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WO1996037626A1 - Integration de vecteurs de recombinaison dans une position specifique du genome d'un eucaryote, assuree par une proteine chimere du type integrase - Google Patents

Integration de vecteurs de recombinaison dans une position specifique du genome d'un eucaryote, assuree par une proteine chimere du type integrase Download PDF

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WO1996037626A1
WO1996037626A1 PCT/US1996/006727 US9606727W WO9637626A1 WO 1996037626 A1 WO1996037626 A1 WO 1996037626A1 US 9606727 W US9606727 W US 9606727W WO 9637626 A1 WO9637626 A1 WO 9637626A1
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gene
vector construct
chimeric
eukaryotic
cells
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PCT/US1996/006727
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James G. Respess
Suzanne B. Sandmeyer
Douglas J. Jolly
Virginia W. Bilanchone
Sandra L. Dildine
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Chiron Corporation
The Regents Of The University Of California
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Priority to EP96915714A priority Critical patent/EP0830455A1/fr
Priority to JP8535720A priority patent/JPH11505128A/ja
Priority to AU57419/96A priority patent/AU5741996A/en
Publication of WO1996037626A1 publication Critical patent/WO1996037626A1/fr

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/13043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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    • C12N2830/00—Vector systems having a special element relevant for transcription
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/60—Vector systems having a special element relevant for transcription from viruses
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    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/44—Vectors comprising a special translation-regulating system being a specific part of the splice mechanism, e.g. donor, acceptor

Definitions

  • the nucleoprotein complex enters the nucleus, where the viral DNA is then integrated pseudo-randomly by staggered cleavage of the host cell genome and joining of the viral DNA to the host cell DNA by strand transfer, creating a "provirus 1 form of the retroviral genome which is inherited by daughter cells.
  • the processing and joining reactions are mediated by IN and the position of pseudo-random integration appears to be determined mainly by host DNA accessibility, not by nucleotide sequence (Kulkosky, et al. (1994), Pharmac. Then, vol. 61 : 185-203).
  • the resultant chimeric integrase may optionally be isolated or purified, for instance by affinity chromatography using an antibody reactive against an epitope of the chimeric integrase.
  • a chimeric integrase protein so produced is incorporated into a gene delivery vehicle assembled by an in vitro process.
  • host cells are packaging cells for the production of recombinant viral particles.
  • packaging cells also produce the chimeric integrase protein which is incorporated into the viral particles.
  • eukaryotic cell genomes (and the corresponding transduced eukaryotic cells) comprising a vector construct integrated into a defined region are provided.
  • the defined region into which the vector construct is integrated is a region adjacent to a eukaryotic gene transcribed by RNA polymerase DI.
  • methods for introducing a vector construct into a defined region of a eukaryotic cell genome such that there is decreased variation in expression of a gene of interest from the vector construct in eukaryotic cells into which the vector construct is introduced as compared to expression of the gene of interest in eukaryotic cells wherein the vector construct is introduced using a wild type integrase protein, the method comprising introducing the vector construct into the eukaryotic cell genome using a chimeric integrase protein of the invention.
  • Other aspects of the invention relate to methods of treating a disease selected form the group consisting of a genetic disease, a cancer, an infectious disease, a degenerative disease, an inflammatory disease, a cardiovascular disease, and an autoimmune disease.
  • Figure 1 is a graphic representation of the Ty-3 and MoMLV genomes.
  • Figure 2 is a graphic representation of the "A”, "B”, and “C” domains of the Ty3 and MoMLV integrase proteins. Amino acid numbers are presented at the boundaries of each domain.
  • Figure 3 is an amino acid alignment between the primary sequences of Ty3 integrase (above) and MoMLV integrase (below) . Boundaries for the "A”, “B”, and “C” domains are represented by arrows. The conserved H-H-C-C and D-D-E motifs in each protein are shown.
  • Figure 4 shows the plasmids BAG-EX, pRgpKan, and pgpChimNeo, the latter generically referencing the expression vector used to make the chimeric integrase proteins described in Example 1.
  • Figure 7 provides the nucleotide sequences of the oligonucleotides used to generate five of the seven chimeric integrase proteins described in Example 1. Above each oligonucleotide sequence are the complementary sequences of the MLV and Ty3 integrase genes. "//" indicates where the loop-out occurred in each mutagenesis reaction.
  • Figure 8 is a illustrates the pseudotyping procedure used to produce infectious retroviral particles containing the chimeric integrase proteins in NC10 cells.
  • Figure 9 illustrates how the integration library described in Example 1 is made.
  • a multivalent vector construct i.e., a vector construct coding for more than one gene of interest
  • a promoter to direct the expression of each heterologous sequence.
  • expression of the gene(s) downstream of the first is mediated by an internal ribosome entry site ("IRES") sequence.
  • the recombinant retroviral vector may also include a signal which directs polyadenylation, selectable markers such as resistance to neomycin, hygromycin, phleomycin, histidinol, or proteins such as DHFR (confers methotrexate resistance) and HS VTK (confers gancilovir sensitivity), as well as one or more restriction sites and a translation termination sequence.
  • selectable markers such as resistance to neomycin, hygromycin, phleomycin, histidinol, or proteins such as DHFR (confers methotrexate resistance) and HS VTK (confers gancilovir sensitivity)
  • DHFR confers methotrexate resistance
  • HS VTK confers gancilovir sensitivity
  • the retroviral vector must include at least one transcriptional promoter/enhancer or locus defining element(s), or other elements which control gene expression by other means such as alternate splicing, nuclear RNA export, post-translational modification of messenger, or post-transcriptional modification of protein.
  • Such vector constructs must also include sequence encoding a packaging signal, long terminal repeats (LTRs) or portion thereof, and positive and negative strand primer binding sites appropriate to the retrovirus used (if these are not already present in the retroviral vector).
  • a “gene delivery vehicle” refers to a composition capable of delivering a vector construct to an eukaryotic cell which further comprises a chimeric IN protein according to the invention to promote position-specific integration of the vector construct into the genome of the eukaryotic cell.
  • Representative examples of gene delivery vehicles include recombinant viral vectors (e.g. alphaviruses such as Sindbis), physical systems (e.g. ELVS), other viral systems (e.g.
  • the desirable properties include the ability to express a desired substance, such as a protein, enzyme, or antibody, and/or the ability to provide a biological activity, which is where the nucleic acid molecule carried by the GDV is itself the active agent without requiring the expression of a desired substance.
  • a desired substance such as a protein, enzyme, or antibody
  • a biological activity is where the nucleic acid molecule carried by the GDV is itself the active agent without requiring the expression of a desired substance.
  • a desired substance such as a protein, enzyme, or antibody
  • a biological activity is where the delivered nucleic acid molecule incorporates into a specified gene so as to inactivate the gene and "turn ofi" the product the gene was making.
  • the nucleic acid sequence is a antisense molecule that binds to mRNA and inhibits translation. If the nucleic acid sequence encodes a rybozyme the ribozyme binds and cleaves mRNA thereby inhibiting translation.
  • a "defined region of a target eukaryotic genome” is a region into which a vector construct of the invention is integrated due to the position-specificity of the integrase mediating the integration event.
  • a representative example is a region adjacent to a eukaryotic gene transcribed by RNA polymerase m.
  • the enzyme responsible for catalyzing the integration event is the retroviral IN which mediates removal of a 3' dinucleotide from the ends of the extrachromosomal retroviral vector DNA, cleavage of the target site generating a characteristic (4 to 6) bp 5' overhang, and ligation of the 3' ends of the retroviral vector DNA to the 5' ends of the host chromosomal DNA (Fujiwara, et al. (1988), Cell, vol. 54: 497-504; Brown, et al. (1989), Proc. Natl. Acad. Sci. USA, vol. 86: 2525-2529).
  • the MoMLV intracellular viral core particle containing replicated DNA was shown to be competent for integration (Brown, et al. (1987), Cell, vol. 49:347-356; Fujiwara, et al. (1989), Proc. Natl. Acad. Sci. USA, vol. 86:3065-3069). More recently, purified, recombinant IN together with linear molecules representing replicated viral DNA or oligonucleotide duplexes representing the ends of the replicated virus in the presence of buffer and divalent cations was shown to be sufficient to catalyze nicking and ligation, mimicking an integration reaction (Katz, et al. (1990), Cell, vol. 63:87-95; Craigie, et al.
  • IN can perform a reversal of the integration reaction on an oligonucleotide substrate DNA (Chow, et al. (1992), Science, vol. 255:723-726).
  • the internal domain contains two ORFs, GAG3 and POLS, corresponding to the retroviral gag and pol genes.
  • the GAG3 gene encodes the precursor polyprotein Pr38" ⁇ G j , w i c is processed into a 26 kDa capsid (CA) species and a 9 kDa nucleocapsid (NC) species. These species have conserved motifs found in their retroviral counterparts and are functionally equivalent to those proteins.
  • the GAG3-POL3 173 fusion polypeptide is processed to the GAG3 proteins described above, a 16 kDa aspartyl protease (PR), a pi 15 POL3 species composed of reverse transcriptase (RT) which includes IN domains, a 55 kDa RT, and 61 and 58 kDa IN species.
  • These proteins together with Ty3 RNA form virus-like particles (VLPs) about 50 nm in diameter and 156S in size.
  • VLPs virus-like particles
  • the particle fraction displays RT activity and contains the full-length, replicated Ty3 DNA.
  • the primer for Ty3 replication is initiator tRNA Met which is complementary to the Ty3 primer binding site which begins 2 bp downstream of the U5 internal domain junction.
  • this DNA ends in the short, conserved, inverted repeats and has two bp terminal extensions compared to the integrated form.
  • it is structurally analogous to the retrovirus replicated extrachromosomal intermediate.
  • the position specificity of the yeast Ty3 element is conferred to the integrase (IN) of a Moloney murine leukemia virus-based retroviral vector.
  • Integrase proteins are known to comprise at lease three domains, an amino terminal domain, a core domain ("B" in FIG. 2) with strand transfer and metal chelation activity, while the carboxyl terminal domain ("C" in FIG. 2) is involved in DNA binding.
  • B amino terminal domain
  • C carboxyl terminal domain
  • One or more of these discrete domains of an integrase known to confer position- specific integration, e.g., the Ty3 IN can be substituted for the corresponding domain(s) of the integrase to be incorporated into a gene delivery vehicle according to the invention.
  • Vector constructs of the invention which code for a chimeric integrase carry substitutions of the coding region for the amino terminal region, the core region, and the carboxyl terminal region of Ty3 IN for the analogous coding regions for the nonspecific integrase activity of MoMLV.
  • Example 1 Seven representative chimeric constructs are described in Example 1, below. Because Ty3 inserts within a few nucleotides of the transcription initiation site and RNA pol m gene sequences are highly conserved and which are not regulated in a tissue-specific manner, predictable and less variable expression levels can be achieved. Northern analysis and PCR are among the techniques that can be employed to assess levels of expression of the gene(s) of interest.
  • Ty3 integrates with position specificity which is not observed for any retrovirus.
  • Ty3 integrates specifically in the region of transcription initiation of genes transcribed by RNA polymerase in, e.g., 5S, U6, and tRNA genes.
  • the tRNA class of polymerase HI transcribed genes is distinguished by the boxA and boxB internal promoter elements (Chalker, et al. (1990), Genetics, vol. 126:837-850). These regions of the tRNA gene- coding sequence direct binding of transcription factor TFi ⁇ C which subsequently directs binding of 1F1-UB to the 5' flanking region of the tRNA gene.
  • T ll-lB bound to the DNA template is sufficient to direct RNA pol ID to initiate multiple rounds of transcription (Chalker, et al. (1992), Genes Dev., vol 6: 117-128). No consensus DNA binding sequence for TFillfcJ has been observed and the region upstream of the gene does not contain conserved promoter elements.
