WO1996017072A2 - Vecteurs d'alphavirus de recombinaison - Google Patents

Vecteurs d'alphavirus de recombinaison Download PDF

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WO1996017072A2
WO1996017072A2 PCT/US1995/015490 US9515490W WO9617072A2 WO 1996017072 A2 WO1996017072 A2 WO 1996017072A2 US 9515490 W US9515490 W US 9515490W WO 9617072 A2 WO9617072 A2 WO 9617072A2
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Prior art keywords
vector
alphavims
sequence
alphavirus
viral
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PCT/US1995/015490
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WO1996017072A3 (fr
WO1996017072A9 (fr
Inventor
Thomas W. Dubensky, Jr.
John M. Polo
Carlos E. Ibanez
Stephen M. W. Chang
Douglas J. Jolly
David A. Driver
Barbara A. Belli
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Chiron Viagene, Inc.
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Priority to AU45949/96A priority Critical patent/AU4594996A/en
Priority to EP95944045A priority patent/EP0797679A2/fr
Priority to JP8519023A priority patent/JPH11504802A/ja
Publication of WO1996017072A2 publication Critical patent/WO1996017072A2/fr
Publication of WO1996017072A9 publication Critical patent/WO1996017072A9/fr
Publication of WO1996017072A3 publication Critical patent/WO1996017072A3/fr

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    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
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    • 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
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
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    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
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    • C12N2840/206—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES having multiple IRES

Definitions

  • the present invention relates generally to use of recombinant viruses as vectors, and more specifically, to recombinant alphaviruses which are capable of expressing a heterologous sequence in target cells.
  • Alphaviruses comprise a set of ser ologically related arthropod-borne viruses of the Togavirus family. Briefly, alphaviruses are distributed worldwide, and persist in nature through a mosquito to vertebrate cycle. Birds, rodents, horses, primates, and humans are among the defined alpha virus vertebrate reservoir hosts.
  • HI test segregates the 26 alphaviruses into three major complexes: the Venezuelan encephalitis (VE) complex, the Semliki Forest (SF) complex, and the western encephalitis (WE) complex.
  • VE Venezuelan encephalitis
  • SF Semliki Forest
  • WE western encephalitis
  • EE eastern encephalitis
  • Barmah Forest Barmah Forest
  • Middelburg Ndumu
  • alphaviruses associated primarily with encephalitis and alphaviruses associated primarily with fever, rash, and polyarthritis. Included in the former group are the VE and WE complexes, and EE. In general, infection with this group can result in permanent sequelae, including behavior changes and learning disabilities, or death. In the latter group is the SF complex, comprised of the individual alphaviruses Chikungunya, O'nyong-nyong, Sindbis, Ross River, and Mayaro. With respect to this group, although serious epidemics have been reported, infection is in general self-limiting, without permanent sequelae.
  • Sindbis virus is the prototype member of the alphavirus genus of the Togavirus family. Although not usually apparent, clinical manifestations of Sindbis virus infection include fever, arthritis, and rash. Sindbis vims is distributed over Europe, Africa, Asia, and Australia, with the best epidemiological data coming from South Africa where 20% of the population is seropositive. (For a review, see Peters and Dalrymple, Fields Virology (2d ed). Fields et al. (eds ), B.N. Raven Press, New York, NY, chapter 26, pp. 713-762). Infectious Sindbis virus has been isolated from human serum only during an outbreak in Kenya and in a single case from Central Africa.
  • alphavirus genus The morphology and morphogenesis of the alphavirus genus is generally quite uniform.
  • the enveloped 60-65 nm particles infect most vertebrate cells, where productive infection is cytopathic.
  • infection of invertebrate cells for example, those derived from mosquitoes, does not result in any overt cytopathology.
  • alphaviruses are propagated in BHK-21 or vero cells, where growth is rapid, reaching a maximum yield within 24 hours of infection.
  • Field strains are usually isolated on primary avian embryo, for example chick, fibroblast cultures.
  • the genomic RNA (49S RNA) of alphaviruses is unsegmented, of positive polarity, approximately 1 1-12 kb in length, and contains a 5' cap and a 3' polyadenylated tail.
  • Infectious enveloped virus is produced by assembly of the viral nucleocapsid proteins onto viral genomic RNA in the cytoplasm, and budding through the cell membrane embedded with viral encoded glycoproteins. Entry of virus into cells occurs by endocytosis through clatherin-coated pits, fusion of the viral membrane with the endoso e, release of the nucleocapsid and uncoating of the viral genome.
  • the genomic 49S RNA serves as template for synthesis of the complementary negative strand.
  • the negative strand in turn serves as template for genomic RNA and for an internally initiated 26S subgenomic RNA.
  • the non-structural proteins are translated from the genomic RNA.
  • Alphaviral structural proteins are translated from the subgenomic 26S RNA. All viral genes are expressed as polyproteins and processed into individual proteins by proteolytic cleavage post translation.
  • recombinant alphavirus vectors to treat individuals requires that they be able to be transported and stored for long periods at a desired temperature, such that infectivity and viability of the recombinant virus is retained.
  • Current methods for storing recombinant viruses generally involve storage as liquids and at low temperatures. Such methods present problems in Third World countries, which typically do not have adequate refrigeration capabilities. For example, each year in Africa, millions of children die from infectious diseases such as measles. Vaccines necessary for the prevention of these diseases cannot be distributed to the majority of these countries because refrigeration is not readily accessible.
  • present viral formulations often contain media components that are not desirable for injection into patients. Consequently, there is a need in the art for a method of preserving purified recombinant viral vector (and in particular, alphavirus vectors) in a lyophilized form at elevated temperatures, and for this form to be suitable for injection into patients.
  • the present invention discloses recombinant alphavirus vectors which are suitable for use in a variety of applications, including for example, gene therapy, and further provides other related advantages.
