WO1995013081A1 - Agents et vecteurs lymphotropes - Google Patents

Agents et vecteurs lymphotropes Download PDF

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Publication number
WO1995013081A1
WO1995013081A1 PCT/US1994/012715 US9412715W WO9513081A1 WO 1995013081 A1 WO1995013081 A1 WO 1995013081A1 US 9412715 W US9412715 W US 9412715W WO 9513081 A1 WO9513081 A1 WO 9513081A1
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hhv
cells
vector
treatment
nucleic acid
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PCT/US1994/012715
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English (en)
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Nitza Frenkel
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Ramot University Authority For Applied Research And Industrial Development Ltd.
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Priority to AU10885/95A priority Critical patent/AU1088595A/en
Priority to US08/646,242 priority patent/US6503752B1/en
Publication of WO1995013081A1 publication Critical patent/WO1995013081A1/fr

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    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16511—Roseolovirus, e.g. human herpesvirus 6, 7
    • C12N2710/16522—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16511—Roseolovirus, e.g. human herpesvirus 6, 7
    • C12N2710/16541—Use of virus, viral particle or viral elements as a vector
    • C12N2710/16543—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16511—Roseolovirus, e.g. human herpesvirus 6, 7
    • C12N2710/16541—Use of virus, viral particle or viral elements as a vector
    • C12N2710/16545—Special targeting system for 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
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
    • C12N2810/60—Vectors comprising as targeting moiety peptide derived from defined protein from viruses