  • Ty3 integration events occurred so that the gene-proximal member of the staggered nicks at the integration site were within one or two bp of the position of transcription initiation and immediately downstream of the position of TFIHB.
  • Ty3 integration into a target tRNA gene requires functional tRNA gene promoter elements (Natsoulis, et al. (1989), Genetics, vol.
  • RNA pol IQ promoter elements are internal, integration of recombinant DNA molecules according to the invention by a chimeric integrase having the position-specificity of the Ty3 IN just upstream of the pol ID initiation site will not disrupt tRNA gene expression.
  • the Ty3 VLP contain, in addition to other components, IN and full-length Ty3 DNA, and c-an donate the Ty3 DNA to a plasmid target.
  • VLPs are isolated from cells overexpressing Ty3 and mixed with plasmid to which RNA pol DI transcription extract or purified factors (IDC and mB) and polymerase HI is added.
  • the plasmid contains a modified SUP2 tRNA gene with either a wild-type or mutant (G56) boxB promoter element.
  • the components of the integration reaction are mixed together on ice in buffer with 5-20 mM MgCl 2 and then incubated at 30°C for 30 min.
  • the DNA is then extracted and quantitated by a fluorometric assay.
  • Integration of Ty3 DNA into the target plasmid containing the tRNA gene can be monitored by a PCR assay. Integration of the Ty3 DNA into the initiation region of the tRNA gene target results in a product which can be amplified into a diagnostic fragment.
  • one embodiment of the present invention provides recombinant retroviruses which are constructed to deliver one or more selected nucleic acid molecules, or "genes,” of interest in a position-specific manner to the genome of a eukaryotic cell. Position-specific integration is mediated by the chimeric IN protein incorporated into the recombinant retroviral particle.
  • numerous retroviral gene delivery vehicles may be utilized within the context of the present invention, including for example those described in EP 0,415,731; WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; U.S. Patent No.
  • Retroviral gene delivery vehicles of the present invention may be readily constructed from a wide variety of retroviruses, including for example, B, C, and D type retroviruses as well as spumaviruses and lentiviruses (see RNA Tumor Viruses, Second Edition, Cold Spring Harbor Laboratory, 1985).
  • Preferred retroviruses for the preparation or construction of retroviral gene delivery vehicles of the present invention include retroviruses selected from the group consisting of Avian Leukosis Virus, Bovine Leukemia Virus, Murine Leukemia Virus, Mink-Cell Focus-Inducing Virus, Murine Sarcoma Virus, Reticuloendotheliosis virus and Rous Sarcoma Virus.
  • Murine Leukemia Viruses include 4070A and 1504A (Hartley and Rowe, J. Virol 19: 19-25, 1976), Abelson (ATCC No. VR-999), Friend (ATCC No. VR-245), Graffi, Gross (ATCC No. VR-590), Kirsten, Harvey Sarcoma Virus and Rauscher (ATCC No. VR-998), and Moloney Murine Leukemia Virus (ATCC No. VR-190).
  • retroviruses may be readily obtained from depositories or collections such as the American Type Culture Collection ("ATCC”; Rockville, Maryland), or isolated from known sources using commonly available techniques.
  • retroviral gene delivery vehicles Any of the above retroviruses may be readily utilized in order to assemble or construct retroviral gene delivery vehicles given the disclosure provided herein, and standard recombinant techniques (e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, 1989; Kunkle, PNAS 52:488, 1985).
  • portions of the retroviral gene delivery vehicles may be derived from different retroviruses.
  • retroviral vector LTRs may be derived from a Murine Sarcoma Virus, a tRNA binding site from a Rous Sarcoma Virus, a packaging signal from a Murine Leukemia Virus, and an origin of second strand synthesis from an Avian Leukosis Virus.
  • recombinant retroviruses useful in the practice the invention may be made by introducing a vector construct as discussed above, into a cell (termed a "packaging cell") which contains those elements necessary for production of infectious recombinant retrovirus which mediate position-specific integration of the recombinant retroviral genome, but which are lacking in the vector construct from which the recombinant retroviral genome is transcribed.
  • a wide variety of retroviral vector constructs may be utilized within the present invention in order to prepare recombinant retroviruses.
  • retroviral vector constructs comprising a 5' LTR, a tRNA binding site, a packaging signal, one or more heterologous sequences, an origin of second strand DNA synthesis and a 3' LTR, wherein the vector construct lacks gag/pol or env coding sequences.
  • LTRs Long Terminal Repeats
  • U5, R and U3 These elements contain a variety of signals which are responsible for the biological activity of a retrovirus, including for example, promoter and enhancer elements which are located within U3. LTRs may be readily identified in the provirus due to their precise duplication at either end of the genome.
  • a 5' LTR should be understood to include a 5' promoter element and sufficient LTR sequence to allow reverse transcription and integration of the DNA form of the vector.
  • the 3' LTR should be understood to include a polyadenylation signal, and sufficient LTR sequence to allow reverse transcription and integration of the DNA form of the vector.
  • the tRNA binding site and origin of second strand DNA synthesis are also important for a retrovirus to be biologically active, and may be readily identified by one of skill in the art.
  • retroviral tRNA binds to a tRNA binding site by Watson- Crick base pairing, and is carried with the retrovirus genome into a viral particle.
  • the tRNA is then utilized as a primer for DNA synthesis by reverse transcriptase.
  • the tRNA binding site may be readily identified based upon its location just downstream from the 5' LTR.
  • the origin of second strand DNA synthesis is, as its name implies, important for the second strand DNA synthesis of a retrovirus.
  • This region which is also referred to as the poly-purine tract, is located just upstream of the 3' LTR.
  • the retroviral vector construct include sequences which, when positioned in the reverse transcribed double stranded form of the retroviral genome to be integrated, are processed by intact chimeric IN protein. Following reverse transcription of the retroviral RNA genome, LTRs are present at each end of the linear, double stranded DNA molecule. Several nucleotides, typically two, are removed from the 3'-OH ends of the retroviral DNA by the chimeric IN.
  • the substrate DNA has a cytosine, adenosine (5'-CA-3') dinucleotide two base pairs from the 3' terminus of each of the two complementary DNA strands.
  • cytosine, adenosine (5'-CA-3') dinucleotide two base pairs from the 3' terminus of each of the two complementary DNA strands.
  • resultant retroviral DNAs which contain a substitution of either or both nucleotides or where the dinucleotide is more recessed from one or both ends of the DNA can also be employed, although processing and joining (to the cleaved eukaryotic genome) of the retroviral DNA will likely be less efficient.
  • retroviral vector constructs which are provided herein also comprise a packaging signal, as well as one or more nucleic acid molecules (e.g., heterologous sequences), each of which is discussed in more detail below.
  • retroviral vector constructs are provided which lack both gag/pol and env coding sequences.
  • the phrase "lacks gag/pol or env coding sequences" should be understood to mean that the retroviral vector does not contain at least 20, preferably at least 15, more preferably at least 10, and most preferably at least 8 consecutive nucleotides which are found in gag/pol or env genes, and in particular, within gag/pol or env expression cassettes that are used to construct packaging cell lines for the retroviral vector construct.
  • Packaging cell lines suitable for use with the above-described retroviral vector constructs may be readily prepared (see U.S. Serial No. 08/240,030, filed May 9, 1994; see also WO 92/05266), and utilized to create producer cell lines (also termed vector cell lines or "VCLs") for the production of recombinant vector particles which mediate position-specific integration.
  • producer cell lines also termed vector cell lines or "VCLs”
  • packaging cell lines are derived from cell lines obtained from the same species as will be treated with the resulting recombinant retroviral particles.
  • recombinant retroviral particles made from human (e.g., HT 1080 cells) cell lines are capable of surviving inactivation in human serum. See U.S.S.N. 08/367,071, filed December 30, 1994.
  • packaging cell lines that produce recombinant retroviral particles at titers greater than 10 6 or 10? cfu/ml (in crude supernatant) may readily be obtained.
  • titers are generally obtained from titer assays on HT1080 cells, which produce a three-fold lower titer than titers obtained on murine 3T3 cells.
  • nucleic acid molecules may be carried and/or expressed by the recombinant vector constructs of the present invention.
  • the nucleic acid molecules which are described herein do not occur naturally in the gene delivery vehicle that carries it, and provides some desirable benefit, typically an ability to fight or prevent a disease, or other pathogenic agent or condition.
  • pathogenic agent refers to a cell that is responsible for a disease state.
  • pathogenic agents include tumor cells, autoreactive immune cells, hormone secreting cells, cells which lack a function that they would normally have, cells that have inappropriate gene expression which does not normally occur in that cell type, and cells infected with bacteria, viruses, or other intracellular parasites.
  • a nucleic acid molecule is considered to be biologically active when the molecule itself provides the desired benefit without requiring the expression of a substance.
  • the biologically active nucleic acid molecule may be an inactivating sequence that incorporates into a specified intracellular nucleic acid molecule and inactivates that molecule, or the molecule may be a tRNA, rRNA or mRNA that has a configuration that provides a binding capability.
  • Substances which may be encoded by the nucleic acid molecules described herein include proteins (e.g., antibodies, including single chain molecules), immunostimulatory molecules (such as antigens, immunosuppressive molecules, blocking agents, palliatives (such as toxins, antisense ribonucleic acids, ribozymes, enzymes, and other material capable of inhibiting a function of a pathogenic agent) cytokines, various polypeptides or peptide hormones, their agonists or antagonists, where these hormones can be derived from tissues such as the pituitary, hypothalamus, kidney, endothelial cells, liver, pancreas, bone, hematopoietic marrow, and adrenal.
  • proteins e.g., antibodies, including single chain molecules
  • immunostimulatory molecules such as antigens, immunosuppressive molecules, blocking agents, palliatives (such as toxins, antisense ribonucleic acids, ribozymes, enzymes, and other material capable
  • Such polypeptides can be used for induction of growth, regression of tissue, suppression of immune responses, apoptosis, gene expression, blocking receptor-ligand interaction, immune responses and can be treatment for certain anemias, diabetes, infections, high blood pressure, abnormal blood chemistry or chemistries (e.g., elevated blood cholesterol, deficiency of blood clotting factors, elevated LDL with lowered HDL), levels of Alzheimer associated amaloid protein, bone erosion/calcium deposition, and controlling levels of various metabolites such as steroid hormones, purines, and pyrimidines.
  • the palliative when "capable of inhibiting a function" is utilized within the context of the present invention, it should be understood that the palliative either directly inhibits the function or indirectly does so, for example, by converting an agent present in the cells from one which would not normally inhibit a function of the pathogenic agent to one which does.
  • functions for viral diseases include adsorption, replication, gene expression, assembly, and exit of the virus from infected cells.
  • functions for cancerous diseases include cell replication, susceptibility to external signals (e.g., contact inhibition), and lack of production of anti-oncogene proteins.
  • cardiovascular disease examples include inappropriate growth or accumulation of material in blood vessels, high blood pressure, undesirable blood levels of factors such as cholesterol or low density lipoprotein that predispose to disease, localized hypoxia, and inappropriately high and tissue-damaging levels of free radicals.
  • functions for neurological conditions include pain, lack of dopamine production, inability to replace damaged cells, deficiencies in motor control of physical activity, inappropriately low levels of various peptide hormones derived from neurological tissue such as the pituitary or hypothalamus, accumulation of Alzheimer's Disease associated amyeloid plaque protein, and inability to regenerate damaged nerve junctions.
  • autoimmune or inflammatory diseases include inappropriate production of cytokines and lymphokines, inappropriate production and existence of autoimmune antibodies and cellular immune responses, inappropriate disruption of tissues by proteases and collagenases, lack of production of factors normally supplied by destroyed cells, and excessive or aberrant regrowth of tissues under autoimmune attack.