  • alphavirus vector constructs comprising a 5' promoter which is capable of initiating the synthesis of viral RNA in vitro from cDNA, a 5' sequence which is capable of initiating transcription of an alphavirus, a nucleotide sequence encoding alphavirus non-structural proteins, a viral junction region which has been inactivated such that viral transcription of the subgenomic fragment is prevented, and an alphavirus RNA polymerase recognition sequence.
  • the viral junction region has been modified such that viral transcription of the subgenomic fragment is reduced.
  • alphavirus vector constructs comprising a 5' promoter which is capable of initiating the synthesis of viral RNA in vitro from cDNA, a 5' promoter which is capable of initiating the synthesis of viral RNA in vitro from cDNA 5* sequence which is capable of initiating transcription of an alphavirus, a nucleotide sequence encoding alphavirus non-structural proteins, a first viral junction region which has been inactivated such that viral transcription of the subgenomic fragment is prevented, a second viral junction region which has been modified such that viral transcription of the subgenomic fragment is reduced, and an alphavirus RNA polymerase recognition sequence.
  • alphavirus cDNA vector constructs comprising a 5' promoter which is capable of initiating the synthesis of viral RNA in vitro from cDNA, followed by a 5' sequence which is capable of initiating transcription of an alphavirus, a nucleotide sequence encoding alphavirus non-structural proteins, a viral junction region which has been inactivated such that viral transcription of the subgenomic fragment is prevented, an alphavirus RNA polymerase recognition sequence, and a 3* sequence which controls transcription termination.
  • alphavirus cDNA vector constructs comprising a 5' promoter which is capable of initiating the synthesis of viral RNA from cDNA followed by a 5' sequence which is capable of initiating transcription of an alphavirus, a nucleotide sequence encoding alphavirus non-structural proteins, a viral junction region which has been modified such that viral transcription of the subgenomic frag ent is reduced, an alphavirus RNA polymerase recognition sequence, and a 3' sequence which controls transcription termination.
  • alphavirus cDNA vector constructs comprising a promoter which is capable of initiating the synthesis of viral RNA from cDNA followed by a 5' sequence which is capable of initiating transcription of an alphavirus, a nucleotide sequence encoding alphavirus non-structural proteins, a first viral junction region which has been inactivated such that viral transcription of the subgenomic fragment is prevented, followed by a second viral junction region which has been modified such that viral transcription of the subgenomic fragment is reduced, an alphavirus RNA polymerase recognition sequence, and a 3* sequence which controls transcription termination.
  • eukaryotic layered vector initiation systems comprising a promoter which is capable of initiating the 5' synthesis of
  • RNA from cDNA a construct which is capable of autonomous replication in a cell, the construct being capable of expressing a heterologous nucleic acid sequence, and a 3' sequence which controls transcription termination.
  • eukaryotic layered vector initiation systems comprising a DNA promoter which is capable of initiating the 5' synthesis of RNA from cDNA, a construct which is capable of autonomous replication in a cell, the construct being capable of expressing a heterologous ribonucleic acid sequence, and a 3' DNA sequence which controls transcription termination.
  • the construct within the eukaryotic layered vector initiation systems of the present invention is an alphavirus vector construct.
  • the construct is derived from a virus selected from the group consisting of poliovirus, rhinovirus, coxsackieviruses, rubella, yellow fever, HCV, TGEV, IBV, MHV, BCV, parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus, influenza virus, RSV, MoMLV, HIV, HTLV, hepatitis delta virus and Astrovirus.
  • the promoter which is capable of initiating the 5' synthesis of RNA from cDNA is selected from the group consisting of the MoMLV promoter, metallothionein promoter, glucocorticoid promoter, SV40 promoter, and the CMV promoter.
  • the eukaryotic layered vector initiation systems further comprise a polyadenylation sequence.
  • the vector ⁇ e.g., alphavirus vector construct, alphavirus cDNA vector construct, or eukaryotic layered vector initiation system
  • the vectors described above contain a heterologous sequence.
  • such vectors contain a heterologous nucleotide sequence of greater than 100 bases, generally the heterologous nucleotide sequence is greater than 3 kb, and sometimes greater than 5 kb, or even 8 kb.
  • the heterologous sequence is a sequence encoding a protein selected from the group consisting of IL-1, IL-2, IL-3, IL-4, IL- 5, IL-6, IL-7, IL-8, IL-9, IL-10, IL- 1 1. IL-12, IL-13, IL-14, IL-15, and ⁇ -IFN, G-CSF, and GM-CSF.
  • the heterologous sequence may encode a lymphokine receptor. Representative examples of such receptors include receptors for any of the lymphokines set forth above.
  • the vectors described above include a selected heterologous sequence which may be obtained from a virus selected from the group consisting of influenza virus, HPV, HBV, HCV, EBV, HIV, HSV, FeLV, FIV, Hanta virus, HTLV I, HTLV II and CMV.
  • the heterologous sequence obtained from HPV encodes a protein selected from the group consisting of E5, E6, E7 and LI.
  • the vectors described above include a selected heterologous sequence encoding an HIV protein selected from the group consisting of HIV gpl20 and gag.
  • the selected heterologous sequences described above may also be an antisense sequence, noncoding sense sequence, or ribozyme sequence.
  • the antisense or noncoding sense sequence is selected from the group consisting of sequences which are complementary to influenza virus, HPV, HBV, HCV, EBV, HIV, HSV, FeLV, FIV, Hanta virus, HTLV I, HTLV II, and CMV sequences.
  • the vectors described above contain no alphavirus structural protein genes.
  • the selected heterologous sequence is located downstream from a viral junction region.
  • the selected heterologous sequence may, within certain embodiments, be located downstream from the second viral junction region.
  • the vector construct may further comprise a polylinker located subsequent to the viral junction region.
  • polylinkers do not contain a wild-type alphavirus virus restriction endonuclease recognition sequence.