Definitions

  • the present invention is generally in the field of targeted therapeutic agents.
  • the present invention concerns an agent which specifically binds to receptors on certain cells.
  • the present invention concerns vectors specifically targeted to certain cells.
  • a specific aspect of the present invention concerns the prophylaxis and treatment of AIDS.
  • HHV-6 Human herpes virus-6 was first isolated from peripheral blood mononucleur cells (PBMC) of patients with lympho proliforative disorders as well as from patients suffering from acquired immune deficiency syndrome (AIDS) (Salahuddin et al.. 1986).
  • PBMC peripheral blood mononucleur cells
  • AIDS acquired immune deficiency syndrome
  • HHV-6 strains Two types of HHV-6 strains are recognized today and designated as variant A and variant B which differ as regards to their growth properties, restriction enzyme patterns and antigenicity and they are also distinct epidemiologically (Schirmer et al.. 1991). Only the HHV-6 B variant appears to be associated with human disease and has been found to be the causative agent of exanthem subitum (ES, roseola infantum). HHV-6 is shown to replicate only in interluken-2 (IL-2) activated T cells (Frenkel et al.. 1990) and is inhibited by very high concentrations of IL-2. After the initial infection process, the HHV-6 virus undergoes a latency period in the infected cells (Kondo et al.. 1991).
  • IL-2 interluken-2
  • HHV-6 may effect the efficiency of expression of the human immunodeficiency virus- 1 (HIV-1) when the two viruses have infected a single cell (Lusso et al.. 1989).
  • Human herpes virus-7 (HHV-7) is a DNA virus first isolated in the laboratory of the inventor of the present invention from activated T cells expressing the CD4 antigen (see U.S. Serial No. 07/553,798 and
  • CD4- cells Cells expressing this antigen on their membrane will hereinafter be referred to as "CD4- cells”.
  • HHV-7 was found to be distinct, both molecularly and antigenically, from all previously identified herpes viruses. HHV-7 replicates well in lymphocytes and particularly in T cells including CD4 T cells and possibly other cells carrying the CD4 marker.
  • the HHV-7 virions specifically target the T cells wherein the viral DNA is synthesized in the nucleus as concatemers which are tlgen cleaved and packaged into structural infectious particles. It has been shown recently that HHV-7 binds specifically to the CD4 receptor by which it infects CD4 cells.
  • HHV-7 is found in sera of more than 95% of humans (Wyatt et al.. 1991). In addition, the virus is very often found in human saliva
  • HHV-6 and HHV-7 DNA comprise a long unique sequence which is flanked by terminal direct repeats (TR) on each side (Pellet et al.. 1990, Lawrence et al.. 1990, Lindquester et al.. 1991 and Martin et al.. 1991).
  • TR terminal direct repeats
  • Each TR contains on one side a sequence which is heterogenous in size, designated het.
  • the het sequence was thought to be a variable and unstable sequence but later was found to be a unique sequence for each virus strain and to remain stable in a single strain over many passages of the virus (Schirmer et al.. 1991).
  • On the other side of the TR there is a repeated telomeric-like sequence having repeated units of the sequence GGGTTA.
  • CD4 + cells are also the target cells of the human immunodefi- ciency virus (HTV) which is the cause of acquired immuno deficiency syndrome (AIDS).
  • HTV human immunodefi- ciency virus
  • HIV binds to the CD4 receptor on the target cell with a high affinity, integrates itself into the host cells' genome and is believed to undergo a long latency period during which it is virtually undetectable. Activation of the virus to induce the disease may occur at different time periods after the first infection.
  • CD4-ligand a ligand capable of binding to the CD4 receptor
  • Such a ligand is usefiil for inhibiting the infectious process of viruses which infect the CD4 cells and which enter the cells by first binding to the CD4 receptors.
  • Another possible use of the CD4-ligand is as an immunomodulating agent.
  • lymphotropic vector a DNA vector specifically intended for lymphatic cells
  • Specific applications of the lymphotropic vector are in the treatment of AIDS as well as in the treatment of lymphatic malignancies, various autoimmune disorders, as well as a variety of T-cell pathologies.
  • CD4-ligand selected from the group consisting of:
  • HHV-7 human herpes virus 7
  • (c) a virus particle of the virus of (a) or (b); (d) a virion polypeptide of (c) capable of binding to CD4 recep ⁇ tor; (e) a fusion protein of a fragment of (d) and another protein or peptide, which is capable of binding the CD4 receptor; (f) derivatives of any of (c), (d) or (e) obtained by chemical modification, addition, deletion or replacement of one or more amino acid residues from the protein or peptides of (c), (d) or
  • a lymphotropic vector comprising a recombinant DNA molecule having: (i) a DNA sequence derived from HHV-6 or HHV-7 and com ⁇ prising an origin of DNA replication, a promoter sequence capable of inducing expression in a lymphatic host cell of a downstream nucleic acid sequence and a cleavage and packag ⁇ ing signal; (ii) a foreign nucleic acid sequence downstream to an expression control of said promotor sequence.
  • said lymphotropic vector is incorporated into a delivery vehicle.
  • delivery vehicle which are viral- derived particles are generally preferred in view of the specificity of such particles to certain cells which facilitate the targeting of the genetic material to such cells.