  • methods are provided for administration of a gene delivery vehicle, the nucleic acid of which encodes and directs the expression of at least one palliative.
  • the palliative may be a DNA molecule, a RNA molecule, some combination of the two, or a protein.
  • palliatives that act directly to inhibit the growth of cells include toxins such as ricin (Lamb et al., Eur. J. Biochem. 148:265-270, 1985), abrin (Wood et al., Eur. J. Biochem. 705:723-732, 1991; Evensen et al., J. of Biol. Chem. 2-5.5:6848-6852, 1991; Collins et al., ./. of Biol Chem. 255:8665-8669, 1990; Chen et al., Fed of Eur. Biochem Soc. 309: 115- 118, 1992), diphtheria toxin (Tweten et al., J. Biol.
  • the vector construct carried by the gene delivery vehicle directs the expression of a substance capable of activating an otherwise inactive precursor into an active inhibitor of a pathogenic agent, or a conditional toxic palliative, which are palliatives that are toxic for the cell expressing the pathogenic condition.
  • a wide variety of inactive precursors may be converted into active inhibitors of a pathogenic agent.
  • antiviral nucleoside analogues such as AZT or ddl are metabolized by cellular mechanisms to the nucleotide triphosphate form in order to specifically inhibit retroviral reverse transcriptase, and thus viral replication (Furmam et al., Proc. Natl Acad.
  • Retroviruses which direct the expression of a gene product (e.g., a protein) such as Herpes Simplex Virus Thymidine Kinase (HS VTK) or Varicella Zoster Virus Thymidine Kinase (VZ VTK) which assists in metabolizing antiviral nucleoside analogues to their active form are therefore useful in activating nucleoside analogue precursors (e.g., AZT or ddC) into their active form.
  • nucleoside analogue precursors e.g., AZT or ddC
  • the vector construct may direct the expression of a HSVTK gene under the transcriptional control of an HIV promoter known to be transcription ⁇ illy silent except when activated by HTV tat protein. Briefly, expression of the tat gene product in human cells infected with HTV and carrying the vector construct would lead to production of HSVTK.
  • alkaline phosphatase which will convert inactive phosphorylated compounds such as mitomycin phosphate and doxorubicin-phosphate to toxic dephosphorylated compounds
  • fiingal e.g., Fusarium oxysporum
  • bacterial cytosine deaminase which will convert 5- fluorocytosine to the toxic compound 5-fluorouracil
  • carboxypeptidase G2 which will cleave the glutamic acid from para-N-bis (2-chloroethyl) aminobenzoyl glutamic acid, thereby creating a toxic benzoic acid mustard
  • Penicillin- V amidase which will convert phenoxyacetabide derivatives of doxorubicin and melphalan to toxic compounds.
  • Conditionally lethal gene products of this type have application to many presently known purine- or pyrimidine-based anticancer drugs, which often require intracellular ribosylation or phosphorylation in order to become effective cytotoxic agents.
  • the conditionally lethal gene product could also metabolize a nontoxic drug, which is not a purine or pyrimidine analogue, to a cytotoxic form (see Searle et al., Brit. J. Cancer 55:377-384, 1986).
  • vector constructs may be constructed to take advantage of the fact that mammalian viruses in general tend to have "immediate early" genes, which are necessary for subsequent transcriptional activation of other viral promoter elements. Gene products of this nature are excellent candidates for intracellular signals (or "identifying agents") of viral infection. Thus, conditionally lethal genes transcribed from transcriptional promoter elements that are responsive to such viral "immediate early" gene products could specifically kill cells infected with any particular virus.
  • vector constructs for incorporation into a gene delivery vehicle are provided that produce substances such as inhibitor palliatives, that inhibit viral assembly.
  • the recombinant DNA molecule codes for defective gag, pol, env or other viral particle proteins or peptides which inhibit in a dominant fashion the assembly of viral particles. Such inhibition occurs because the interaction of normal subunits of the viral particle is disturbed by interaction with the defective subunits.
  • inhibitory palliatives One way of increasing the effectiveness of inhibitory palliatives is to express inhibitory genes, such as viral inhibitory genes, in conjunction with the expression of genes which increase the probability of infection of the resistant cell by the virus in question. The result is a nonproductive "dead-end" event which would compete for productive infection events.
  • a vector construct may be administered that inhibits HTV replication (by expressing anti-sense tat, etc., as described above) and also overexpress proteins required for infection, such as CD4.
  • a relatively small number of vector-infected HTV-resistant cells act as a "sink” or "magnet” for multiple nonproductive fusion events with free virus or virally infected cells.
  • vector constructs are provided for the expression substances such as inhibiting peptides or proteins specific for viral protease.
  • Viral protease cleaves the viral gag and gag/pol proteins into a number of smaller peptides. Failure of this cleavage in all cases leads to complete inhibition of production of infectious retroviral particles.
  • the HTV protease is known to be an aspartyl protease, and these are known to be inhibited by peptides made from amino acids from protein or analogues.
  • Vector constructs that inhibit HTV will express one or multiple fused copies of such peptide inhibitors.
  • Administration of the vector constructs discussed above in an appropriate gene delivery vehicle should be effective against many virally linked diseases, cancers, or other pathogenic agents.
  • vector constructs are provided that code for the expression of a palliative, wherein the palliative has a membrane anchor and acts as an anti-tumor agent(s).
  • a palliative may be constructed, for example, as an anti-tumor agent - membrane anchor fusion protein.
  • the membrane anchor aspect of the fusion protein may be selected from a variety of sequences, including, for example, the transmembrane domain of well known molecules.
  • membrane anchor sequences are regions of a protein that bind the protein to a membrane.
  • anchor sequences that attach a protein to the outer surface of a cell membrane: (1) transmembrane regions that span the lipid bilayer of the cell membrane, and interact with the hydrophobic center region (proteins containing such regions are referred to integral membrane proteins), and (2) domains which interact with an integral membrane protein or with the polar surface of the membrane (such proteins are referred to as peripheral, or extrinsic, proteins).
  • a protein can also be covalently anchored directly to a lipid component of the cell membrane.
  • Membrane anchors for use within the present invention may contain transmembrane domains which span the membrane one or more times.
  • the membrane binding region spans the membrane once
  • the transmembrane domain of rhodopsin spans the membrane seven times
  • that of the photosynthetic reaction center of Rhodopseudomonas viridis spans the membrane eleven times (see Ross et al., J. Biol. Chem. 257:4152, 1982; Garters, Pharmac. Ther. 50:337-345, 1991; Engelman et al., Proc. Natl Acad. Sci.
  • the membrane spanning regions typically have a similar structure. More specifically, the 20 to 25 amino-acid residue portion of the domain that is located inside the membrane generally consists almost entirely of hydrophobic residues (see Eisenberg et al., Ann. Rev. Biochem. 55:595-623, 1984). For example, 28 of the 34 residues in the membrane spanning region of glycophorin are hydrophobic (see Ross et al., supra; Tomita et al., Biochemistry / 7:4756-4770, 1978).
  • membrane spanning regions typically have an alpha helical structure, as determined by X-ray diffraction, crystallography and cross-linking studies (see Eisenberg et a ⁇ .,supra; Heijne and Manoil, supra).
  • the location of these transmembrane helices within a given sequence can often be predicted based on hydrophobicity plots. Stryer et al., Biochemistry, 3rd. ed. 304, 1988.
  • Particularly preferred membrane anchors for use within the present invention include naturally occurring cellular proteins (that are non-immunogenic) which have been demonstrated to function as membrane signal anchors (such as glycophorin).
  • a vector construct which encodes a membrane anchor - gamma interferon fusion protein.
  • this fusion protein may be constructed by genetically fusing the sequence which encodes the membrane anchor of the gamma-chain of the Fc receptor, to a sequence which encodes gamma-interferon.
  • vector constructs which have a therapeutic effect by encoding one or more ribozymes (RNA enzymes) (H.aseloff and Gerlach, Nature 55*585, 1989) which will cleave, and hence inactivate, RNA molecules corresponding to a pathogenic function.
  • ribozymes function by recognizing a specific sequence in the target RNA and this sequence is normally 12 to 17 bp, this allows specific recognition of a particular RNA sequence corresponding to a pathogenic state, such as HTV tat, and toxicity is specific to such pathogenic state. Additional specificity may be achieved in some cases by making this a conditional toxic palliative, as discussed above.
  • vector constructs comprising a biologically active nucleic acid molecule that is an antisense sequence
  • an antisense sequence may also be encoded by a nucleic acid sequence and then produced within a host cell via transcription.
  • the antisense sequence is selected from the group consisting of sequences which encode influenza virus, HIV, HSV, HPV, CMV, and HB V.
  • the antisense sequence may also be an antisense RNA complementary to
  • the biologically active nucleic acid molecule may be a sense RNA (or DNA) complementary to RNA sequences necessary for pathogenicity.
  • the biologically active nucleic acid molecule may be an antisense sequence.
  • antisense sequences are designed to bind to RNA transcripts, and thereby prevent cellular synthesis of a particular protein, or prevent use of that RNA sequence by the cell.
  • Representative examples of such sequences include antisense thymidine kinase, antisense dihydrofolate reductase (Maher and Dolnick, Arch. Biochem. ⁇ Biophys.
  • antisense Myc (Stanton et al., Nature 570:423-425, 1984) and antisense ras, as well as antisense sequences which block any of the enzymes in the nucleotide biosynthetic pathway.
  • the substances of the invention include a surface protein that is itself therapeutically beneficial.
  • a surface protein that is itself therapeutically beneficial.
  • expression of the human CD4 protein specifically in HIV-infected cells may be beneficial in two ways: 1. Binding of CD4 to HTV env intracellularly could inhibit the formation of viable viral particles much as soluble CD4 has been shown to do for free virus, but without the problem of systematic clearance and possible immunogenicity, since the protein will remain membrane bound and is structurally identical to endogenous CD4 (to which the patient should be immunologically tolerant).
  • CD4/HTV env complex has been implicated as a cause of cell death
  • additional expression of CD4 in the presence of excess HIV-env present in HIV-infected cells
  • monocytes and macrophages which act as a reservoir for virus production as a result of their relative refractility to HTV-induced cytotoxicity (which, in turn, is apparently due to the relative lack of CD4 on their cell surfaces).
  • vector constructs capable of immunostimulation Briefly, the ability to recognize and defend against foreign pathogens is essential to the function of the immune system.
  • An immune response can also be achieved by transferring to an appropriate immune cell (such as a T lymphocyte) (a) the gene for the specific T-cell receptor that recognizes the antigen of interest (in the context of an appropriate MHC molecule if necessary), (b) the gene for an immunoglobulin which recognizes the antigen of interest, or (c) the gene for a hybrid of the two which provides a CTL response in the absence of the MHC context.
  • an appropriate immune cell such as a T lymphocyte
  • vector constructs may also be used as an immunostimulant, immunomodulator, or vaccine, etc.
  • This approach should be effective against many virally associated diseases or cancers where a characteristic antigen (which does not need to be a membrane protein) is expressed, such as in HPV and cervical carcinoma, HTLV-I- induced leukemias, prostate-specific antigen (PSA) and prostate cancer, mutated p53 and colon carcinoma and melanoma, melanoma specific antigens (MAGEs), and melanoma, mucin and breast cancer.
  • a characteristic antigen which does not need to be a membrane protein
  • substances which are carried and/or expressed by the vector constructs of the present invention may also include "immunomodulatory factors," many of which are set forth above.
  • Immunomodulatory factors refer to factors that, when manufactured by one or more of the cells involved in an immune response, or, which when added exogenously to the cells, causes the immune response to be different in quality or potency from that which would have occurred in the absence of the factor.
  • the factor may also be expressed from a gene delivery vehicle derived gene, but the expression is driven or controlled by the recombinant retrovirus.
