EP0863988A1 - Vih recombine, cellules d'encapsidation modifiees et methode de traitement du syndrome d'immunodeficience acquise - Google Patents

Vih recombine, cellules d'encapsidation modifiees et methode de traitement du syndrome d'immunodeficience acquise

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Publication number
EP0863988A1
EP0863988A1 EP96942059A EP96942059A EP0863988A1 EP 0863988 A1 EP0863988 A1 EP 0863988A1 EP 96942059 A EP96942059 A EP 96942059A EP 96942059 A EP96942059 A EP 96942059A EP 0863988 A1 EP0863988 A1 EP 0863988A1
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EP
European Patent Office
Prior art keywords
hiv
modified
gene
gene product
tat
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EP96942059A
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German (de)
English (en)
Inventor
Syed Zaki Salahuddin
Nickolas Chelyapov
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Clinical Technology Inc
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Clinical Technology Inc
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Priority claimed from US08/563,360 external-priority patent/US6063374A/en
Priority claimed from US08/563,597 external-priority patent/US5753490A/en
Application filed by Clinical Technology Inc filed Critical Clinical Technology Inc
Publication of EP0863988A1 publication Critical patent/EP0863988A1/fr
Withdrawn legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/45—Transferases (2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A61K31/522—Purines, e.g. adenine having oxo groups directly attached to the heterocyclic ring, e.g. hypoxanthine, guanine, acyclovir
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • A61K31/708—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid having oxo groups directly attached to the purine ring system, e.g. guanosine, guanylic acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • 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.
  • HIV acquired immune deficiency syndrome
  • HIV-1 human immunodeficiency virus
  • Numerous treatments for this disease have been proposed and tested and some are currently in use.
  • AIDS is commonly treated by administering the drug AZT to AIDS patients.
  • ⁇ bozyme 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.
  • 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.
  • 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.
  • r HIV human immunodeficiency virus
  • modified T-cell line having a functional regulatory gene from HIV
  • the modified human immuno deficiency virus includes in its genome a modification (e.g. a deletion) of one of the HIV regulatory genes and the inclusion of a foreign gene.
  • the foreign gene is a viral kinase enzyme such as a viral thymidine kinase (tk) from the Herpes simplex virus (HSV-1) .
  • tk viral thymidine kinase
  • HSV-1 Herpes simplex virus
  • 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.
  • 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.
  • this functional regulatory gene product of HIV is the same functional regulatory gene product which the rHIV cannot produce or express.
  • 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.
  • 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.
  • the nucleoside analog is Acyclovir or Gancyclovir.
  • 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.
  • 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.
  • 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.
  • 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.
  • Acyclovir or Gancyclovir which is a cytotoxic substance that will kill the HIV-1 infected cell.
  • normal cellular thymidine kinase phosphorylates the nucleoside analog very minimally with no consequences.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • HIV-1 tat/rev mRNA labeled 101
  • tat/rev cDNA 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.
  • human T or B cells e.g. the Jurkat T cell line
  • a modified T-cell or a modified B-cell such as T-cell 107 or B-cell 108.
  • 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
  • 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 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) .
  • a sample of 10 7 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 960 ⁇ F. 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.
  • 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.
  • 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.
  • 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.
  • the rev gene bay be knocked out in addition to the removal or knock out of the tat gene.
  • transfection operations are performed in order to introduce the respective plasmid into normal T-cell lines or modified T-cell lines.
  • transfection operations may be performed on normal B-cell lines or modified B- cell lines.
  • 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.
  • the electroporation technique is used to cause transfection in order to efficiently introduce the DNA construct into the packaging cells.
  • alternative methods of transfection may also be performed.
  • a DNA construct such as plasmid 209
  • T-cells e.g. modified Jurkat tat+ cells
  • T-cells e.g. modified Jurkat tat+ cells
  • 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.
  • this plasmid 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • plasmid 203 is pNL 43, shown m figure 4.
  • 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.
  • FIG. 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.
  • 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.
  • a foreign gene such as the HSV-1 thymidine kinase gene is inserted into the proviral HIV construct created step 803.
  • 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.
  • step 809 many copies of the recombinant virus [(for example, rHIV (tat-, tk+) ] .
  • step 811 this recombinant virus is harvested and then injected into a human patient suffering from infection by wild-type HIV-1.
  • 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.
  • 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.
  • 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) .
  • further injections of the recombinant virus into the patient may be performed followed by further administrations of the nucleoside analog.
  • 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.
  • 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 (5 1 ) 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.
  • 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.
  • a portion of the HSV-1 genome, containing the tk gene 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.
  • Plasmids 209 and 210 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 transc ⁇ ptase) , observing cytopathic effect (CPE) and cell lysis in the presence of Acyclovir.
  • p24 an HIV-1 structural protein
  • RT reverse transc ⁇ ptase
  • CPE cytopathic effect
  • 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.
  • PCR polymerase chain reaction
  • 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-cell ⁇ with pNL43 and pNL43tk and the desired recombinant virus rHIV-1 (tat-, tk+) produced by transfection of T- cells with pNL43dBMtk] .
  • 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.
  • PBMC peripheral blood mononuclear cells
  • CPE were observed in the infected cells within 3 to 5 days and all cells in these cultures died by 12-15 days.
  • 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 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.
  • 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.
  • plasmids and cell lines described above have been deposited with the American Type Culture Collection (ATCC) .
  • ATCC American Type Culture Collection
  • 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 have been deposited with the ATCC, 12301 Parklawn Drive, Rockville, MD. USA on October 3, 1995 under ATCC Accession No. CRL 11988.
  • 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.
  • 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.
  • 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 ⁇ NL43dBM plasmid, such as pNL43dBMtk.
  • Applicant hereby desires to avail itself of the benefit of the expert option under Rule 28(4) EPC.
  • Ad ress ofdeposttary institution (ineludrng postal code and s uuntry)
  • 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 P NL43dBMtk which may then be used to create a modified HIV ("rHIV").
  • 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.
  • 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/ ) .
  • ADRESSEE Blakely, Sokoloff, Taylor & Zafman
  • GCCTAAAACT 180 GCTTGTACCA ATTGCTATTG TAAAAAGTGT TGCTTTCATT GCCAAGTTTG