  • the lymphotropic vector of the invention is derived from HHV-6 or HHV-7
  • the preferred viral particle for use as a delivery vehicle is derived from these two respective viruses.
  • HHV-7 may activate HHV-6 replication (Frenkel et al.. 1992), and accordingly, it is also possible in accordance with the invention to use an HHV-7 particle as a delivery vehicle for an HHV-6 derived lymphatic vector.
  • HHV-6 or HHV-7 particles have an affinity to specific cell types.
  • the HHV-7 binds to the CD4 receptor and accordingly the particle derived from the HHV-7 is particularly useful for the delivery of said lymphotropic vector to CD4 + cells.
  • the HHV-6 particles have an affinity
  • lymphotropic vector to a variety of cells and mainly to both CD4 and CD8 cells, as well as to some other lymphatic cells, e.g. EBV infected B-cells, and may thus be useful for the targeting of said lymphotropic vector to such cells.
  • the preferred delivery vehicle in accordance with the present invention is a member selected from the group consisting of: (a) an HHV-6 or HHV-7 particle;
  • lymphatic vector there are non-lymphatic cells which are CD4 , e.g. fibroblasts or various brain cells, and said vector, using HHV-7 as a delivery vehicle is thus useful also as a therapeutic agent targeted at such non-lymphatic CD4 + cells.
  • HHV-6 is known to be capable of infecting also non- lymphatic cells, e.g. fibroblasts, CD4 brain cells, endothelial and epithilial cells and accordingly may be used as a therapeutic agent targeted also at such non-lymphatic cells.
  • said ligand or said lymphotropic vectors is in the treatment and/or prophylaxis of viral diseases which infect lymphatic, specifically CD4 or CD8 , cells.
  • An example of such an application is in the treatment and/or prophylaxis of HIV infections, in the treatment and/or prophylaxis of lymphomas or various autoimmune-related diseases or disorders, in the treatment of various T-cell pathologies, etc.
  • the present invention thus provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and, as an active ingredient, either said CD4-ligand or said lymphotropic vector.
  • the present invention still further provides a method for the treatment of a disease or disorder comprising administering said CD4-ligand or said lymphotropic vector to an individual in need.
  • Said CD4-ligand may be used in order to inhibit infections of
  • CD4 + cells which progress through the binding of the infectious agent to the CD4 receptor.
  • said CD4-ligand may also be used as an immuno- modulator, e.g. as a general immunosuppressor in autoimmune diseases.
  • Said lymphotropic vector is useful as an agent for genetic therapy in the treatment of various malignancies, viral infections, enzyme deficiencies and others, of lymphatic cells as well as other cells capable of being infected by HHV-6 or HHV-7.
  • Two kinds of vectors are provided by the present invention: a vector which is capable of autonomous replication (hereinafter: "ARV” (autonomously replicating vector)); a vector which is not capable of self replication (hereinafter: "Tamplicon").
  • ARV autonomous replicating vector
  • Tamplicon a vector which is not capable of self replication
  • a Tamplicon is administered together with a helper virus which provides the transactivation factors for replication of the Tamplicon.
  • a helper virus is typically a self-replicating HHV-6 or HHV-7.
  • the choice of the helper virus may typically be based on the nature of the Tamplicon: in case of a Tamplicon derived from HHV-6, a self-replicating HHV-6 will typically be used, and in the case of a Tamplicon derived from HHV-7, a self-replicating HHV-7 will typically be used.
  • a self-replicating HHV-7 may be used as a helper virus for an HHV-6 derived Tamplicon.
  • HHV-7 has no known pathology and therefore its use as a helper virus is generally preferred where possible over the use of HHV-6.
  • use of HHV-7 is limited in view of the fact that it infects primarily CD4 + cells and accordingly use of HHV-6 is at times preferred.
  • measures should be taken to neutralize this virus after such period of time.
  • a mutant HHV-6 may be used, the expression of which may be controlled by changes in various factors such as, a change in temperature (i.e. a tempera ⁇ ture sensitive mutant).
  • Fig. 1 is a schematic representation of the genome of HHV-6 or HHV-7. The figure shows a unit length of the genome as well as cleavage sides enabling to cut the expressed DNA into unit length sequences and the packaging thereof into a viral particle;
  • Fig. 2 is a schematic representation of an example of an ARV in accordance with the invention
  • Fig. 3 shows the formation of concatemers during replication of the
  • Fig. 4 is a schematic representation of a Tamplicon of the invention and the manner of its use together with a self-replicating virus;
  • Fig. 5 shows pac-1 and pac-2 sequences from a variant of HHV-6;
  • Fig. 6 shows het sequences from the Z29 B substrain of HHV-6; Fig. 6a shows the sequence from the Hindlll site and Fig. 6b shows the sequence from the pst site.
  • Fig. 7 shows the 4 Kb insert of pNF1022, which is a clone of HHV-6;
  • Fig. 8 shows the 2.1 Kb insert of ⁇ NF2009 (HA9), which is a clone of HHV-7;
  • Fig. 9 shows an electrophoretogram of labelled DNAs from cells infected with the parent HHV-7 Z29, cleaved by a number of restriction enzymes.
  • the DNA of HSV (herpes simplex virus) is shown for compari ⁇ son;