  • the quality or potency of a response may be measured by a variety of assays known to one of skill in the art including, for example, in vitro assays which measure cellular proliferation (e.g., 3 H thymidine uptake), and in vitro cytotoxic assays (e.g., which measure 5*Cr release) (see, Warner et al., AIDS Res. and Human Retroviruses 7:645-655, 1991). Immunomodulatory factors may be active both in vivo and ex vivo.
  • cytokines such as IL-1, IL-2 (Karupiah et al., J. Immunology / *290-298, 1990; Weber et al., J. Exp. Med /55.1716-1733, 1987; Gansbacher et al., J. Exp. Med 172: 1217-1224, 1990; U.S. Patent No. 4,738,927), IL-3, IL-4 (Tepper et al., Cell 57:503-512, 1989; Golumbek et al., Science 25*713-716, 1991; U.S. Patent No. 5,017,691), IL-5, IL-6 (Brakenhof et al., J.
  • IL-1 cytokines
  • IL-2 Kerupiah et al., J. Immunology / *290-298, 1990; Weber et al., J. Exp. Med /55.1716-1733, 1987; Gansbacher et al
  • GM-CSF WO 85/04188
  • TNFs tumor necrosis factors
  • CD3 Krissanen et al., Immunogenetics 25:258-266, 1987
  • ICAM-1 Altman et al., Nature 555:512-514, 1989; Simmons et al., Nature 331:624-627, 1988
  • ICAM-2 LFA-1, LFA-3 (Wallner et al., J. Exp.
  • Immunomodulatory factors may also be agonists, antagonists, or ligands for these molecules. For example soluble forms of receptors can often behave as antagonists for these types of factors, as can mutated forms of the factors themselves.
  • An example of an immunomodulatory factor cited above is a member of the B7 family of molecules (e.g., B7.1-.3 costimulatory factor).
  • B7.1-.3 costimulatory factor a member of the B7 family of molecules
  • activation of the full functional activity of T cells requires two signals.
  • One signal is provided by interaction of the antigen-specific T cell receptor with peptides which are bound to major histocompatibility complex (MHC) molecules, and the second signal, referred to as costimulation, is delivered to the T cell by antigen presenting cells.
  • MHC major histocompatibility complex
  • costimulation the second signal
  • the second signal is required for interleukin-2 (IL-2) production by T cells, and appears to involve interaction of the B7.1-.3 molecule on antigen-presenting cells with CD28 and CTLA-4 receptors on T lymphocytes (Linsley et al., J. Exp.
  • B7.1-.3 may be introduced into tumor cells in order to cause costimulation of CD8 + T cells, such that the CD ⁇ +T cells produce enough IL-2 to expand and become fully activated.
  • CD8 + T cells can kill tumor cells that are not expressing B7 because costimulation is no longer required for further CTL function.
  • Vectors that express both the costimulatory B7.1-.3 factor, and, for example, an immunogenic HBV core protein, may be made utilizing methods which are described herein. Cells transduced with these vectors will become more effective antigen presenting cells. The HBV core-specific CTL response will be augmented from the fully activated CD8 + T cell via the costimulatory ligand B7.1-.3.
  • immunomodulatory factor may be based upon known therapeutic effects of the factor, or, experimentally determined.
  • a known therapeutic effector in chronic hepatitis B infections is alpha interferon. This has been found to be efficacious in compensating a patient's immunological deficit, and thereby assisting recovery from the disease.
  • a suitable immunomodulatory factor may be experimentally determined. Briefly, blood samples are first taken from patients with a hepatic disease.
  • Peripheral blood lymphocytes are restimulated in vitro with autologous or HLA matched cells (e.g., EBV transformed cells) that have been transduced with a recombinant retrovirus which directs the expression of an immunogenic portion of a hepatitis antigen and the immunomodulatory factor.
  • HLA matched cells e.g., EBV transformed cells
  • a recombinant retrovirus which directs the expression of an immunogenic portion of a hepatitis antigen and the immunomodulatory factor.
  • These stimulated PBLs are then used as effectors in a CTL assay with the HLA matched transduced cells as targets.
  • the immunomodulatory factor gamma interferon is particularly preferred.
  • the present invention also includes vector constructs which encode immunogenic portions of desired antigens including, for example, viral, bacterial or parasite antigens.
  • desired antigens including, for example, viral, bacterial or parasite antigens.
  • various immunogenic portions of the HBV S antigens may be combined in order to present an immune response when administered by one of the recombinant retroviruses described herein.
  • particular combinations of antigens may be preferred for administration in particular geographic regions.
  • epitopes that are found in all human hepatitis B virus S samples are defined as determinant "a”.
  • mutually exclusive subtype determinants however have also been identified by two-dimensional double immunodiffusion (Ouchterlony, Progr.
  • the immunological variability is due to single nucleotide substitutions in two areas of the hepatitis B virus S antigen open reading frame resulting in the following amino acid changes: (1) exchange of lysine-122 to arginine in the hepatitis B virus S antigen open reading frame causes a subtype shift from dtoy, and (2) exchange of arginine- 160 to lysine causes the shift from subtype r to w.
  • subtype ayw is predominant, whereas in the U.S. and northern Europe the subtype adu>2 is more abundant (Molecular Biology of the Hepatitis B Virus, McLachlan (ed.), CRC Press, 1991).
  • a vector for administration which is appropriate to the particular hepatitis B virus subtype which is prevalent in the geographical region of administration.
  • Subtypes of a particular region may be determined by two-dimensional double immunodiffusion or, preferably, by sequencing the S antigen open reading frame of HBV virus isolated from individuals within that region.
  • HBV pol also presented by HBV are pol ("HBV pol"), ORF 5, and ORF 6 antigens.
  • HBV pol the polymerase open reading frame of HBV encodes reverse transcriptase activity found in virions and core-like particles in infected liver tissue.
  • the polymerase protein consists of at least two domains: the amino terminal domain encodes the protein that primes reverse transcription, and the carboxyl terminal domain which encodes reverse transcriptase and RNase H activity.
  • Immunogenic portions of HBV pol may be administered to a warm-blooded animal by introducing into the animal a recombinant retrovirus which expresses the antigen of interest in order to generate an immune response within the animal.
  • HBV antigens such as ORF 5 and ORF 6, (Miller et al., Hepatology 0:322-327, 1989), may be expressed utilizing vector constructs as described herein.
  • the present invention also includes compositions and methods for treating, as well as vaccines for preventing, various feline diseases, including for example feline leukemia virus (“FeLV”) and feline immunodeficiency virus (“FTV”) infections. These viruses are discussed more fully in PCT application number US 93/09070.
  • feline leukemia virus (FeLV) is a retrovirus of the oncornavirus subfamily. FeLV is presently believed to exist in three subgroups - A, B or C - which are differentiated by their envelope antigens gp70 and pl5E.
  • feline immunodeficiency virus is morphologically and antigenically distinct from other feline retroviruses, including feline leukemia virus, type C oncorna virus (RD- 114), and feline syncytium-fo ⁇ ning virus (FeSFV) (see Yamamoto et al., "Efficacy of experimental FTV vaccines, (Abstract), First International Conference of Feline Immunodeficiency Virus researchers, University of California, Davis, CA, Sep. 4-7, 1991).
  • feline leukemia virus type C oncorna virus
  • FeSFV feline syncytium-fo ⁇ ning virus
  • the vector construct directs the expression of at least one immunogenic portion of an feline immunodeficiency virus antigen selected from the group consisting of plSgag, p24gag, plOgag, pl3pol, p62pol, pl5pol and p3 pol.
  • the vector construct directs the expression of gp68e «v, gp27.?7 ⁇ v and rev.
  • "rev” is understood to refer to the antigen corresponding to the rev open reading frame (see, Phillips et al., First International Conference, supra).
  • Sequences which encode these antigens may be readily obtained by one of skill in the art given the disclosure provided herein (see Phillips et al., J. Vir. 5 (10):4605-4613, 1990; Olmsted et al., PNAS 55:2448-2452, 1989; Talbott et al., PN S 55:5743-5747, 1989). Still other examples include vector constructs which direct the expression of a non-tumorigenic, altered genes such as the ras (ras*) gene (see WO 93/10814; U.S.S.N.
  • MCC and APC gene Both MCC and APC gene (Kinzler et al., Science 257:1366-1370, 1991; Nishiho et al., Science 253:665-669, 1991), an altered cellular receptor, including, for example, neu and mutated or altered forms of the thyroid hormone receptor, the PDGF receptor, the insulin receptor, the Interleukin receptors (e.g., TL-1, -2, -3, etc. receptors), or the CSF receptors, such as the G-CSF, GM-CSF, or M-CSF receptors ( U.S.S.N.
  • an altered cellular receptor including, for example, neu and mutated or altered forms of the thyroid hormone receptor, the PDGF receptor, the insulin receptor, the Interleukin receptors (e.g., TL-1, -2, -3, etc. receptors), or the CSF receptors, such as the G-CSF, GM-CSF, or M-CSF receptors ( U.S.S
  • the altered cellular component is associated with making the cell tumorigenic, then, it is necessary to make the altered cellular component non-tumorigenic.
  • the sequence or gene of interest which encodes the altered cellular component is truncated in order to render the gene product non- tumorigenic.
  • the gene encoding the altered cellular component may be truncated to a variety of sizes, although it is preferable to retain as much as possible of the altered cellular component.
  • any truncation leave intact at least some of the immunogenic sequences of the altered cellular component.
  • multiple translational termination codons may be introduced into the gene which encodes the altered cellular component, downstream of the immunogenic region.
  • E3 encodes a 19 kD transmembrane glycoprotein, E3/19K, transcribed from the E3 region of the adenovirus 2 genome.
  • tissue cells are transformed with a vector construct containing the E3/19K sequence, which upon expression produces the E3/19K protein.
  • the E3/19K protein inhibits the surface expression of MHC class I surface molecules, and cells transformed by the recombinant retrovirus evade an immune response. Consequently, donor cells can be transplanted with reduced risk of graft rejection and may require only a minimal immunosuppressive regimen for the transplant patient. This allows an acceptable donor-recipient chimeric state to exist with fewer complications. Similar treatments may be used to treat the range of so-called autoimmune diseases, including systemic lupus erythematosus, multiple sclerosis, rheumatoid arthritis or chronic hepatitis B infection.
  • Another alternative method of immunosuppression involves the use of antisense message, ribozyme, or other gene expression inhibitor specific for T-cell clones which are autoreactive in nature. These block the expression of the T-cell receptor of particular unwanted clones responsible for an autoimmune response.
  • the antisense, ribozyme, or other gene may be introduced using a gene delivery vehicles.
  • MHC class D presentation proteins that function to inhibit, suppress or down-regulate MHC class I antigen presentation
  • MHC class D presentation proteins that function to inhibit, suppress or down-regulate MHC class I antigen presentation
  • a recombinant retrovirus that expresses a protein or an active portion thereof suspected of being capable of inhibiting MHC class I antigen presentation is transformed into a tester cell line, such as BC.
  • the tester cell lines with and without the sequence encoding the candidate protein are compared to stimulators and/or targets in the CTL assay. A decrease in cell lysis corresponding to the transformed tester cell indicates that the candidate protein is capable of inhibiting MHC presentation.
  • the blocking action of a blocking agent can be mediated either from inside a susceptible cell or by secreting a version of the blocking protein to locally block the pathogenic interaction.
  • the two agents of interaction are the gp 120/gp 41 envelope protein and the CD4 receptor molecule.
  • an appropriate blocker would be an HTV env analogue that blocks HIV entry without causing pathogenic effects, or a CD4 receptor analogue.