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Abstract

On décrit un virus d'immunodéficience acquise, humain et recombiné (VIHr), une lignée cellulaire mammifère recombinée (systèmes d'encapsidation) ainsi qu'une méthode de traitement de l'infection de cellules causée par le virus d'immunodéficience (VIH). Un VIHr, tel que le VIHr-1, comprend une construction de gènes incluant un gène étranger dont l'expression est activée dans les cellules humaines en présence du VIH de type sauvage. Ce produit génique, typiquement une thymidine kinase virale, peut provoquer la mort des cellules en présence d'un médicament approprié, par exemple l'acyclovir. On construit également le VIHr de manière à ce que celui-ci soit incapable de se répliquer par lui-même, par suite de l'absence d'un gène régulateur nécessaire à cette réplication, tel qu'un gène tat ou rev ou les deux. Dans ce système d'encapsidation de lignées cellulaires mammifères recombinées, ces lignées comprennent dans leur génome une construction de gènes recombinés comportant typiquement un gène régulateur fonctionnel provenant du VIH et manquant au VIHr, tel que le gène tat ou le gène rev, ou les deux à la fois. La méthode de traitement de l'infection de cellules par le VIH consiste à administrer une composition comprenant le VIHr, laquelle est produite in vitro à partir d'une lignée cellulaire mammifère recombinée, puis à traiter à l'aide d'un analogue de nucléoside, tel que l'acyclovir ou le gancyclovir.
EP96942059A 1995-11-28 1996-11-25 Vih recombine, cellules d'encapsidation modifiees et methode de traitement du syndrome d'immunodeficience acquise Withdrawn EP0863988A1 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US08/563,360 US6063374A (en) 1995-11-28 1995-11-28 Recombinant HIV and modified packaging cells and method for using
US08/563,597 US5753490A (en) 1995-11-28 1995-11-28 Recombinant HIV and modified packaging cells and method for treating acquired immune deficiency syndrome
US563360 1995-11-28
PCT/US1996/018885 WO1997020052A1 (fr) 1995-11-28 1996-11-25 Vih recombine, cellules d'encapsidation modifiees et methode de traitement du syndrome d'immunodeficience acquise
US563597 2000-05-03

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AU8576298A (en) * 1997-07-18 1999-02-10 Chiron Corporation Lentiviral vectors
JP2002508338A (ja) * 1997-12-12 2002-03-19 ナルディニ，ルイジ レンチウィルスベクターの治療使用
US5994136A (en) * 1997-12-12 1999-11-30 Cell Genesys, Inc. Method and means for producing high titer, safe, recombinant lentivirus vectors
EP1849873B1 (fr) 1999-04-29 2011-10-12 Gbp Ip, Llc Procédé et supports de production de vecteurs de lentivirus recombinant sécurisé à fort dosage
CN109810953A (zh) * 2019-01-07 2019-05-28 西安彤盛生物科技有限公司 去除tk基因的重组天坛株溶瘤痘苗病毒及其制备和应用

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US5306631A (en) * 1987-08-21 1994-04-26 University Of Colorado Foundation, Inc. Compositions and method for inhibition of HIV production
US5206353A (en) * 1988-07-23 1993-04-27 The United States Of America As Represented By The Department Of Health And Human Services CD-4/cytotoxic gene fusions

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