  • Fig. 10 shows an electrophoretogram of the DNA from the A and B substrains of HHV-6 Z29 strain, under different dilutions and following restriction by two restriction enzymes, after various passages in vitro
  • Fig. 11 shows an electrophoretogram of HHV-6 Z29 substrain A and B DNA isolated either from the nucleus (n) or from the cytoplasm (c); and
  • Fig. 12 is a schematic representation showing the size of the terminal fragments and the concatemeric junctions of the Z29 B substrain of HHV-6.
  • novel lymphotropic agents as defined above, are provided, useful for the treatment and/or prophylaxis of various diseases or disorders, particularly lymphatic diseases or disorders.
  • the present invention has two embodiments, a first embodi ⁇ ment involving the use of said CD4-ligand and a second embodiment invovling the use of said lymphotropic vector. Each of these two embodi ⁇ ments will be separately detailed below.
  • Said CD4-ligand is useful for protecting individuals against primary infection by viruses which are targeted to CD4 receptors, notably the HIV virus, the causative virus of AIDS.
  • said CD4- ligand may be administered to individuals which are at high risk of encountering such infections.
  • the CD4-ligand may also be administered to individuals already infected by said virus, in which case it may protect non- infected CD4 + cells from infection by the HTV.
  • HHV-7 has no known pathogenicity, namely, there are no known diseases associated with this virus. Accordingly, the intact HHV-7, or a weakened variant thereof may be used as said CD4-ligand. Addition ⁇ ally, also mutants of HIV-7 may be used as said CD4-ligand.
  • a particle of the virus, as defined in the definitions of said CD4-ligand under (c), may be obtained by various standard methods which are known in the art.
  • Various polypeptides, as defined under (d), are obtainable either by chemical methods or by methods of genetic engineering, namely, by cloning and expressing a gene coding for the polypeptide.
  • Such a polypeptide is typically a portion of the capsule which determines the binding affinity of the capsule to the CD4 receptor.
  • Polypeptides produced by means of genetic engineering are very often obtained as fusion proteins of the desired polypeptide with another protein or peptedic component. Such fusion proteins, defined under (e), may also be useful at times as said CD4-ligand.
  • CD4-ligand under (f) may be obtained by various standard chemical or biochemical methods, or by methods of genetic engineering, such methods being generally known per se.
  • Said agent may be useful for both in vitro or in vivo applica ⁇ tions.
  • In vitro applications may, for example, be in laboratory use, in labelling CD4 + cells, in which case said agent will be conjugated to a detectable marker (fluorescent marker, radioactive marker, enzyme marker, chemiluminescence marker, etc.).
  • a detectable marker fluorescent marker, radioactive marker, enzyme marker, chemiluminescence marker, etc.
  • In vivo applications include various therapeutic applications as already pointed out above.
  • a certain constant level of said agent should be retained in the blood.
  • the advantage of said agent as defined under (a) and some of said agents as defined under (b) is in that they have self- replicating properties and can thus replicate autonomously within the individual. Consequently, said agents (a) and (b) will retain a certain constant level in the blood. against this, said agent under (c)-(f) have to be administered continuously in order to retain a certain level thereof in the blood.
  • said lymphotropic vector is administered to cells for genetic modification of these cells.
  • the said lymphotropic vector may be administered in vivo, or may be applied to cells ex vivo.
  • An example of ex vivo use is in infection of lymphatic cells which are removed from the body.
  • Various therapeutic protocols used today call for the withdrawal of lymphatic cells, modification of the cells ex vivo and then return of the modified cells into the body where the modified length lymphatic cells exert a therapeutic effect.
  • withdrawn lymphatic cells are infected with said lymphatic vector wherby they become genetically modified upon which they may be returned to the body.
  • a patient is administered with said lymphatic vector, typically packaged in a matching viral particle.
  • said vector is an RDV
  • it is administered together with a self-replicating HHV-6 or HHV-7, as the case may be.
  • said foreign nucleic acid sequence depends on the desired therapeutic indication.
  • said foreign DNA sequence may, for example be one of the following:
  • An example is in the treatment of Gaucher disease in which there is a glucocerebrocidase deficiency.
  • Another example is supplementing deficiencies in insulin and insulin-like growth factors, missing in diabetic patients because of the lack of pancreas beta cells.
  • said foreign and nucleic acid sequence may be placed under control of a promoter, the activation of which is contingent on the glucose level.
  • controlling the level of expression of the foreign sequence is necessary in order to prevent situations of harmful over-production of the specific enzyme or protein.
  • control may be achieved by use of a lymphotropic vector comprising a second foreign sequence encoding the Thymidine Kinase (TK) enzyme.
  • HHV-6 and HHV-7 are insensitive to antiviral drugs such as
  • lymphatic vector for use in this indication may either contain a nucleic acid sequence which encodes a product which either destroys or inhibits proliferation of malignant cells or, alternatively, encodes an expression product which protects non- malignant cells from the toxic effect of chemotherapeutic agents or radiation.