  • the CD4 analogue would be secreted and would function to protect neighboring cells, while the gp 120/gp 41 is secreted or produced only intracellularly so as to protect only the vector-containing cell. It may be advantageous to add human immunoglobulin heavy chains or other components to CD4 in order to enhance stability or complement lysis. Delivery of a vector construct encoding such a hybrid-soluble CD4 in a position-specific manner results in a continuous supply of a stable hybrid molecule.
  • Recombinant DNA molecules mediating the expression of HTV env may also be constructed. It will be evident to one skilled in the art which portions are capable of blocking virus adsorption without overt pathogenic side effects (Willey et al., J. Virol 62:139, 1988; Fisher et al., Science 255:655, 1986).
  • Another embodiment of the invention involves the delivery of suppressor genes which, when deleted, mutated or not expressed in a cell type, lead to tumorigenesis in that cell type. Reintroduction of the deleted gene leads to regression of the tumor phenotype in these cells. Since malignancy can be considered to be an inhibition of cellular terminal differentiation compared with cell growth, the delivery and expression of gene products which lead to differentiation of a tumor should also, in general, lead to regression.
  • Sequences which encode the above-described nucleic acid molecules may be obtained from a variety of sources.
  • plasmids which contain sequences that encode altered cellular products may be obtained from a depository such as the American Type Culture Collection (ATCC, Rockville, Maryland), or from commercial sources such as Advanced Biotechnologies (Columbia, Maryland) or British Bio- Technology Limited (Cowley, Oxford England).
  • ATCC American Type Culture Collection
  • cDNA sequences for use with the present invention may be obtained from cells which express or contain the sequences.
  • mRNA from a cell which expresses the gene of interest is reverse transcribed with reverse transcriptase using oligo dT or random primers.
  • the single stranded cDNA may then be amplified by PCR (see U.S. Patent Nos. 4,683,202, 4,683,195 and 4,800,159. See also RCR Technology: Principles and Applications for DNA Amplification, Erlich (ed.), Stockton Press, 1989) utilizing oligonucleotide primers complementary to sequences on either side of desired sequences.
  • PCR see U.S. Patent Nos. 4,683,202, 4,683,195 and 4,800,159. See also RCR Technology: Principles and Applications for DNA Amplification, Erlich (ed.), Stockton Press, 1989) utilizing oligonucleotide primers complementary to sequences on either side of desired sequences.
  • a double stranded DNA is denatured by heating in the presence of heat stable Taq polymerase, sequence specific DNA primers, ATP, CTP, GTP and TTP. Double-stranded DNA is produced when synthesis is complete. This cycle may be repeated many times, resulting in a factorial a
  • genes of known nucleotide sequence useful for practice of the invention may be obtained by cloning the desired gene from an available cDNA or mRNA library using standard techniques.
  • Nucleic acid molecules which are carried and/or expressed by the recombinant DN molecules described herein may also be synthesized, in whole or in part, for example, on an Applied Biosystems Inc. DNA synthesizer (e.g., APB DNA synthesizer model 392 (Foster City, California).
  • high titer recombinant retroviral preparations are employed.
  • cell culture methods as described in U.S.S.N. 08/367,071, supra, are used. Briefly, a wide variety of methods may be utilized, including for example, the use of fermenters or bioreactors, roller bottles, cell hotels or cell factories, and hollow fiber culture.
  • cells are preferably grown on microcarriers (i.e., Cytodex 1 or Cytodex 2; Pharmacia, Piscataway, N.J.) at concentrations ranging from 3 to 15 grams of microcarrier per liter of appropriate media.
  • the volume of media being cultured on the cell side is approximately 10 to 100 fold lower then volumes required for equivalent cell densities cultured in tissue culture dishes or flasks, resulting in a 10-100 fold increase in titer when an individual retroviral producer cell line is amenable to hollow fiber growth conditions.
  • the individual cells must be able to grow in very close proximity and on top of each other. Many cell lines will not grow in this fashion and retroviral packaging cell lines based on these types of cell lines may not achieve 10 fold increases in titer.
  • Cell lines which would grow very well would be non-adherent cell line and it is believed that a retroviral producer line based on a non- adherent cell line may reach 100 fold increases in titer compared to tissue culture dishes and flasks.
  • methods are provided for quantitating gene delivery vehicles utilizing non-denaturing gels (e.g., 4-15% gradient polyacrylamide gels for resolving recombinant retroviral particles), along with methods for estimating or quantitating the resultant products such as, for example, staining with coomassie blue or silver stain, followed by densitometry scanning.
  • non-denaturing gels e.g., 4-15% gradient polyacrylamide gels for resolving recombinant retroviral particles
  • methods for estimating or quantitating the resultant products such as, for example, staining with coomassie blue or silver stain, followed by densitometry scanning.
  • assays are provided for titering recombinant virus, such as a recombinant retrovirus, in a sample.
  • recombinant virus such as a recombinant retrovirus
  • assays may be based upon presence of a selectable marker, or formation of blue colonies.
  • gene delivery vehicles are provided which do not include a gene coding for a selectable marker. Therefore, antibody and PCR assays, the latter of which is described below, may be employed in order to determine titer.
  • appropriate amplification primers are required.
  • PCR primers pairs include those specific for LTR sequences, packaging signal sequences or other regions of the retroviral backbone, and may include primers specific for the gene(s) of interest.
  • a PCR titering assay is used to determine titer of a recombinant retroviral preparation. The assay is performed by growing a known number of cells transduced with the recombinant retrovirus on 6-well plates for at least 16 hr. before harvest.
  • DNA is prepared using a QUIAmp DNA isolation kit (QUIAgen, Inc.Chatsworth, CA). DNAs are resuspended in 5 x 10 cell equivalents/ ⁇ L per .sample.
  • a standard curve is generated using DNA isolated from 5 x 10 6 untransduced HT1080 cells (negative control) and 5 x 10 6 HT1080 cells transduced with a known vector and having one integrated copy of that vector per cell genome (positive control), such as may be prepared from packaging cell lines transduced with a recombinant retrovirus encoding a selectable marker, e.g., neomycin resistance.
  • the standard curve is generated by combining different amounts of the positive and negative control DNA and amplifying specific sequences therefrom by PCR using primers specific to a particular region of the recombinant retrovirus.
  • a representative group of mixtures for generating a standard curve is:
  • Negative Control ( ⁇ L) 0 12.5 25 37.5 45 47.5 50 0
  • PCR reactions 50 ⁇ L total volume are then initiated by adding 45.0 ⁇ L of a reaction mix containing the following components per tube to be tested: 24.5 ⁇ L water, 5 ⁇ L 10X reaction PCR buffer, 4 ⁇ L of 25 mM MgCb, 4 ⁇ L dNTPs (containing 2.5 mM of each of dATP, dGTP, dCTP, and dTTP), 5 ⁇ L of primer mix (100 ng or each primer), 0.25 ⁇ L TaqStart monoclonal antibody (Clontech Laboratories, Inc., Palo Alto, CA), 1.00 ⁇ L TaqStart buffer (Clontech Labs, Inc.), and 0.25 ⁇ L AmpliTaq DNA polymerase (Perkin- Elmer, Inc.
  • thermocycler Just prior to aliquoting the reaction mix to the reaction tubes, 1 ⁇ L of a- 32 P dCTP (250 ⁇ Ci; 3000 C/mmol, 10 mCi mL, Amersham Corp., Arlington Heights, IL) is added into the reaction mix. After aliquoting 45.0 ⁇ L the reaction mix into each of the reaction tubes, the tubes are capped and placed into a thermocycler. The particular denaturation, annealing, elongation times and temperatures, and number of thermocycles will vary depending on size and nucleotide composition of the primer pair used. 20 - 25 amplification thermocycles are then performed.
  • the scanning results are then downloaded and plotted on a log scale as cpm (ordinate) versus percent positive control DNA (abscissa).
  • Titers infectious units/mL
  • Titers are calculated by multiplying the number of cells from which DNA was isolated by the percentage (converted to decimal form) determined from the standard curve based on the detected radioactivity, divided by the volume of recombinant retrovirus used to transduce the cells.
  • other methods of detection such as color-metric methods, may also be employed to label the amplified products.
  • Another aspect of the invention relates to formulation of the gene delivery vehicles according to the invention.
  • the recombinant retrovirus can also be preserved in a purified form. More specifically, prior to the addition of the formulation buffer, the crude recombinant retrovirus described above may be clarified by passing it through a filter, and then concentrated, such as by a cross flow concentrating system (Filtron Technology Corp., Nortborough, MA). Within one embodiment, DNase is added to the concentrate to digest exogenous DNA. The digest is then diafiltrated to remove excess media components and establish the recombinant retrovirus in a more desirable buffered solution. The diafiltrate is then passed over a Sephadex S-500 gel column and a purified recombinant retrovirus is eluted.
  • a cross flow concentrating system Frtron Technology Corp., Nortborough, MA
  • the formulation buffer is an aqueous solution that contains a saccharide, a high molecular weight structural additive, and a buffering component in water.
  • the aqueous solution may also contain one or more amino acids.
  • the crude recombinant retrovirus can also be purified by ion exchange column chromatography. This method is described in more detail in U.S. Patent Application Serial No. 08/093,436.
  • the aqueous suspension in crude or purified form can be dried by lyophilization or evaporation at ambient temperature.
  • lyophilization involves the steps of cooling the aqueous suspension below the glass transition temperature or below the eutectic point temperature of the aqueous suspension, and removing water from the cooled suspension by sublimation to form a lyophilized retrovirus. Briefly, aliquots of the formulated recombinant retrovirus .are placed into an Edwards Refrigerated Chamber (3 shelf RC3S unit) attached to a freeze dryer (Supermodulyo 12K). A multistep freeze drying procedure as described by Phillips et al.
  • the aqueous solutions used for formulation are composed of a saccharide, high molecular weight structural additive, a buffering component, and water.
  • the solution may also include one or more amino acids.
  • the combination of these components act to preserve the activity of the recombinant retrovirus upon freezing and lyophilization, or drying through evaporation.
  • a preferred saccharide is lactose
  • other saccharides may be used, such as sucrose, mannitol, glucose, trehalose, inositol, fructose, maltose or galactose.
  • combinations of saccharides can be used, for example, lactose and mannitol, or sucrose and mannitol.
  • a particularly preferred concentration of lactose is 3%-4% by weight.
  • the concentration of the saccharide ranges from 1% to 12% by weight.
  • the high molecular weight structural additive aids in preventing viral aggregation during freezing and provides structural support in the lyophilized or dried state.
  • structural additives are considered to be of "high molecular weight" if they are greater than 5000 m.w.
  • a preferred high molecular weight structural additive is human serum albumin.
  • other substances may also be used, such as hydroxyethyl-cellulose, hydroxymethyl-cellulose, dextran, cellulose, gelatin, or povidone.
  • a particularly preferred concentration of human serum albumin is 0.1% by weight.
  • the concentration of the high molecular weight structural additive ranges from 0.1% to 10% by weight.
  • buffers may be used, depending on the pH range desired, preferably between 7.0 and 7.8. Suitable buffers include phosphate buffer and citrate buffer. A particularly preferred pH of the recombinant retrovirus formulation is 7.4, and a preferred buffer is tromethamine.
  • the aqueous solution contain a neutral salt which is used to adjust the final formulated recombinant retrovirus to an appropriate iso- osmotic salt concentration.
  • Suitable neutral salts include sodium chloride, potassium chloride or magnesium chloride.
  • a preferred salt is sodium chloride.
  • Aqueous solutions containing the desired concentration of the components described above may be prepared as concentrated stock solutions.
  • Lyophilized or dehydrated recombinant retrovirus may be reconstituted with any convenient volume of water or the reconstituting agents noted above that allow substantial, and preferably total solubilization of the lyophilized or dehydrated sample.
  • methods for treating eukaryotic organisms, particularly warm blooded animals, and especially human, afflicted with a variety of diseases, including a genetic disease, cancer, an infectious disease, an autoimmune disease, and inflammatory disease, a cardiovascular disease, and a degenerative disease.