  • the selected heterologous sequence may be located within the nucleotide sequence encoding alphavirus non-structural proteins.
  • the vectors described above include a viral junction region consisting of the nucleotide sequence as shown in Figure 3, from nucleotide number 7579, to nucleotide number 7597 (SEQ. ID NO. 1).
  • an E3 adenovirus gene may be located downstream from the second viral junction region.
  • Vectors of the present invention may also further comprise a retroviral packaging sequence located between the first viral junction region and the second viral junction region.
  • the present invention provides an isolated recombinant alphavirus vector which does not contain a functional viral junction region, and which in preferred embodiments produces reduced viral transcription of the subgenomic fragment.
  • the present invention provides an alphavirus structural protein expression cassette, comprising a promoter and one or more alphavirus structural protein genes, the promoter being capable of directing the expression of alphavirus structural proteins.
  • the expression cassette is capable of expressing alphavirus structural proteins, such as an alphavirus structural protein selected from the group consisting of C, 6K, E3, E2, and El .
  • the present invention provides an alphavirus structural protein expression cassette, comprising a promoter, one or more alphavirus structural proteins, and a heterologous ligand sequence, the promoter being capable of directing the expression of the alphavirus structural proteins and the heterologous sequence.
  • the heterologous ligand sequence is selected from the group consisting of VSVG, HIV g ⁇ l20, antibody, insulin, and CD4.
  • the expression cassettes described above include a promoter selected from the group consisting of metallothionein, Drosophila actin 5C distal, SV40, heat shock protein 65, heat shock protein 70, Py, RSV, BK, JC, MuLV, MMTV, alphavirus junction region, CMV and VAIRNA.
  • the alphavirus structural protein is derived from an alphavirus selected from the group consisting of Aura, Fort Morgan, Venezuelan Equine Encephalitis, Ross River, Semliki Forest, Sindbis and Mayaro viruses.
  • the present invention provides alphavirus particles which, upon introduction into a BHK cell, produces an infected cell which is viable at least 24 hours and as much as 48, 72, 96 or I week after infection. Also provided are mammalian cells which contain such alphavirus particles.
  • the present invention provides recombinant alphavirus particles which, upon introduction into a BHK cell, produces an infected cell which is viable at least 24 hours after infection, the particle also carrying a vector construct which directs the expression of at least one antigen or modified form thereof in target cells infected with the alphavirus particle, the antigen or modified form thereof being capable of stimulating an immune response within an animal.
  • the expressed antigen or modified form thereof elicits a cell-mediated immune response, preferably an HLA class I- restricted immune response.
  • the present invention provides recombinant alphavirus particles which carry a vector capable of directing the expression of a palliative in cells infected with the alphavirus particle, the palliative being capable of inhibiting a function of a pathogenic agent necessary for pathogenicity.
  • the pathogenic agent is a virus, fungi, protozoa, or bacteria
  • the inhibited function is selected from the group consisting of adsorption, replication, gene expression, assembly, and exit of the pathogenic agent from infected cells.
  • the pathogenic agent is a cancerous cell, cancer- promoting growth factor, autoimmune disorder, cardiovascular disorders such as restenosis, osteoporosis and male pattern baldness, and the inhibited function is selected from the group consisting of cell viability and cell replication.
  • the vector directs the expression of a toxic palliative in infected target cells in response to the presence in such cells of an entity associated with the pathogenic agent; preferably the palliative is capable of selectively inhibiting the expression of a pathogenic gene or inhibiting the activity of a protein produced by the pathogenic agent.
  • the palliative comprises an inhibiting peptide specific for viral protease, an antisense RNA complementary to RNA sequences necessary for pathogenicity, a sense RNA complementary to RNA sequences necessary for pathogenicity, or a defective structural protein of a pathogenic agent, such protein being capable of inhibiting assembly of the pathogenic agent.
  • alphavirus particles described above directing the expression of a palliative more particularly, directs the expression of a gene product capable of activating an otherwise inactive precursor into an active inhibitor of the pathogenic agent, for example, the herpes thymidine kinase gene product, a tumor suppressor gene, or a protein that activates a compound with little or no cytotoxicity into a toxic product in the presence of a pathogenic agent, thereby effecting localized therapy to the pathogenic agent.
  • a gene product capable of activating an otherwise inactive precursor into an active inhibitor of the pathogenic agent
  • the herpes thymidine kinase gene product for example, the herpes thymidine kinase gene product, a tumor suppressor gene, or a protein that activates a compound with little or no cytotoxicity into a toxic product in the presence of a pathogenic agent, thereby effecting localized therapy to the pathogenic agent.
  • the alphavirus particle directs the expression of a protein that is toxic upon processing or modification by a protein derived from a pathogenic agent, a reporting product on the surface of target cells infected with the alphavirus and containing the pathogenic agent, or an RNA molecule which functions as an antisense or ribozyme specific for a pathogenic RNA molecule required for pathogens.
  • the protein in the alphavirus particles described above, is herpes thymidine kinase or CD4.
  • the present invention provides alphavirus particles which direct the expression of a gene capable of suppressing one or more elements of the immune system in target cells infected with the alphavirus, and an alphavirus particle which directs the expression of a blocking element in cells infected with the alphavirus, the blocking element being capable of binding to either a receptor or an agent such that the receptor/agent interaction is blocked.
  • methods are provided for administering any of the above-described alphavirus particles or vectors, for a prophylactic or therapeutic effect.
  • the present invention provides methods of stimulating an immune response to an antigen, comprising the step of infecting susceptible target cells with an alphavirus particle which directs the expression of at least one antigen or modified form thereof in target cells infected with the alphavirus, the antigen or modified form thereof being capable of stimulating an immune response within an animal.
  • the target cells are infected in vivo, although within other embodiments the target cells are removed, infected ex vivo, and returned to the animal.