  • Malignant cells are very actively replicating cells, and although the lymphotropic vector of the invention is not discriminatory, namely, it infects both malignant and non-malignant cells, in view of the activity of the malignant cells, said nucleic acid sequence will be expressed mainly in the cells.
  • Said foreign nucleic acid sequence may encode a toxin protein which will be expressed mainly in the malignant cells and consequently these cells will either be inhibited or die.
  • said foreign nucleic acid sequence may have an antisense sequence of an oncogene active in these cells or may encode a product which will interfere in the cancerous process.
  • said foreign DNA sequence will encode the product which will increase the cells' sensitivity to chemotherapy.
  • a specific example of the latter alternative is the use of a lymphotropic vector as described above wherein said foreign nucleic acid sequence encodes the enzyme Thymidine Kinase (TK).
  • TK Thymidine Kinase
  • the lymphotropic vector of the invention may also be used for infection of lymphocytes ex vivo.
  • Therapeutic techniques involving the withdrawal of lymphocytes, the treatment of withdrawn lymphocytes and the return of these to the body are well known, example being the reintroduction into the body of IL-2 treated T-infiltrating lymphocytes (TIL).
  • TIL T-infiltrating lymphocytes
  • TIL TIL-2 treated T-infiltrating lymphocytes
  • TIL TIL-2 treated T-infiltrating lymphocytes
  • the infected lymphocytes may first be subject to selection whereby only the infected lymphocytes will then be returned to the body.
  • TIL may be achieved, for example, by the use of a marker such as the neo gene which confers ability to grow in a selection medium containing the antibiotic G418. ex vivo infection of TIL may also be useful for various imaging applications.
  • the lymphotropic vector in accordance with such an embodiment, carries a gene encoding a detectable marker. The TIL, after return to the body, are attracted to tumor sites, and consequently the marker will be localized in such sites which may provide a way to identify a tumor and its metastisis.
  • the expression of said first nucleic acid sequence in the infected lymphocyte may be controlled by using a vector comprising a second foreign sequence encoding the TK enzyme, in a similar manner to that described above.
  • lymphotropic vector may be used in the treatment of autoimmune diseases or disorders.
  • autoim ⁇ mune diseases specific clones directed against self antigens are activated and start to divide.
  • the treatment of autoimmune diseases will be similar, in principle, to the treatment of malignancies, e.g. use of said lymphatic vector wherein said nucleic acid sequence encodes the enzyme TK.
  • An example of an autoimmune indication which can be treated by the use of the lymphotropic vector of the invention, is multiple sclerosis (MS).
  • MS multiple sclerosis
  • MBP myelin basis protein
  • TM such patients is intramuscular injection of COP-1 (Teva Pharmaceuticals, Israel) wherein the active ingredient consists of a random assembly of oligopeptides corresponding to MBP.
  • COP-1 Teva Pharmaceuticals, Israel
  • lymphocytes can be treated to express the COP-1 oligopeptides by ex vivo or in vivo infection.
  • the expressed COP-1 oligopeptides will be released to the bloodstream and will then act in a similar manner to
  • control of expression of the foreign sequence in the lymphotropic vector may be achieved by the use of an additional foreign sequence encoding the TK enzyme and treating the individual with Acyclovir, in a similar manner to that described above.
  • the het sequences remain constant in a single variant over many in vitro passages of the virus. Seeing that the het sequence has no involvement in the replication of the virus, it is a preferred target for incorporation of said foreign nucleic acid sequence.
  • the HHV-6 or HHV-7 consist of several genes, represented herein schematically as the unique sequence U, and at its right and left side two terminal repeats, TR ⁇ and TR ⁇ .
  • Each of the teriminal respeats comprises a pac-1 and pac-2 site.
  • the viral genome is expressed as a concatemer comprising a plurality of repeats of the basic sequence and is then cleaved at a site which is formed from the combined pac-1 sequence of LTR ⁇ and the pac-2 sequence of an adjacent T TM .
  • the site required for such a cleavage includes about 42 bases from TRT and 33 bases from TR ⁇ .
  • Fig. 2 shows an ARV in accordance with the invention.
  • a plasmid comprising a foreign nucleic acid sequence, referred to in the figure as "gene”, having an attached selection gene, "neo" in the specific example, is recombined with the HHV-6 genome by cotransfecting the HHV-6 and the plasmid into a host cell, e.g. a T cell.
  • Said gene with the attached selection gene is flanked by its two ends by a het sequence identical to the het sequence of the viral genome and consequently, this nucleic acid section becomes incorporated into the hat sequence of the viral genome.
  • a lymphotropic vector is formed containing said gene in its two het sequences.
  • Fig. 3 is a schematic representation of the manner of replication of an HHV-6 or HHV-7, discovered in accordance with the invention.
  • the viral genome (A) is replicated as a concatemer consisting of a plurality of basic genome units bound to one another. From the concatemer, said genomic units are formed by cleavage occurring between pac-1 and pac-2 in the manner shown in Fig. 1.