  • diseases include but are not limited to; thalassemia, phenylketonuria, Lesch-Nyan syndrome, SOD, hemophilia A and B, cystic fibrosis, Duchenne's muscular dystrophy, inherited emphysema, -familial hypercholesterolemia, and Gaucher's disease.
  • cancers include but are not limited to; solid tumors, leukemias and lymphomas. Representative examples include melanomas, colorectal carcinomas, lung carcinomas (including large cell, small cell, squamous and adeno-carcinomas), renal cell carcinomas, cervical cancer, adult T-cell lymphoma leukemia, and breast adeno-carcinomas.
  • Infectious diseases include but not limited to; hepatitis, tuberculosis, malaria, human immunodeficiency virus, herpes virus, tetanus, dysentery, shigella, FeLV, and FTV.
  • Degenerative diseases include but are not limited to: Alzheimer's disease, multiple sclerosis, muscular dystrophy, amyotrophic lateral sclerosis, Inflammatory diseases include rheumatoid arthritis, spinal meningitis, and pancreatitis.
  • Autoimmune diseases include diabetes, uveitis, HTV, and SC D.
  • Cardiovascular diseases include, chronic rheumatic heart disease, arteriosclerosis, mitral valve and aortic stenosis, myocarditis, pericarditis, Marian's syndrome, Ehlers-Danlos syndrome, Churg-Strauss syndrome, and scleroderma.
  • a “therapeutically effective amount” of a gene product expressed from a vector construct according to the invention is an amount that achieves a desired therapeutic benefit in a patient to an extent greater than that observed when the patient was not treated with the gene product.
  • a “therapeutically effective amount” refers to the amount of factor Vm needed to produce therapeutically beneficial clotting and will thus generally be determined by each patient's attending physician, although serum levels of about 0.2 ng mL (about 0.1% of "normal” levels) or more will typically be therapeutically beneficial.
  • RNA molecule with intrinsic biological activity such an antisense RNA or ribozyme
  • a "therapeutically effective amount” is an amount sufficient to achieve a clinically relevant change in the patient's condition through reduced expression of the targeted gene product, most often a protein.
  • the RNA molecule with intrinsic biological activity i.e., antisense RNA or ribozyme
  • Expression levels of the heterologous and targeted RNAs can be determined by various assays, e.g., by PCR analysis.
  • Typical dosages for ex vivo treatment of hematopoietic stem cells will generally range from about 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 ⁇ to 10 12 infectious gene delivery vehicles, with dosages of 10 7 to 10 10 infectious particles being preferred.
  • the gene delivery vehicles of the present invention may be administered to a wide variety of locations including, for example, into sites such as the cerebral spinal fluid, bone marrow, joints, arterial endothelial cells, rectum, buccal/sublingual, vagina, the lymph system, to an organ selected from the group consisting of lung, liver, spleen, skin, blood and brain, or to a site selected from the group consisting of tumors and interstitial spaces.
  • the recombinant retrovirus may be administered intraocularly, intranasally, sublingually, orally, topically, intravesically, intrathecally, topically, intravenously, intraperitoneally, intracranially, intramuscularly, or subcutaneously.
  • Other representative routes of administration include gastroscopy, ECRP and colonoscopy, which do not require full operating procedures and hospitalization, but may require the presence of medical personnel. Considerations for administering the compositions of the present invention include the following:
  • Oral administration is easy and convenient, economical (no sterility required), safe (over dosage can be treated in most cases), and permits controlled release of the active ingredient of the composition (the gene delivery vehicle).
  • the gene delivery vehicle may be subject to "first pass effect" by hepatic metabolism and gastric acid and enzymatic degradation.
  • efficient plasma levels may not be reached, a patient's cooperation is required, and food can affect absorption.
  • Preferred embodiments of the present invention include the oral administration of gene delivery vehicles that express genes encoding erythropoietin, insulin, GM-CSF cytokines, various polypeptides or peptide hormones, their agonists or antagonists, where these hormones can be derived from tissues such as the pituitary, hypothalamus, kidney, endothelial cells, liver, pancreas, bone, hemopoetic marrow, and adrenal.
  • Such polypeptides can be used for induction of growth, regression of tissue, suppression of immune responses, apoptosis, gene expression, blocking receptor-ligand interaction, immune responses and can be treatment for certain anemias, diabetes, infections, high blood pressure, abnormal blood chemistry or chemistries (e.g., elevated blood cholesterol, deficiency of blood clotting factors, elevated LDL with lowered HDL), levels of Alzheimer associated amaloid protein, bone erosion/calcium deposition, and controlling levels of various metabolites such as steroid hormones, purines, and pyrimidines.
  • the gene delivery vehicles are first lyophilized, then filled into capsules and administered.
  • Buccal/sublingual administration is a convenient method of administration that provides rapid onset of action of the active component(s) of the composition, and avoids first pass metabolism. Thus, there is no gastric acid or enzymatic degradation, and the absorption of gene delivery vehicle is feasible. There is high bioavailability, and virtually immediate cessation of treatment is possible. Conversely, such administration is limited to relatively low dosages (typically about 10-15 mg), and there can be no simultaneous eating, drinking or swallowing.
  • Preferred embodiments of the present invention include the buccal/sublingual administration of gene delivery vehicles that contain genes encoding self and or foreign MHC, or immune modulators, for the treatment of oral cancer; the treatment of Sjogren's syndrome via the buccal/sublingual administration of such gene delivery vehicles that contain IgA or IgE antisense genes; and, the treatment of gingivitis and periodontitis via the buccal/sublingual administration of IgG or cytokine antisense genes.
  • Rectal administration provides a negligible first pass metabolism effect (there is a good blood lymph vessel supply, and absorbed materials drain directly into the inferior vena cava), and the method is suitable of children, patients with emesis, and the unconscious.
  • the method avoids gastric acid and enzymatic degradation, and the ionization of a composition will not change because the rectal fluid has no buffer capacity (pH 6.8; charged compositions absorb best).
  • pH 6.8 charged compositions absorb best
  • lipidophilic and water soluble compounds are preferred for absorption by the rectal mucosa, and absorption enhancers (e.g., salts, EDTA, NSADD) may be necessary.
  • absorption enhancers e.g., salts, EDTA, NSADD
  • Preferred embodiments of the present invention include the rectal administration of gene delivery vehicles that contain genes encoding colon cancer antigens, self and/or foreign MHC, or immune modulators.
  • Nasal administration avoids first pass metabolism, and gastric acid and enzymatic degradation, and is convenient.
  • nasal administration is useful for gene delivery vehicle administration wherein the gene delivery vehicle carries nucleic acid capable of expressing a polypeptide with properties as described herein. Conversely, such administration can cause local irritation, and absorption can be dependent upon the state of the nasal mucosa.
  • Pulmonary administration also avoids first pass metabolism, and gastric acid and enzymatic degradation, and is convenient. Further, pulmonary administration permits localized actions that minimize systemic side effects and the dosage required for effectiveness, and there can be rapid onset of action and self-medication.
  • Preferred embodiments of the present invention include the pulmonary administration of vehicles that express genes encoding IgA or IgE for the treatment of conditions such as asthma, hay fever, allergic alveolitis or fibrosing alveolitis, the CFTR gene for the treatment of cystic fibrosis, and protease and collagenous inhibitors such as a-1-antitrypsin for the treatment of emphysema.
  • vehicles that express genes encoding IgA or IgE for the treatment of conditions such as asthma, hay fever, allergic alveolitis or fibrosing alveolitis, the CFTR gene for the treatment of cystic fibrosis, and protease and collagenous inhibitors such as a-1-antitrypsin for the treatment of emphysema.
  • CFTR gene for the treatment of cystic fibrosis
  • protease and collagenous inhibitors such as a-1-antitrypsin for the treatment of emphysema.
  • Ophthalmic administration provides local action, and permit prolonged action where the administration is via inserts. Further, avoids first pass metabolism, and gastric acid and enzymatic degradation, and permits self-administration via the use of eye- drops or contact lens-like inserts. Conversely, the administration is not always efficient, because the administration induces tearing.
  • Preferred embodiments of the present invention include the ophthalmic administration of gene delivery vehicles that express genes encoding IgA or IgE for the treatment of hay fever conjunctivitis or vernal and atomic conjunctivitis; and ophthalmic administration of gene delivery vehicles that contain genes encoding melanoma specific antigens (such as high molecular weight-melanoma associated antigen), self and/or foreign MHC, or immune modulators.
  • Transdermal administration permits rapid cessation of treatment and prolonged action leading to good compliance. Further, local treatment is possible, and avoids first pass metabolism, and gastric acid and enzymatic degradation. Conversely, such administration may cause local irritation, is particularly susceptible to tolerance development, and is typically not preferred for highly potent compositions.
  • Preferred embodiments of the present invention include transdermal administration to express genes encoding IgA or IgE for the treatment of conditions such as atopic dermatitis and other skin allergies; and transdermal administration of gene delivery vehicles encoding genes encoding melanoma specific antigens (such as high molecular weight-melanoma associated antigen), self and/or foreign MHC, or immune modulators.
  • Vaginal administration provides local treatment and one preferred route for hormonal administration. Further, such administration avoids first pass metabolism, and gastric acid and enzymatic degradation, and is preferred for administration of compositions wherein the gene delivery vehicles express peptides.
  • Preferred embodi ents of the present invention include vaginal administration to express genes encoding self and/or foreign MHC, or immune modulators.
  • Other preferred embodiments include the vaginal administration of genes encoding the components of sperm such as histone, flagellin, etc., to promote the production of sperm-specific antibodies and thereby prevent pregnancy. This effect may be reversed, and/or pregnancy in some women may be enhanced, by delivering gene delivery vehicles carrying vectors encoding immunoglobulin antisense genes, which genes interfere with the production of sperm- specific antibodies.
  • Intravesical administration permits local treatment for urogenital problems, avoiding systemic side effects and avoiding first pass metabolism, and gastric acid and enzymatic degradation. Conversely, the method requires urethral catheterization and requires a highly skilled staff.
  • Preferred embodiments of the present invention include intravesical administration for delivering antitumor genes such as a prodrug activation gene such thymidine kinase or various immunomodulatory molecules such as cytokines.
  • Endoscopic retrograde cystopancreatography (ERCP) (goes through the mouth; does not require piercing of the skin) takes advantage of extended gastroscopy, and permits selective access to the biliary tract and the pancreatic duct.
  • ERCP Endoscopic retrograde cystopancreatography
  • the method requires a highly skilled staf- ⁇ and is unpleasant for the patient.
  • routes of administration described herein may be accomplished simply by direct administration using a needle, catheter or related device.
  • one or more dosages may be administered directly in the indicated manner: into the cerebral spinal fluid at dosages greater than or equal to 10 , 10 , 10 , 10 , 10 or 10 cfu; into bone marrow at dosages greater than or equal to 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 or 10 11 cfu; into joint(s) at dosages greater than or equal to 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 or 10 11 cfu; intravenously at dosages greater than or equal to 10 8, 109, 1010 or 1011 cfu; intra-arterially at dosages greater than or equal to 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 or 10 11 cfu; intra-cranially at dosages greater than or equal to 10 , 10 or 10 cfu; intra-muscularly at dosages greater than or equal to 10
  • Colony forming unit means the number of cells transduced in vitro in some detectable fashion (e.g. drug resistance, gene expression detected by reaction with an antibody, PCR for transduced genes etc.)
  • the gene delivery vehicles of the invention may also be delivered in an ex vivo format.