  • methods of stimulating an immune response to a pathogenic antigen comprising the step of infecting susceptible target cells with an alphavirus particle which directs the expression of a modified form of a pathogenic antigen in target cells infected with the alphavirus, the modified antigen being capable of stimulating an immune response within an animal but having reduced pathogenicity relative to the pathogenic antigen.
  • methods of stimulating an immune response to an antigen comprising infecting susceptible target cells with a alphavirus particle which directs the expression of a peptide having multiple epitopes, one or more of the epitopes derived from different proteins.
  • methods of stimulating an immune response within a warm-blooded animal comprising infecting susceptible target cells associated with a warm-blooded animal with nucleic acid sequences coding for either individual class I or class II MHC protein, or combinations thereof, and infecting the cells with an alphavirus particle which directs the expression of at least one antigen or modified form thereof in target cells infected with the alphavirus particle, the antigen or modified form thereof being capable of stimulating an immune response within the animal.
  • methods of inhibiting a pathogenic agent comprising infecting susceptible target cells with an alphavirus particle which directs the expression of a palliative in cells infected with the alphavirus particle, the palliative being capable of inhibiting a function of a pathogenic agent necessary for pathogenicity.
  • vector or vector constructs which direct the expression of a heterologous sequence of interest in fact refers to transcribed vector RNA directs the expression of the heterologous sequence of interest.
  • mammals are generally referred to, it should be understood that the present invention may be readily applied to a wide variety of animals (both mammalian and non-mammalian), including for example, humans, chimps, macaques, cows, horses, sheep, dogs, birds, cats, fish, rats, and mice.
  • alphaviruses such as Sindbis may be specifically described herein, it should be understood that a wide variety of other alphaviruses may also be utilized including, for example.
  • methods for delivering a heterologous nucleic acid sequence to an animal comprising the steps of administering to the warm-blooded animal a eukaryotic layered vector initiation system as described above.
  • the eukaryotic layered vector initiation system may be introduced into the target cells directly as a DNA molecule by physical means, as a complex with various liposome formulations, or as a DNA ligand complex including the vector molecule ⁇ e.g., along with a polycation compound such as polylysine, a receptor specific ligand, or a psoralen inactivated virus such as Sendai or Adenovirus).
  • a polycation compound such as polylysine, a receptor specific ligand, or a psoralen inactivated virus such as Sendai or Adenovirus.
  • the present invention also provides packaging cell lines and producer cell lines suitable for producing recombinant alphavirus particles.
  • packaging or producer cell lines may be either mammalian or non-mammalian ⁇ e.g., insect cells such as mosquito cells).
  • packaging cell lines are provided which, upon introduction of a vector construct, produce alphavirus particles capable of infecting human cells.
  • the packaging cell line produces alphavirus particles in response to one or more factors.
  • alphavirus inhibitory protein is not produced within the packaging cell line.
  • retroviral-derived packaging cell lines are provided which are suitable for packaging and production of an alphavirus vector.
  • a retroviral-derived producer cell line suitable for packaging and production of an alphavirus vector comprising an expression cassette which directs the expression of gag/pol, an expression cassette which directs the expression of em, and alphavirus cDNA vector construct containing a retroviral packaging sequence.
  • a VSV-G derived packaging cell suitable for packaging and production of an alphavirus vector, comprising a stably integrated expression cassette which directs the expression of VSV-G.
  • packaging cell lines comprise a stably integrated expression cassette which directs the expression of one or more alphavirus structural proteins.
  • producer cell lines are provided based upon the above packaging cell lines.
  • such producer cells produce alphavirus particles in response to a differentiation state of the producer cell line.
  • alphavirus producer cell line refers to a cell line which is capable of producing recombinant alphavirus particles.
  • the producer cell line should include an integrated alphavirus structural protein expression cassette capable of directing the expression of alphavirus structural protein(s), and also, an alphavirus vector construct.
  • the alphavirus vector construct is a cDNA vector construct. More preferably, the alphavirus vector construct is an integrated cDNA vector construct.
  • the alphavirus vector construct may, in some instances, function only in response to one or more factors, or the differentiation state of the alphavirus producer cell line.
  • ex vivo cells are infected with any of the above-described recombinant alphaviruses are provided.
  • recombinant alphavirus particles are provided which are resistant to inactivation in serum.
  • recombinant alphavirus particles are considered to be resistant to inactivation in serum if the ratio of surviving particles to input/starting particles in a complement inactivation assay is greater in a statistically significant manner, preferably at least 5-fold, and as much as 10- to 20-fold, as compared to a reference sample produced in BHK cells.
  • pharmaceutical compositions are provided comprising any of the above-described vectors, or recombinant alphavirus particles, in combination with a physiologically acceptable carrier or diluent.
  • Figure 1 is a schematic illustration of Sindbis virus genomic organization.
  • Figure 2 is an illustration which depicts a method for amplification of a Sindbis RNA genome by RT-PCR.
  • Figures 3A-H set forth the sequence of a representative Eukaryotic Layered Vector
  • Figure 4 is a schematic illustration of a Sindbis Basic Vector and a Sindbis-luciferase Vector.
  • Figure 5 is an illustration of Sindbis Helper Vector Construction.
  • Figure 6 is a graph which illustrates expression and rescue of a Sindbis-luciferase
  • Figure 7 is an illustration of one method for modifying a Sindbis junction region.
  • Figure 8 is a schematic illustration of a representative embodiment of a Eukaryotic Layered Vector Initiation System.
  • Figure 9 is a graph which shows a time course for luciferase expression from ELVIS-
  • Figure 10 is a bar graph which depicts the level of vector reporter gene expression for several different vector constructs.
  • Figure 1 1 is a schematic illustration of Sindbis Packaging Expression Cassettes.
  • Figure 12 is a bar graph which shows SIN-luc vector packaging by representative packaging cell lines.