  • Fig. 4 is a schematic representation of the manner of use of a Tamplicon in combination with a helper virus.
  • the Tamplicon comprises an origin of replication in combina ⁇ tion with a pac site which comprises pac-1 and pac-2 sequences.
  • the Tamplicon When introduced into the cell a Tamplicon will not replicate in view of the fact that it lacks the transactivating factors encoded by the viral gene. Thus, in order to activate said gene, the Tamplicon is co-transfected with a helper virus. Following such co-transfection, the gene with its origin of replication becomes amplified in view of the formation of a concatemer and can then be expressed in large amounts. The presence of the pac sites ensures the cleavage into individual units, which can then either combine into the hosts' cells' genome or be packed in a viral particle encoded by the helper virus.
  • Tamplicon embodiment is at times advanta ⁇ geous seeing that it brings to the formation of many copies of said gene.
  • a preferred virus for use as a helper virus is the HHV-7, as already pointed out above, in view of its non-pathogenic nature.
  • HHV-6 When the HHV-6 is used as the helper virus, means should be provided for its neutralization or destruction at some stage.
  • HHV-7 is purified from infected cells, homogenized and centrifiiged through dextran gradient. The resultant purified HHV-7 is cross-linked to magnetic beads and the beads carrying the HHV-7 are then incubated with each of the following cells:
  • cells from a B cell line e.g. Raji cells
  • cells of the human cell line HeLa e.g.
  • HHV-6 virions bound to magnetic beads are incubated with the above cells.
  • CD4 cells which are the HeLa cells transfected with the CD4 gene, remain bound to the magnetic beads by virtue of their binding to the HHV-7.
  • the binding of these cells to the HHV-7, can be inhibited by the use of anti-CD4 antibodies.
  • HHV-7 virions are radioactively labelled by infecting cells with HHV-7 and labelling them with S35 methionine. HHV-7 is extracted from the labelled cells, homogenized and centrifuged through dextran gradients. Magnetic beads are cross-linked with a CD4 protein or with soluble CD4 (sCD4) and the CD4-bound beads are incubated with the purified labelled HHV-7 virions.
  • a non-ionic detergent NP40 is added to the beads-virion mixture to solubilize the envelope glycoproteins of the viruses.
  • the beads are then washed and all virion proteins excluding the attached glycoprotein interacting with the CD4 receptor on the beads are washed away.
  • This glycoprotein is useful as a CD4-ligand of the invention.
  • CD4 cells are purified from cord blood by methods known per se (Frenkel et al., 1990).
  • the CD4 cells are activated for a day with phytohemaglutinin (PHA) and interluken-2 (E 2).
  • PHA phytohemaglutinin
  • E 2 interluken-2
  • the CD4 cells are incubated for 20 minutes with anti-CD4 antibodies (e.g. anti- Leu3A) or with anti-CD8 antibodies (e.g. anti-Leu2A).
  • Antibody concentra ⁇ tions have to be in the range of 0.01 ⁇ g/ml to 5.0 ⁇ g/ml.
  • the CD4 + cells are then incubated with HHV-7 or HHV-6 in the presence of the antibodies. Viral infection of the cells is monitored by methods known in the art, e.g. cytopathic effect (CPE) immunofluorescence assay (IF A), polymerase chain (PCR) or hybridization with various probes (such as pNF 2001
  • An autonomously replicating vector was constructed as follows:
  • HHV-6 U1102 was cotransfected into Jurkat T cells together with a bacterial plasmid construct comprising the inserted gene and the neo selection gene both situated between two flanking heterogenous (het) sequences of HHV-6.
  • the cotransfection was carried out by methods known in the art, e.g. electroporation. Inside the Jurkat cells recombination occurred at a high frequency between the het sequences of the bacterial plasmids and the HHV-6 resulting in the insertion of the gene and neo gene into the HHV-6 virus within its het region.
  • the Jurkat T cells were further grown in a selection medium containing G418 enabling only cells compris ⁇ ing the recombinant HHV-6 virus containing the neo gene to grow.
  • the recombinant viruses were then isolated from the cells, purified by terminal dilutions, tittered and analyzed by southern blot hybridization using methods known per se and suitable probes.
  • the resulting recombinant viruses (autonomously replicating vectors - ARV) comprised the gene of interest and the neo gene in their left and right heterogenous sequences.
  • other T cell lines may be used such as Jjhan or Molt 3 cells.
  • the selection gene may be any one of several known selection genes (e.g. hygramycin or tynidine kinase (TK)).
  • the cotransfection of the bacterial plasmid and the viral DNA may be carried out any one of several known methods, e.g. electroporation, the CaC ⁇ method or DEAE dextran method, depending on the type of transfected cells used.
  • a replication deficient vector (Tamplicon) was constructed by methods known in the art comprising a bacterial replication origin (ORI in
  • a drug selection gene the cis acting sites of replication including the HHV-6 or HHV-7 replication origin, their cleavage packaging sequences (pac), additional genes with expression capabilities (e.g. a promoter and polyA signal) and a foreign nucleic acid sequence.
  • the constructed Tamplicon was cotransfected into a T cell line (Jurkat, Molt 3 Jjahn or cord blood cells) together with either an HHV-6 or HHV-7 helper virus DNA. Alternatively, the constructed Tamplicon was first transfected into the T cells and only later the same cells were infected with HHV-6 or HHV-7 as the case may be.
