTCAA 300

GACAGTCAGA CTCATCAAGT TTCTCTATCA AAGCAGTAAG TAGTACATGT A 351

Claims

1. A method for treating acquired immune deficiency disease in a human caused by human

immunodeficiency virus (HIV), said method comprising:

administering a composition comprising a modified human immunodeficiency virus (modified HIV) having a gene which encodes a viral thymidine kinase, said modified HIV being unable to express at least one functional regulatory gene product; and

administering a nucleoside analog.

2. A method as in claim 1 wherein said

nucleoside analog comprises one of Acyclovir or

Gancyclovir.

3. A method as in claim 1 wherein said

functional regulatory gene product is the expressed gene product of the tat gene of the HIV genome.

4. A method as in claim 1 wherein said

functional regulatory gene product is the expressed gene product of the rev gene of the HIV genome.

5. A method as in claim 1 wherein said

nucleoside analog is selected from the group comprising Acyclovir and Gancyclovir, and wherein said modified HIV is unable to express functional gene products of the tat and rev genes of the HIV genome.

6. A method as in claim 1 wherein said modified HIV is unable to replicate in a cell without the presence of a wild-type HIV.

7. A method as in claim 1 wherein said modified HIV is harvested from a modified mammalian cell having a tat HIV regulatory gene in the genome of said

modified mammalian cell.

8. A method as in claim 7 wherein said modified mammalian cell is a human T-cell.

9. A method as in claim 7 wherein said modified mammalian cell is a human B-cell.

10. A method as in claim 1 wherein said modified HIV infects human T-cells which have been previously infected by wild-type HIV.

11. A method of treating human immumodeficiency virus (HIV) infection in a human, said method

comprising:

administering a composition comprising a modified human immunodeficiency virus (modified HIV) having a gene, which when expressed in a cell which is infected by HIV, causes at least a portion of cells which are infected by HIV to be killed.

12. A method as in claim 11 further comprising: administering an agent which in combination with an expressed product of said gene causes said portion of cells to be killed.

13. A method as in claim 12 wherein said agent is a nucleoside analog and wherein said gene encodes a kinase enzyme.

14. A method as in claim 11 wherein said modified HIV is unable to replicate in a cell without the presence of a wild-type HIV.

15. A method as in claim 11 wherein said modified HIV is unable to express at least one functional regulatory gene product.

16. A method as in claim 15 wherein said

functional regulatory gene product comprises one of the tat and the rev genes of the HIV genome.

17. A method as in claim 16 further comprising: harvesting said modified HIV from a modified mammalian cell-line having said one of the tat and the rev genes in the genome of said modified mammalian cell-line.

18. A modified human immunodeficiency virus (modified HIV) comprising in its genome a chimeric recombinant gene construction including a foreign gene, said chimeric recombinant gene construction being effective in human cells infected with a human

immunodeficiency virus (HIV) to express a gene product coded by the foreign gene, said gene product being capable of causing at least a portion of human cells infected by HIV to be killed.

19. A modified HIV as in claim 18 wherein said modified HIV is unable to express at least one

functional regulatory gene product of the genome of HIV.

20. A modified HIV as in claim 19 wherein said at least one functional regulatory gene product is

selected from the group consisting of the tat and the rev genes in the genome of HIV.

21. A modified HIV as in claim 18 wherein said gene product is a kinase enzyme.

22. A modified HIV as in claim 21 wherein said kinase enzyme is thymidine kinase.

23. A modified HIV as in claim 21 wherein the genome of said modified HIV does not include a

functional regulatory gene which codes for a functional regulatory gene product of the genome of HIV.

24. A modified HIV as in claim 23 wherein said functional regulatory gene is selected from the group consisting of the tat and the rev genes in the genome of HIV.

25. A modified HIV as in claim 20 wherein said gene product is a kinase enzyme and wherein said modified HIV is unable to replicate in a cell without the presence of a wild type HIV.

26. A modified HIV as in claim 20 wherein said chimeric recombinant gene construction includes

promoter and control sequences.

27. A modified HIV as in claim 20 wherein said modified HIV is harvested from a modified mammalian cell having at least one of the tat and the rev genes in the genome of said modified mammalian cell.

28. A modified HIV as in claim 27 wherein said gene product is thymidine kinase enzyme and wherein said gene product kills said portion of human cells wherein combined with a nucleoside analog.

29. A method for treating acquired immune deficiency disease in a human caused by human

immunodeficiency virus (HIV), said method comprising:

administering to said human a composition comprising one of Acyclovir or Gancyclovir; and

administering a composition capable of providing viral thymidine kinase in said human.

30. A method as in claim 29 wherein said

composition capable of providing viral thymidine kinase comprises a modified human immunodeficiency virus

(modified HIV) having a gene which encodes said viral thymidine kinase, said modified HIV being unable to express at least one functional regulatory gene

product.

31. A method as in claim 29 wherein said

composition capable of providing viral thymidine kinase comprises a saline solution of viral thymidine kinase, and wherein said step of administering comprises injecting said solution into a patient.

32. A method as in claim 30 wherein said at least one functional regulatory gene product is the expressed gene product of the tat gene of the HIV genome.

33. A method as in claim 30 wherein said modified HIV is unable to replicate in a cell without the presence of a wild-type HIV.

34. A method for producing a modified human immunodeficiency virus (modified HIV) comprising:

producing a plurality of modified HIV in a modified mammalian cell, said modified HIV having a gene which encodes a foreign gene product and being unable to express at least one functional regulatory gene product, said modified mammalian cell having a gene which encodes said at least one functional

regulatory gene product.

35. A method as in claim 34 further comprising harvesting said plurality of modified HIV from said modified mammalian cell.

36. A method as in claim 35 wherein said step of producing is performed in tissue culture and wherein said modified mammalian cell is from a human T cell-line.

37. A method as in claim 36 wherein said foreign gene product is a kinase enzyme and said functional regulatory gene product is a tat protein of a human immunodeficiency virus.

38. A method as in claim 37 wherein said kinase enzyme is thymidine kinase.

39. A modified human immunodeficency virus

(modified HIV) produced by a process comprising: culturing a modified mammalian cell, said modified mammalian cell having a gene which encodes at least one functional regulatory gene product of HIV;

producing a plurality of modified HIV in said modified mammalian cell, said modified HIV having a gene which encodes a viral thymidine kinase and being unable to express said at least one functional

regulatory gene product of HIV.

40. A modified HIV produced by a process as in claim 39, wherein prior to culturing said modified mammalian cell, said modified mammalian cell was transfected with a vector containing the genome of said modified HIV.

41. A modified HIV produced by a process as in claim 39 further comprising harvesting said plurality of modified HIV from said modified mammalian cell.

42. A modified HIV produced by a process as in claim 39 wherein said modified mammalian cell is from a human T cell line.

43. A modified HIV produced by a process as in claim 39 wherein said at least one functional

regulatory gene product of HIV is the tat gene product.

44. A modified HIV produced by a process as in claim 39 wherein said modified HIV is unable to

replicate in a mammalian cell without the presence of wild-type HIV.