  • Figure 13 is a bar graph which shows SIN-luc vector packaging by PCL clone #18 over time.
  • Figure 14 is a bar graph which depicts the level of expression by several different luciferase vectors in BHK cells and undifferentiated F9 cells.
  • Figure 15 is a schematic illustration of how Astroviruses or other heterologous viruses may be used to express Sindbis structural proteins.
  • Figure 16A is a bar graph which shows Sindbis BV-HBe expression and packaging in
  • Figure 16B is a bar graph which shows Sindbis BV-HBe expression and packaging in BHK cells (supernatant).
  • Figure 17 is a bar graph which shows Sindbis BV-HB core expression and packaging in BHK cells.
  • Figure 18 is a bar graph which shows a comparison of HB core expressed from
  • Figure 19 is a bar graph which shows ELVIS-HBe vector expression in BHK cells.
  • Figure 20A-D is a schematic illustration of several representative mechanisms for activating a disabled viral junction region by "RNA loop-out.”
  • Alphavirus vector construct refers to an assembly which is capable of directing the expression of a sequence(s) or gene(s) of interest.
  • the vector construct should include a 5' sequence which is capable of initiating transcription of an alphavirus, as well as sequence(s) which, when expressed, code for biologically active alphavirus non-structural proteins ⁇ e.g.,
  • NSP1, NSP2, NSP3, and NSP4 an alphavirus RNA polymerase recognition sequence.
  • the vector construct should include a viral junction region which may, in certain embodiments, be modified in order to prevent, increase, or reduce viral transcription of the subgenomic fragment and an alphavirus RNA polymerase recognition sequence.
  • the vector may also include nucleic acid molecule(s) which are of a size sufficient to allow production of viable virus, a 5* promoter which is capable of initiating the synthesis of viral RNA in vitro from cDNA, as well as one or more restriction sites, and a polyadenylation sequence.
  • Alphavirus cDNA vector construct refers to an assembly which is capable of directing the expression of a sequence(s) or gene(s) of interest.
  • the vector construct should include a 5' sequence which is capable of initiating transcription of an alphavirus, as well as sequence(s) which, when expressed, code for biologically active alphavirus non-structural proteins ⁇ e.g., NSP1, NSP2, NSP3, and NSP4), and an alphavirus RNA polymerase recognition sequence.
  • the vector construct should include a 5* promoter which is capable of initiating the synthesis of viral RNA from cDNA, a viral junction region which may, in certain embodiments, be modified in order to prevent, increase, or reduce viral transcription of the subgenomic fragment, an alphavirus RNA polymerase recognition sequence, and a 3' sequence which controls transcription termination.
  • the vector may also include nucleic acid molecule(s) which are of a size sufficient to allow production of viable virus, splice recognition sequences, a catalytic ribozyme processing sequence, as well as a polyadenylation sequence.
  • "Expression cassette” refers to a recombinantly produced molecule which is capable of expressing alphavirus structural protein(s).
  • the expression cassette must include a promoter and a sequence encoding alphavirus structural protein(s).
  • the expression cassette may include transcription termination, splice recognition, and polyadenylation addition sites.
  • Preferred promoters include the CMV and adenovirus VAIRNA promoters.
  • the expression cassette may contain selectable markers such as Neo, SV2 Neo, hygromycin, phleomycin, histidinol, and DHFR.
  • Recombinant alphavirus particle refers to a capsid which contains an alphavirus vector construct.
  • the alphavirus capsid is contained within a lipid bilayer, such as a cell membrane, in which viral encoded proteins are embedded.
  • a variety of vectors may be contained within the alphavirus particle, including the alphavirus vector constructs of the present invention.
  • the present invention provides alphavirus vector constructs, alphavirus particles containing such constructs, as well as methods for utilizing such vector constructs and particles.
  • sequences encoding wild-type alphavirus suitable for use in preparing the above-described vector constructs and particles may be readily obtained given the disclosure provided herein from naturally-occurring sources, or from depositories (e.g., the American Type Culture Collection, Rockville, Maryland).
  • suitable alphaviruses include Aura (ATCC VR-368),
  • Bebaru virus (ATCC VR-600, ATCC VR-1240), Cabassou (ATCC VR-922), Chikungunya virus (ATCC VR-64, ATCC VR-1241), Eastern equine encephalomyelitis virus (ATCC VR- 65, ATCC VR-1242), Fort Morgan (ATCC VR-924), Getah virus (ATCC VR-369, ATCC VR-1243), Kyzylagach (ATCC VR-927), Mayaro (ATCC VR-66), Mayaro virus (ATCC VR-1277), Middleburg (ATCC VR-370), Mucambo virus (ATCC VR-580, ATCC VR- 1244), Ndumu (ATCC VR-371), Pixuna virus (ATCC VR-372, ATCC VR-1245), Ross River virus (ATCC VR-373, ATCC VR-1246), Semliki Forest (ATCC VR-67, ATCC VR- 1247), Sindbis virus (ATCC VR-68, ATCC VR-1248), Tonate (ATCC VR-925), Triniti (
  • the sequences which encode wild-type alphavirus may be obtained from the Sindbis virus.
  • a Sindbis cDNA clone may be obtained by linking the 5' end of a Sindbis virus cDNA clone to a bacteriophage RNA polymerase promoter, and the 3' end of the cDNA clone to a poly- adenosine (poly A) tract of at least 25 nucleotides.
  • synthesis of the first cDNA strand from the viral RNA template may be accomplished with a 3' oligonucleotide primer having a consecutive sequence comprising an enzyme recognition sequence, a sequence of 25 deoxythymidine nucleotides, and a stretch of approximately 18 nucleotides which is complementary to the viral 3' end and with a 5' primer containing buffer nucleotides, an enzyme recognition sequence, a bacteriophage promoter, and a sequence complimentary to the viral 5' end.