  • This example describes how to confer the position specificity of the yeast Ty3 element (U.S. Patent No. 5,292,662) to the integrase (IN) protein derived from Moloney murine leukemia virus. Under appropriate conditions, the resulting chimeric integrase can then be packaged into a variety of gene delivery vehicles to confer on a vector construct according to the invention position-specific integration into the genome of a eukaryotic cell transduced or otherwise transformed therewith.
  • Ty3 IN is a functional and structural analog of retroviral IN proteins, including MoMLV IN.
  • An alignment of the amino acid sequences of Ty3 and MoMLV see FIG. 3) using the UWGCG Bestfit algorithm (Devereux, et al. (1984), Nucl.
  • Acids Res., vol. 12:387-395) revealed roughly 25% identity within the core region which contains the D-D-E motif conserved among retrovirus IN proteins.
  • MoMLV IN which comprises 408 amino acids
  • the core region corresponds to approximately 220 residues
  • the amino-terminal domain comprises about the first 40 amino acids
  • the carboxy terminus the remaining 140 or so amino acids.
  • Ty3 IN which is comprised of 536 amino acids
  • the amino-terminal domain spans about the first 60 residues
  • the core domain about the next 240 amino acids, with the carboxy-terminal domain comprising approximately 230 residues.
  • each protein the amino-terminal domain is referred to as the "A" domain, the carboxyl end as the “C” domain, and the core domain as "B", as designated in FIG. 2.
  • the single upper case designation therefor may be immediately followed by "m” for a domain derived from MoMLV IN or "t” for a domain derived from Ty3.
  • “At” designates the A domain derived from Ty3, and a chimeric IN protein or gene therefor (or vector harboring such gene) referred to as AmBmCt, for example, is a chimera comprising the A and B domains from MoMLV IN and the C domain from Ty3.
  • Each chimeric IN retroviral vector is based on the retroviral vector pRgpKan, which is illustrated in FIG. 4.
  • pRgpKan contains all cis elements of a high titer retroviral vector. In addition, it expresses functional gag and pol gene products and, when introduced into a cell line expressing appropriate env gene products, results in the production of recombinant retroviral particles containing pRgpKan genomic RNA.
  • pRgpKan contains the neomycin phosphotransferase gene from transposon Tn5; the gene is expressed as kanamycin resistance in bacteria and G418 resistance in mammalian cells (utilizing the SV40 early promoter).
  • a negative control retroviral vector designated pRgpAmBtCt(-)
  • pRgpAmBtCt(-) was prepared by inserting the AmBtCt DNA sequence in the antisense orientation.
  • pRgpAmBtCt(-) is introduced into a suitable packaging cell line, e.g., NC10 or 2922-3, recombinant retroviral particles are produced, although these particles are replication incompetent and do not contain functional IN.
  • pRgpKan from which the MoMLV IN coding region was initially derived, was used as the positive control.
  • cultures producing recombinant retroviral particles carrying chimeric integrase may be produced as described hereinafter.
  • 293 2-3 cells AKA 293-GP, Bums, etal, Proc.Natl.Acad.Sci. 00:8033 1993, see WO 92/05266) generated from the human adenovirus 5-transformed embryonal kidney cell line 293 (ATCC #CRL1573) and which express MoMLV gag and pol genes, are produced.
  • NC10 cells which express the 4070A amphotropic envelope (see WO 92/05266), are generated from the HT1080 human fibrosarcoma line (ATCC #CCL 121). Both HT1080 and 292 cells have been demonstrated to lack DNA sequences that hybridize to the MoMLV genome.
  • a plasmid construct, pMLP-G expresses vesicular stomatitis virus G- protein and is used to complement MoMLV gag-pol proteins to pseudotype retroviral vector particles.
  • FIG. 8 illustrates the method for generating producer cultures. In each case, the plasmid encoding the chimeric IN retroviral vector is co- transfected (Graham, et al. (1973), Virology, vol.
  • the retroviral RNA genome encoding the chimeric IN is encapsidated along with pol proteins into particles formed by gag gene products. These particles also contain VS V-G protein on their outer surface.
  • filtered (.45 ⁇ m, cellulose acetate) supernatant fluid is placed onto NC10 cells.
  • the transduced (Emi, et al. (1991), J. Virol., vol. 65:1202-1207.) NC10 culture is placed under G418 selection (600 ⁇ g/ml).
  • NC10 control cultures no longer contain viable cells.
  • the resulting cultures produce retroviral particles which consist of: a RNA genome coding for, among other things, a chimeric IN protein; gag and pol gene products expressed from the chimeric retroviral vector construct; an amphotropic envelope; and chimeric IN protein.
  • retroviral particles Functionality of these retroviral particles is subsequently assessed by placing filtered supernatant fluid onto target cells, e.g., HT1080, and selecting for G418 resistant transductants. All of the chimeric retroviral vector particles produced have been used successfully to generate NC10 producer cells, i.e., cells which produce infectious recombinant retroviral particles. As judged by relative transient titers, retroviral genomes coding for a chimeric IN protein are packaged at the .same rate as observed for RgpKan.
  • NC10 producer cells To further characterize the NC10 producer cells to ensure that the biological activity of the gag and pol components, NC10 cell lysates and supernatants are tested by Northern analysis (Sambrook, et al., supra) to determine the level of expression and packaging of the retroviral constructs coding for the chimeric integrases, as compared to the expression levels detected for the control RgpKan construct. Because no cis acting sequences required for transcription, packaging, or replication are modified, the results for the chimeric retroviral vectors are expected to be similar to those determined for the control.
  • NC10 cell lysates and supernatants are tested by Western blot analysis for production of MoMLV gag and IN proteins using anti- p30, anti-MoMLV IN, .and anti-Ty3 IN rabbit polyclonal antibodies prepared separately according to standard techniques. Because the levels of gag protein is comparable for control RgpKan and the seven chimeric constructs, they serve as an internal control for the levels of pol-de ⁇ ved IN protein. The mobility and detected amount of chimeric integrase produced from each construct indicates if correct processing by the MoMLV protease occurred at the MoMLV IN processing site three amino acids in front of the junctions with the Ty3 sequence, for those chimeras containing an At domain.
  • NCIO cell supernatants are concentrated by centrifugation and tested for reverse transcriptase activity (Gof ⁇ et al. (1981), J. Virol, vol. 38:329-248.) to determine if incorporation of the heterologous IN domain into the retroviral particle disrupts reverse transcriptase function.
  • Activity of test constructs is compared to activity in the RgpKan supernatants and both are normalized to levels of gag protein.
  • a transduction assay using a eukaryotic cell line e.g., HT1080
  • HT1080 cells are susceptible to infection by amphotropic retroviral vectors, however, here transduction (as determined by G418 resistance) depends on successful integration mediated by a chimeric IN incorporated into the retroviral particle during virion assembly in the NCIO producer cell, where the integrase function is encoded in NCIO cells by the retroviral vector introduced by the VSV-g pseudotype virion produced in the 2922-3 cells.
  • RNA polymerase ID transcribed genes which include tRNA genes
  • animal cell tRNA is purified, labeled (Goddard, et al. (1983), Nuc. Acids Res., vol. 11:2551-62.), and used to probe Southern blots of pooled plasmid DNA from the kanamycin resistant bacteria. Parallel blots probed with LTR specific probes reveal the relative abundance of proviral DNA and adjacent tRNA genes.
  • Nonspecific integration yields a frequency of association provirus and a tRNA gene less than 1 in 1,000 clones, based on an average size of cloned integration fragment of 3 kb and a total genomic DNA of 10 ⁇ kb harboring about 1,300 tRNA genes (Hatlen, et al. (1971), J. Mol. Biol., vol. 56:535-553). Pooled DNA and colonies can also be screened in a similar manner using 5S and U6 sequence probes, since these genes are conserved in sequence but repeated in the genome, 1,000 to 2,000 for the 5S gene (Sorensen, et al. (1991), Nuc. Acids Res., vol. 19, 4147-51) and 200 for U6 (Hayashi, K. (1981), Nuc. Acids Res, vol. 9:3379-88).
  • Clones and pools from the integration library may also be probed with a radiolabeled Alu-specific probe.
  • Alu elements are generally not transcriptionally active, they contain pol ID promoter elements.
  • at least ten plasmids for each construct are analyzed by sequencing to identify potential target genes independent of probe reactivity (Pavesi, et al. (1994), Nuc. Acids Res, vol. 22: 1247-56).
  • Sequencing from the LTRs allows a determination of provirus orientation with respect to the pol ID-transcribed element, .(fan insertion is within 500 bp, preferably within 100 bp, more preferably within 20 bp of the 5' end of the mature tRNA coding region (or coding region for another RNA pol ID gene), integration is deemed to have involved position- specificity. However, only after approximately 10-20 position-specific events are analyzed is a chimeric IN function be considered position-specific. In addition, the level of expression of a gene of interest from a provirus integrated in a position-specific manner by a chimeric integrase according to the invention can also be analyzed.
  • expression levels will vary less than 100-fold, more preferably less than 50-fold, and most preferably less than 10-fold for a given gene of interest integrated in a position-specific manner, as compared to the same gene randomly integrated in a comparable eukaryotic cell genome.
  • target cells are tested by quantitative Northern blot analysis for the expression levels of transcripts produced from the integrated provirus. To perform a statistical analysis, at least ten independent clones are examined per original construct
  • the N2R5 construct is mutated by site-directed in vitro mutagenesis to change the ATG start codon to ATT preventing gag expression.
  • This mutagenized fragment is 200 base pairs (bp) in length and flanked by Pst I restriction sites.
  • the Pst I- Pst I mutated fragment is purified from the SK + plasmid and inserted into the Pst I site of N2 MoMLV 5' LTR in plasmid pUC31 to replace the non-mutated 200 bp fragment.
  • the plasmid pUC31 is derived from pUC19 (Stratagene, La Jolla, CA) in which additional restriction sites Xho I, Bgl D, BssH D and Nco I are inserted between the EcoR I and Sac I sites of the polylinker. This construct is designated pUC3 l/N2R5gM.
  • a 1.0 kilobase (Kb) MoMLV 3' LTR EcoR I-EcoR I fragment from N2 is cloned into plasmid SK + resulting in a construct designated N2R3 ⁇ .
  • a 1.0 Kb Cla I-Hind ID fragment is purified from this construct.
  • KT-3B or KT-1 vectors are constructed by a three part ligation in which the Xho I-Cla I fragment containing the gene of interest and the 1.0 Kb MoMLV 3' LTR Cla I-KGnd ID fragment are inserted into the Xho I-Hind ID site of ⁇ UC3 l N2R5gM plasmid. This gives a vector designated as having the KT-1 backbone.
  • the 1.3 Kb Cla I- BstB I neo gene fragment from the pAFVXM retroviral vector is then inserted into the Cla I site of this plasmid in the sense orientation to yield a vector designated as having the KT-3B backbone.
  • a gene encoding full length factor VID can be obtained from a variety of sources.
  • One such source is the plasmid pCIS-F8 (see EP 0260 148), which contains a full length factor VID cDNA whose expression is under the control of a CMV major immediate-early (CMV MIE) promoter and enhancer.
  • CMV MIE CMV major immediate-early
  • the factor VID cDNA contains approximately 80 bp of 5' untranslated sequence from the factor VID gene and a 3' untranslated region of about 500 bp.
  • a CMV intron sequence or "cis" element.
  • a plasmid vector encoding a truncation of about 80% (approximately 370 bp) of the 3' untranslated region of the factor VID cDNA, designated pND-5, is constructed in a pKT-1 vector as follows: As described for pJW-2, the pKT-1 vector employed has its Xho I restriction site replaced by that for Not I.
  • the factor VID insert is generated by digesting pCIS-F8 with Cla I and Xba I, the latter enzyme cutting 5' of the factor VDI stop codon. The approximately 7 kb fragment containing all but the 3' coding region of the factor VDI gene is then purified.