  • transfection procedure as well as the later infection of the transfected cells was carried out according to any one of several known methods.
  • transactivating factors encoded by the helper virus enable the replication of the Tamplicon from its origin resulting in a concatemer of the foreign nucleic acid sequence.
  • the purification of the replicated Tamplicon from the cells was carried out by methods known per se.
  • the pac-2 sequence of the HHV-6 pNF1022 DNA was analyzed and the sequence is shown in Fig. 5B. As seen, the sequence is the junction between the TR and the unique sequence.
  • the pac-1 sequence of HHV-6 pNF1022 was analyzed and the sequence is shown in Fig. 5C
  • the pac-1 signal away from the terminus was derived by fill in and flush ligation.
  • the heterogenous sequence of one clone derived from the Z29 B substrain was subjected to several restriction enzymes and their sequence was analyzed by and is shown in Fig. 6.
  • the HHV-6 pNF1022 DNA probe was subjected to restriction by the Sail restriction enzyme and the resulting 4 kb insert, spanning the junction of the left TR and the unique sequence, was sequenced.
  • the full 20 nucleic acid sequence of this fragment is shown in Fig. 7.
  • the first and second origins of replication (ORF1 and ORF2) as well as the telomeric sequence (TEL) comprising 51 repeats of TAACCC and the pac-2 sequence which immediately follows the telomeric sequence and is further followed by the unique sequence are indicated in the figure.
  • the pNF2009 (HA9) of HHV-7 DNA is a 2.1 kb insert resulting from restriction of the virus by the restriction enzyme Hindlll and containing the two origins of replication.
  • the nucleotide sequence of H49 is shown in Fig. 8.
  • Example 13 The Z29 strain of human herpesvirus-6 (HHV-6) (Lopez et al..).
  • HHV-6 Z29 was propagated in peripheral blood mononuclear cells (PBL) and in cord blood mononuclear cells (CBMC) (DiLuca et al.. 1990 and Frankel et al. 1990, respectively).
  • PBL peripheral blood mononuclear cells
  • CBMC cord blood mononuclear cells
  • the cell extract containing the Z29 virus was purified by filtration and by terminal dilution resulting in pure cultures containing the A or B substrains of the virus.
  • Each viral isolate was propagated for a number of times in two separate CBMC.
  • the het sequence in each of the isolates propagated in each CBMC were compared and identical het sequences served as proof of the virus isolate originating from a certain patient.
  • the DNA replication of the infected cells was shut off after infection with the virus (DiLuca et al.. 1990) and 32P-orthophosphate was added, enabling a significant fraction of 32P-orthophosphate to be preferen- tially incorporated into viral DNA.
  • the 32P-labelled, virus infected, cell DNA was subjected to restriction by several restriction enzymes. Represen ⁇ tative patterns of the resulting fragments are shown in Fig. 9.
  • the parental (P in the figure) stock contains a mixture of two variants, A and B substrains which differ from one another by their restriction pattern.
  • Example 14 The A and B substrains of the Z29 HHV-6 virus were propagated for 17 passages under different dilution modes (1:4, 1:10, 1:100, 1 :1000). The various viruses were restricted by several endonuclease restriction enzymes. As seen in Fig. 10, the het BamHl fragment in the A substrain is 10 kb long and the het BamHl fragment in the B substrain is 7.5 kb long. The het size remained stable from passage 2 through passage 17 in each one of the substrains.
  • DNA was isolated from either the nucleus ("n” in Fig. 11) or the cytoplasm ("c" in Fig. 11) of cells infected with either Z29 A substrain or Z29 B substrain.
  • the isolated DNA was cleaved with EcoRI, southern blotted and hybridized with a HHV-6 pNF1022 DNA probe (Frenkel et al.. 1990, Schirmer et al.. 1991).
  • the pNF1022 probe is a 4 kb clone which contains the Sail L fragment of U1102 spanning the junction between the left terminal repeat (TR j ) and the unique sequence two origin of replication and the telomeric sequence (see Fig. 9). This probe detects the concatemeric junctions and the terminal sequence of linear DNA.
  • the nuclear DNA of cells infected both with the A substrain and the B substrain contained three fragments which hybridize with the HHV-6 U1102 probe.
  • the three fragments represent linear packaged DNA having the length of a single unit, concatemeric nuclear DNA of which junctions comprise only a single terminal repeat (TR) and concatemeric nuclear DNA comprising two adjacent terminal repeat sequences.
  • TR terminal repeat
  • the concatemeric DNA comprising only a single TR most likely arose after recombination of the adjacent TRs (see Fig. 3).
  • the concatemeric nuclear DNA comprising two adjacent TRs most likely arose as a result of rolling circle replication and is the species where cleavage is predicted to occur.
  • the cytoplasmic DNA of cells infected with the A or B substrain contains two fragments which hybridize with the HHV-6 Ul 102 probe. These two fragments represent concatemeric nuclear DNA containing a single TR sequence and DNA containing two adjacent TRs.
  • the species comprising two adjacent TRs (in which the EcoRI fragment is 20 kb long) is a minor component of the concatemeric DNA both in the nucleus and in the cytoplasm.
  • FIG. 12 A schematic representation of the concatemeric junctions of the HHV-6 Z29 B substrain is shown in Fig. 12.