  • the enzyme recognition sites present on each of these primers should be different from each other, and not found in the Sindbis virus.
  • the first nucleotide linked to the 3' end of the bacteriophage RNA polymerase promoter may be the authentic first nucleotide of the RNA virus, or may contain one or more additional non-viral nucleotides.
  • RNA transcribed in vitro from the viral cDNA clone, having the construction described above and linearized by digestion with the unique dT:dA 3' distal restriction enzyme will, after introduction into the appropriate eukaryotic cell, initiate the same infection cycle which is characteristic of infection by the wild-type virus from which the cDNA was cloned.
  • This viral cDNA clone which yields RNA able to initiate infection after in vitro transcription, is referred to below as an "infectious cDNA clone.”
  • alphavirus vector constructs of the present invention comprising a 5' sequence which is capable of initiating transcription of an alphavirus, a nucleotide sequence encoding alphavirus non-structural proteins, a viral junction region which has been inactivated such that viral transcription of the subgenomic fragment is prevented, and an alphavirus RNA polymerase recognition sequence.
  • alphavirus vector constructs which have inactivated viral junction regions do not transcribe the subgenomic fragment, making them suitable for a wide variety of applications.
  • alphavirus vector constructs which contain a 5' promoter which is capable of initiating the synthesis of viral RNA in vitro from cDNA.
  • preferred 5' promoters include both eukaryotic and prokaryotic promoters, such as, for example, the ⁇ -galactosidase promoter, trpE promoter, lacZ promoter, T7 promoter, T3 promoter, SP6 promoter, SV40 promoter, CMV promoter, and MoMLV LTR.
  • the alphavirus vector constructs of the present invention contain a 5' sequence which is capable of initiating transcription of an alphavirus.
  • Representative examples of such sequences include nucleotides 1-60 of the wild- type Sindbis virus ⁇ see Figure 3), nucleotides 10-75 for tRNA Asparagine (Schlesinger et al., U.S. Patent No. 5,091,309), and 5' sequences from other Togaviruses which initiate transcription.
  • Alphavirus vector constructs of the present invention should also contain sequences which encode Alphavirus Non-Structural Proteins (NSP).
  • NSP Alphavirus Non-Structural Proteins
  • NSP1-NSP3 Sindbis non-structural proteins 1 through 3
  • NSP4 is, within one embodiment, encoded by nucleotides 5928 to 7579 ⁇ see Figure 3).
  • non-structural protein derivatives may be made, including for example, various substitutions, insertions, or deletions, the net result of which do not alter the biological activity of the alphavirus non-structural proteins.
  • alphavirus non-structural proteins are deemed to be "biologically active" in toto if they promote the self-replication of the vector construct.
  • Self-replication which refers to replication of viral nucleic acids and not the production of infectious virus, may be readily determined by RNase protection assays performed over a course of time. Methods for making such derivatives may be readily accomplished by one of ordinary skill in the art given the disclosure provided herein ⁇ see also, Molecular Cloning: A Laboratory Manual (2d. ed.), Cold Spring Harbor Laboratory Press).
  • the alphavirus vector constructs also include a viral junction region which has been inactivated, such that viral transcription of the subgenomic fragment is prevented.
  • the alphavirus viral junction region normally controls transcription initiation of the subgenomic fragment.
  • the normal viral junction region typically begins at approximately nucleotide number 7579 and continues up through at least nucleotide number 7612 (and possibly beyond).
  • nucleotides 7579 to 7602 (5'- ATC TCT ACG GTG GTC CTA AAT AGT - SEQ. ID NO. 2) are believed necessary for transcription of the subgenomic fragment.
  • This region (nucleotides 7579 to 7602) is hereinafter referred to as the "minimal junction region core.”
  • the viral junction region is inactivated in order to prevent viral transcription of the subgenomic fragment.
  • inactivated means that the fragment corresponding to the initiation point of the subgenomic fragment, as measured by a RNase protection assay, is not detected. (Representative assays are described by Melton et al., Nuc. Acids Res. 72:7035-7056, 1984; Calzon et al.. Methods in E . J 52:611-632, 1987; and Kekule et al.. Nature 573:457-461, 1990.)
  • the viral junction region is inactivated by truncating the viral junction region at nucleotide 7597 ⁇ i.e., the viral junction region will then consist of the sequence as shown in Figure 3, from nucleotide 7579 to nucleotide 7597). This truncation prevents transcription of the subgenomic fragment, and additionally permits synthesis of the complete NSP4 region (which is encoded by nucleotides 5928 to 7579).
  • deletions, substitutions or insertions may also be made in order to inactivate the viral junction region.
  • the viral junction region may be further truncated into the region which encodes NSP4, thereby preventing viral transcription from the subgenomic fragment while retaining the biological activity of NSP4.
  • nucleotide substitutions may be made in the sequence encoding NSP4, the net effect of which does not alter the biological activity of NSP4 yet, nevertheless, prevents transcription of the subgenomic fragment. 5.
  • ALPHA VIRI IS RNA PC ILYMERASE REC( JNITK IN SEQI ⁇ ENCE. AND POLY-A TAIL
  • alphavirus vector constructs of the present invention should also include an alphavirus RNA polymerase recognition sequence (also termed "alphavirus replicase recognition sequence").
  • the alphavirus RNA polymerase recognition sequence provides a recognition site at which the virus begins replication by synthesis of the negative strand.
  • a wide variety of sequences may be utilized as an alphavirus RNA polymerase recognition sequence.
  • Sindbis vector constructs of the present invention include a Sindbis polymerase recognition sequence which is encoded by nucleotides 1 1,647 to 1 1,703 ⁇ see Figure 3).
  • the Sindbis polymerase recognition is truncated to the smallest region which can still function as a recognition sequence ⁇ e.g., nucleotides 1 1,684 to 1 1,703 of Figure 3).
  • the vector construct may additionally contain a poly-A tail.