  • pCIS-F8 is also digested with Xba I and Pst I to release a 121 bp fragment containing the gene's termination codon. This fragment is also purified and then ligated in a three way ligation with the larger fragment encoding the rest of the factor VDI gene and Cla I Pst I restricted BLUESCRIPT® KS + plasmid (Stratagene, supra) to produce a plasmid designated pND-2. The unique Sma I site in pND-2 is then changed to a Cla I site by ligating Cla I linkers (New England Biolabs, Beverly, MA) under dilute conditions to the blunt ends created by a Sma I digest. After recircularization and ligation, plasmids containing two Cla I sites are identified and designated pND-3.
  • the precursor DNA for the B-deleted FVID is obtained from Miles Laboratory.
  • This expression vector is designated p25D and has the exact backbone as pCISF8 above.
  • the Hpa I site at the 3' of the FVID8 cDNA in p25D is modified to Cla-I by oligolinkers.
  • An Ace I to Cla I fragment is clipped out from the modified p25D plasmid. This fragment spans the B-domain deletion and includes the entire 3' two-thirds of the cDNA
  • An Ace I to Cla I fragment is removed from the retroviral vector JW-2 above, and replaced with the modified B-domain deleted fragment just described. This is designated B-del-1.
  • MdH culture supernatant is passed through a 0.45 ⁇ cellulose-acetate filter and transferred to a well of a 6-well plate containing 5.0 x 10* Mus dunni target cells in 2 ml standard culture medium containing polybrene.
  • supernatants are replaced with standard culture media containing 250 ⁇ g/ml of hygromycin B and subsequently replaced on days 2 and 5 with media containing 200 ⁇ g/ml of hygromycin B. Colonies resistant to hygromycin B appear and are visualized on day 9 post-selection, by staining with 0.2% Coomassie blue.
  • the intestinal epithelium is an attractive site for gene delivery due to its rapidly proliferating tissue mass and the known location of stem cells in the crypts of Lieberkuhn.
  • the deep location of the stem cells in the crypts and the protective role of the mucus gel layer makes the retrovirus relatively inaccessible to the tissue cells.
  • the accessibility of the retroviral vector to these stem cells can be improved in animal models by the in vivo mucus removal method of Sandberg, J, et ⁇ l,( Human Gene Therapy 5:3232-329, 1994).
  • the color reaction is performed with peroxidase-conjugated streptavidin (Boehringer Mannheim, Indianapohs, IN.) and o- phenylenediamine dihydrochloride as substrate.
  • peroxidase-conjugated streptavidin Boehringer Mannheim, Indianapohs, IN.
  • o- phenylenediamine dihydrochloride as substrate.
  • the human factor VDI:c standard from the National Institute for Biological Standards and Control, Hertfordshire, U.K.
  • normal rat plasma are used as references.
  • Lyophilized recombinant retrovirus containing the gene for Factor VDI expression is formulated into an enteric coated tablet or gel capsule according to known methods in the art. These are described in the following patents: US 4,853,230, EP 225,189, AU 9,224,296, AU 9,230,801, and WO 92/14452.
  • the capsule is administered orally to be targeted to the jejunum.
  • expression of Factor VID is measured in the plasma and blood by the Coatest R Factor VID assay as described in Example 2B1.
  • a 1.2 Kb Cla I Eco Rl 5' LTR and W fragment from N2 is subcloned into the same sites of an SK vector.
  • This vector is designated pN2CR5.
  • the 5' LTR containing a 6 bp deletion of the sphce donor sequence (Yee et al, Cold Spring Harbor, Quantitative Biology, 51:1021, 1986) is subcloned as a 1.8 Kb Eco Rl fragment into pUC31.
  • This vector is designated p31N25D[+], Figure 6.
  • HSV-1 thymidine kinase gene (HSVTK) are isolated as a 1.8 Kb Bgl D Pvu D fragment from plasmid 322TK (3.5 b Bam HI fragment of HSV-1 (McKnight etal.) cloned into Bam HI of pBR322 (ATCC No. 31344)) and cloned into Bgl D/Sma I-digested pUC31. This construct is designated pUCTK.
  • the plasmid pPrTKDA ( Figure 8), which contains the HSVTK promoter and coding sequence (lacking an (A) signal), is constructed as follows.
  • TK-1 Figure 9
  • Vector pDHF828 containing the fuU-length human growth hormone gene is constructed essentiaUy as foUows. Briefly, plasmid pDHF811, was constructed by removing the Xhol- Clal fragment of the KT-1 retroviral vector described above, and inserting the following oUgonucleotide Unkers by Ugation of the cohesive ends:
  • the linkers were annealed at 65°C for 20 minutes, 42°C for 20 minutes, 37°C for 20 minutes, and room temperature for 2 hours.
  • the concentrations of both oUgonucleotides was 18mM and the salt concentration was 100 mM NaCl.
  • 50ml of 1.8 mM annealed linker was digested with Clal overnight to generate Clal ends.
  • 3nM of KT-1 Xhol - Clal fragment was mixed with 90nM of linker, and the resultant mixture incubated at 15°C for 3 hours.
  • the Ugated DNA sample was transformed into DH-5a competent cells, foUowed by screening of transformants.
  • Plasmid chGH 800 containing the fuU length cDNA of the hGH gene (Martial, R.A. et al. Science 205:602, 1979) was digested with Hind ID, blunt-ended with the Klenow fragment enzyme, and cloned into the Srfl site of pDHF811.
  • the resultant plasmid was designated pDHF828 and can be introduced into an appropriate packaging ceU line to produce recombinant retroviral particles incorporating a chimeric integrase protein which confers position-specific integration into the targeted genome.
  • Crude and purified solutions of recombinant retrovirus particles may be separated on gradient polyacrylamide gels utilizing, for example, the PHASTGEL system (Pharmacia Biotech). Briefly, samples are place on 4-15% polyacrylamide gels without pretreatment and electrophoresed for 35 minutes at 250V. The gels are then removed and stained with coomassie blue in order to detect virus and other protein components. The gels are then scanned by laser densitometry in order to determine the content of virus and other components.
  • Virus bands may be identified by their relative molecular weight and by reverse transcriptase activity (RT).
  • RT reverse transcriptase activity
  • the purpose of this assay is to quantify the activity of reverse transcriptase (RT), an enzyme exclusively associated with all retroviruses.
  • the relative amount of retrovirus in a sample can be determined by measuring the activity of this enzyme in a given preparation.
  • Moloney murine leukemia virus reverse transcriptase (Pharmacia, Newark, NJ) is diluted to a concentration of 1 ⁇ g/ml by addition of lx Tris/EDTA buffer solution containing 10 mM Tris-HCl and ImM EDTA, pH 8.0.
  • Val Leu Leu Thr Thr Pro Thr Ala Leu Lys Val Asp Gly lie Ala Ala
  • Trp lie His Ala Ala His Val Lys Ala Ala Asp Pro Gly Gly Gly Pro

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Abstract

L'invention concerne des compositions et des procédés pour diriger un vecteur de recombinaison vers un site d'intégration spécifique, afin d'insérer un ou plusieurs gènes souhaités dans une région spécifique du génome d'une cellule eucaryote cible. Ces compositions et ces procédés sont utiles pour effectuer une thérapie génique sur des cellules somatiques ou des cellules germinales. Parmi les avantages d'une intégration dans un site spécifique, on peut citer des niveaux d'expression plus uniformes du ou des gènes souhaités et une diminution substantielle des risques de mutation inhérents aux insertions.
PCT/US1996/006727 1995-05-22 1996-05-10 Integration de vecteurs de recombinaison dans une position specifique du genome d'un eucaryote, assuree par une proteine chimere du type integrase WO1996037626A1 (fr)

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EP96915714A EP0830455A1 (fr) 1995-05-22 1996-05-10 Integration de vecteurs de recombinaison dans une position specifique du genome d'un eucaryote, assuree par une proteine chimere du type integrase
JP8535720A JPH11505128A (ja) 1995-05-22 1996-05-10 キメラインテグラーゼタンパク質に媒介される真核生物ゲノム中へのベクター構築物の位置特異的組み込み
AU57419/96A AU5741996A (en) 1995-05-22 1996-05-10 Position-specific integration of vector constructs into eukaryotic genomes mediated by a chimeric integrase protein

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US6139833A (en) * 1997-08-08 2000-10-31 Lexicon Genetics Incorporated Targeted gene discovery
WO2000075342A1 (fr) 1999-06-04 2000-12-14 Nippon Institute For Biological Science Nouveau vecteur plasmidique
US6776988B2 (en) 1998-03-27 2004-08-17 Lexicon Genetics Incorporated Vectors for gene mutagenesis and gene discovery
US6855545B1 (en) 1996-10-04 2005-02-15 Lexicon Genetics Inc. Indexed library of cells containing genomic modifications and methods of making and utilizing the same
EP1546322A4 (fr) * 2002-07-24 2006-07-19 Univ Vanderbilt Vecteurs et procedes d'integration d'acide nucleique bases sur des transposons
US7332338B2 (en) 1996-10-04 2008-02-19 Lexicon Pharmaceuticals, Inc. Vectors for making genomic modifications
WO2008020335A2 (fr) 2006-06-09 2008-02-21 Novartis Ag Compositions immunogènes pour streptococcus agalactiae
EP1935979A2 (fr) 1999-07-14 2008-06-25 Novartis Vaccines and Diagnostics S.r.l. Peptides de méningococcie antigène
EP2210945A2 (fr) 1998-01-14 2010-07-28 Novartis Vaccines and Diagnostics S.r.l. Antigènes de Neisseria meningitidis
EP2251424A1 (fr) 1999-05-19 2010-11-17 Novartis Vaccines and Diagnostics S.r.l. Peptides antigeniques de Neisseria
EP2261346A2 (fr) 1998-05-01 2010-12-15 Novartis Vaccines and Diagnostics, Inc. Antigènes de Neisseria meningitidis et compositions
EP2267005A1 (fr) 2003-04-09 2010-12-29 Novartis Vaccines and Diagnostics S.r.l. Toxine ADP-ribosylante de Listeria monocytogenes
EP2270175A1 (fr) 2001-03-27 2011-01-05 Novartis Vaccines and Diagnostics S.r.l. Acides nucléiques et protéines de Streptococcus pneumoniae
EP2275129A2 (fr) 2000-01-17 2011-01-19 Novartis Vaccines and Diagnostics S.r.l. Vaccin omv complèté contre la méningocoque
EP2275552A2 (fr) 1999-10-29 2011-01-19 Novartis Vaccines and Diagnostics S.r.l. Peptides antigéniques de Neisseria
EP2277895A1 (fr) 2000-10-27 2011-01-26 Novartis Vaccines and Diagnostics S.r.l. Acides nucléiques et protéines dérivés des groupes de streptocoques A et B
EP2278006A2 (fr) 1997-11-06 2011-01-26 Novartis Vaccines and Diagnostics S.r.l. Antigènes de Neisseria
EP2278007A1 (fr) 1999-04-30 2011-01-26 Novartis Vaccines and Diagnostics S.r.l. Antigènes conservés de Neisseria
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EP2335724A1 (fr) 2001-12-12 2011-06-22 Novartis Vaccines and Diagnostics S.r.l. Immunisation contre la Chlamydia trachomatis
WO2013178559A1 (fr) 2012-05-28 2013-12-05 Universitätsklinikum Hamburg-Eppendorf Procédé de détection de glycanes faisant appel à des polypeptides comprenant le domaine de reconnaissance des glucides des cd301
EP2792742A1 (fr) 2013-04-17 2014-10-22 Universitätsklinikum Hamburg-Eppendorf (UKE) Vecteurs de thérapie génique permettant de traiter la myocardiopathie
CN104975353A (zh) * 2015-06-08 2015-10-14 浙江大学 Hiv-1整合酶突变体库的构建法

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