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Abstract

L'herpès humain (HVH) 7 peut se lier à l'antigène CD4 et ce virus, ou une protéine de liaison qui en est dérivée, se révèle donc utile comme ligand du CD4 pour différentes applications thérapeutiques. Le HVH 6 et le HVH 7 sont lymphotropes et donc utiles comme vecteurs lymphotropes permettant un apport d'ADN dans les lymphocytes.
PCT/US1994/012715 1993-11-10 1994-11-09 Agents et vecteurs lymphotropes WO1995013081A1 (fr)

Priority Applications (2)

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AU10885/95A AU1088595A (en) 1993-11-10 1994-11-09 Lymphotropic agents and vectors
US08/646,242 US6503752B1 (en) 1993-11-10 1994-11-09 Lymphotropic agents and vectors

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IL10755793A IL107557A (en) 1993-11-10 1993-11-10 Lymphotropic agents and vectors

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008047656A1 (fr) 2006-10-12 2008-04-24 Kaneka Corporation Procédé de fabrication d'un acide l-amino

Citations (3)

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Publication number Priority date Publication date Assignee Title
EP0507179A2 (fr) * 1991-04-05 1992-10-07 Bayer Ag Virus de l'herpès équin (EHV) avec de l'ADN étranger, procédé pour leur préparation et leur utilisation comme vaccins
US5230997A (en) * 1990-07-19 1993-07-27 The United States Of America As Represented By The Department Of Health And Human Services Methods of detecting the presence of human herpesvirus-7 infection
US5242820A (en) * 1986-06-18 1993-09-07 American Registry Of Pathology Pathogenic mycoplasma

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US5242820A (en) * 1986-06-18 1993-09-07 American Registry Of Pathology Pathogenic mycoplasma
US5230997A (en) * 1990-07-19 1993-07-27 The United States Of America As Represented By The Department Of Health And Human Services Methods of detecting the presence of human herpesvirus-7 infection
EP0507179A2 (fr) * 1991-04-05 1992-10-07 Bayer Ag Virus de l'herpès équin (EHV) avec de l'ADN étranger, procédé pour leur préparation et leur utilisation comme vaccins

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JOURNAL OF VIROLOGICAL METHODS, Volume 19, issued 1988, FUNG et al., "Detection and Purification of a Recombinant Human B Lymphotropic Virus (HHV-6) in the Baculovirus Expression System by Limiting Dilution and DNA Dot-blot Hybridization", pages 33-42. *
MEDICAL HYPOTHESES, Volume 39, issued 1992, GLINSKY, "The Blood Group Antigen-related Glycoproteins: Key Structural Determinants in Immunogenesis and AIDS Pathenogenesis", pages 212-224. *
THE JOURNAL OF IMMUNOLOGY, Volume 146, Number 9, issued 01 May 1991, CRUIKSHANK et al., "Lymphocyte Chemoattractant Factor Induces CD4-dependent Intracytoplasmic Signaling in Lymphocytes", pages 2928-2934. *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008047656A1 (fr) 2006-10-12 2008-04-24 Kaneka Corporation Procédé de fabrication d'un acide l-amino

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