  • the poly-A tail may be of any size which is sufficient to promote stability in the cytoplasm, thereby increasing the efficiency of initiating the viral life cycle.
  • the poly-A tail comprises at least 10 adenosine nucleotides, and most preferably, at least 25 adenosine nucleotides.
  • alphavirus vector constructs which are generally described above, a wide variety of other alphavirus vector constructs may also be prepared utilizing the disclosure provided herein.
  • modified viral junction regions should be understood to include junction regions which have wild-type activity, but a non-wild-type sequence, as well as junction regions with increased, decreased, or no activity.
  • alphavirus vector constructs are provided wherein the viral junction region has been modified, such that viral transcription of the subgenomic fragment is reduced. Briefly, infection of cells with wild- type alphavirus normally results in cell death as a result of abundant viral transcription of the subgenomic fragment initiated from the viral junction region.
  • RNA molecules can overwhelm the transcriptional machinery of the infected cell, ultimately resulting in death of the cell.
  • a therapeutic effect e.g., strand scission of a target nucleic acid or prolonged expression of a heterologous protein
  • modifications to the alphavirus vector construct may be made in order to reduce the level of viral transcription of the subgenomic fragment, and thereby prolong the life of the vector infected target cell.
  • viral transcription of the subgenomic fragment is considered to be "reduced” if it produces less subgenomic fragment than a standard wild-type alphavirus ⁇ e.g., Sindbis virus ATCC No. VR-1248) as determined by a RNase protection assay.
  • a standard wild-type alphavirus e.g., Sindbis virus ATCC No. VR-1248
  • Viral junction regions may be modified by a variety of methods in order to reduce the level of viral transcription of the subgenomic fragment. For example, within one embodiment of the invention, due to the redundancy of the genetic code nucleotide substitutions may be made in the viral junction region 7579 to 7597, the net effect of which does not alter the amino acid sequence NSP4 (or, within other embodiments, the biological activity of NSP4), and yet reduces the level of viral transcription of the subgenomic fragment. If the modified vector construct includes nucleotides beyond 7597 ⁇ e.g., to 7602 or 7612), further nucleotide substitutions may likewise be made, although, since NSP4 terminates at 7597, such substitutions need not be based upon genetic redundancy. Representative examples of modified viral junction regions are described in more detail below in Example 3.
  • alphavirus vector constructs which comprise a 5' sequence which is capable of initiating transcription of an alphavirus, a nucleotide sequence encoding alphavirus non-structural proteins, a first viral junction region which has been inactivated such that viral transcription of the subgenomic fragment is prevented, a second viral junction region which has been modified such that viral transcription of the subgenomic fragment is reduced, and an alphavirus RNA polymerase recognition sequence.
  • tandem vector constructs are referred to as "tandem" vector constructs because they comprise a first inactivated (or “disabled”) viral junction region, as well as a second modified (or “synthetic") viral junction region.
  • the inactivated junction region is followed directly by the second modified viral junction region.
  • a minimal junction region core may be inserted downstream in tandem to the inactivated junction region.
  • sequences corresponding to the entire junction region may be added to the in-tandem junction region, in increments.
  • an adenovirus E3 gene is inserted into a tandem vector construct following the second viral junction region, in order to down- regulate HLA expression in alphavirus infected cells.
  • repeated inoculations of a gene therapeutic into the same individual is desirable.
  • repeated inoculations of alphaviruses such as the Sindbis virus may lead to the development of specific antibodies or cell-mediated immune response against Sindbis viral non-structural protein (NSP).
  • NSP Sindbis viral non-structural protein
  • products of the Adenovirus type 2 early region gene 3 are utilized in order to down-regulate the expression of integral histocompatibility antigens expressed on the surface of infected cells.
  • the E3 19,000 dalton (E3/19K) protein binds to, and forms a molecular complex with, class I H-2/HLA antigens in the endoplasmic reticulum, preventing terminal glycosylation pathways necessary for the full maturation and subsequent transport of the class I H-2/HLA antigens to the cell membrane.
  • target cells infected with an alphavirus vector encoding the Ad 2 E3 protein co-expression of the viral non-structural proteins in the context of class I antigens will not occur.
  • the Human Cytomegalovirus (“HCMV”) H301 gene is cloned into an alphavirus vector construct, preferably immediately following the second viral junction region in a tandem vector, in order to inhibit host CTL response directed against viral specific proteins expressed in vector infected cells.
  • HCMV Human Cytomegalovirus
  • 2-Microglobulin (2m) protein binds to the 1, 2 and 3 domains of the a-chain of the class I major histocompatibility molecules of higher eukaryotes. Preventing the interaction between 2m and MHC class I products renders infected cells unrecognizable by cytotoxic T cells. Therefore, as described in greater detail below in Example 4B, expression of the HCMV H30I gene product as a component of a therapeutic palliative may be utilized in order to mitigate the host immune response to viral NSP.
  • a retroviral packaging sequence is inserted into a tandem vector and positioned between the first (inactivated) viral junction region and the second, modified viral junction region.
  • retroviral packaging sequences signal the packaging of an RNA genome into a retroviral particle.
  • a retroviral packaging sequence may be utilized in order to package an alphavirus vector into a retroviral particle using a retroviral packaging cell line. This is performed in order to increase the efficiency of alphavirus vector transfer into an alphavirus packaging cell line.
  • the genomic length and subgenomic length of mRNAs transcribed in wild-type alphavirus infected cells are polycistronic, coding for, respectively, the viral four non- structural proteins (NSPs) and four structural proteins (SPs).
  • NSPs non- structural proteins
  • SPs structural proteins
  • the genomic and subgenomic mRNAs are translated as polyproteins, and processing into the individual non-structural and structural proteins is accomplished by post translational proteolytic cleavage, catalyzed by viral encoded NSP- and SP- specific proteases.
  • the expression of more than one heterologous gene is desired.
  • multiple administrations of alphavirus vectors or particles may be required, since duration of the therapeutic palliative may be limited. Therefore, with certain embodiments of the invention it may be desirable to co-express in a target cell the Ad 2 E3 gene (.see Example 4), along with a therapeutic palliative, such as the glucocerebrosidase gene ⁇ see Example 17).
  • the structural protein (“SP") polycistronic message is translated into a single polyprotein which is subsequently processed into individual proteins by cleavage with SP encoded proteases.
  • alphavirus vectors may be constructed by placing appropriate signals either ribosome readthrough or internal ribosome entry between cistrons.
  • the placement of signals promoting either ribosome readthrough or internal ribosome entry immediately downstream of the disabled junction region vector pKSSINBVdlJR is described ⁇ see Examples 3 and 5).
  • synthesis of subgenomic message cannot occur; however, the heterologous proteins are expressed from genomic length mRNA by either ribosomal readthrough (scanning) or internal ribosome entry. Relative to wild-type, the low level of viral transcription with this alphavirus vector would prolong the life of the infected target cell.
  • placement of signals promoting either ribosome readthrough or internal ribosome entry immediately downstream of the pKSSINBVdlJRsjr or pKSSINBV vectors is described. Briefly, since synthesis of subgenomic mRNA occurs in cells infected with the pKSSINBVdlJRsjr and pKSSINBV vectors, placement of either a ribosome readthrough sequence or an internal ribosome entry sequence between the two heterologous genes permits translation of both proteins encoded by the subgenomic mRNA polycistronic message.
  • heterologous genes can be placed in the subgenomic mRNA region, provided that a suitable translation initiation signal resides at the 5' end of the translational AUG start codon.
  • the number of heterologous gene(s) which can be inserted into the subgenomic mRNA region, as described here, is limited only by the packaging constraints of the vector. Different sequences which allow either ribosome readthrough, cap-independent translation, or internal ribosome entry may be placed into Sindbis vectors pKSSINBVdlJR, pKSSINBV, or pKSSINBVdlJRsjrc in the configurations as discussed above.
  • the source of these translation control sequences are the picornaviruses polio and EMCV, the 5' noncoding region of the human immunoglobulin heavy-chain binding protein, and a synthetic sequence of at least 15 bps corresponding in part to the Kozak consensus sequence for efficient translational initiation.
  • these signals which affect translation initiation can also be placed downstream of the junction region and between heterologous genes in all of the modified junction region vectors described in Example 3.
  • the alphavirus cDNA vector construct also includes a 3' sequence which controls transcription termination.
  • a representative example of such a sequence is set forth in more detail below in Examples 2 and 3.
  • alphavirus vector constructs are provided which are capable of expressing a desired heterologous sequence only in a selected tissue.
  • Figure 20 One such representative example is shown in Figure 20.
  • Figure 20A a recombinant alphavirus vector is constructed such that upon introduction of the vector ( Figure 20A) into a target cell, internal inverted repeat sequences which flank the transcriptional control regions ⁇ e.g., modified junction region) loop out ⁇ see Figure 20B), thereby preventing viral transcription of subgenomic sequences ("G.O.I. ”) from the synthetic junction region.
  • activation of the vector can be attained if the inverted repeats are designed to also hybridize to a specific cellular RNA sequence which is characteristic of a selected tissue or cell type.
  • cellular RNA disrupts the disabling stem loop structure, thereby allowing the formation of a more stable secondary stem loop structure ( Figures 20C and 20D).
  • This secondary stem loop structure allows transcription of the sub-genomic message by placing the junction region back into its correct positional configuration.
  • Full-length alphavirus vectors can also be transcribed using the secondary stem loop structure by taking advantage of the ability of the viral polymerase to switch templates during synthesis of the negative strand using a strand hopping mechanism termed copy choice (King, RNA genetics II, CRC Press, Inc., Boca Raton Fla., Domingo et al. (ed.), pp. 150-185, 1988).
  • copy choice a strand hopping mechanism termed copy choice
  • the resulting RNA transcript does not contain inverted repeats because they are deleted as a result of the polymerase copy choice event.
  • This newly synthesized RNA molecule now functions as the primary RNA vector transcript which will transcribe and express as any other non-disabled genomic alphavirus vector previously described.
  • tissue or cell-specific activation of the disabled Sindbis vector can be achieved if specific RNA sequences, present only in the targeted cell or tissue types, are used in the design of the inverted repeats.
  • alphaviruses such as Sindbis can be engineered to be tissue- specific expression vectors using similar inverted sequences described above.
  • tumor-specific markers such as the carcinoembryonic tumor specific antigen (CEA) and the alpha- fetoprotein tumor marker.
  • CEA carcinoembryonic tumor specific antigen
  • utilizing such tumor-specific RNA to target specific tumors allows for the tumor-specific expression of toxic molecules, lymphokines or pro- drugs discussed below.
  • Such methods may be utilized for a wide variety of tumors, including for example, colorectal, lung, breast, ovary, bladder and prostate cancers because all these tumors express the CEA.
  • One representative illustration of vectors suitable for use within this aspect of the present invention is set forth in more detail below in Example 16.
  • CEA was one of the first tumor-specific markers to be described, along with the alpha-fetoprotein tumor marker.
  • CEA is a normal glycoprotein in the embryonic tissue of the gut, pancreas and liver during the first two trimesters of fetal development ⁇ Pathologic Basis of Disease, 3rd edition 1984, Robbins et al. (eds.)).
  • CEA was believed to be specific for adenocarcinomas of the colon, however, with the subsequent development of more sensitive radioimmunoassays it became apparent that CEA was presented in the plasma with many endodermally derived cancers, particularly pancreatic, gastric and broncogenic.
  • alphavirus cell-specific expression vectors may be constructed to express viral antigens, ribozyme, antisense sequences or immunostimulatory factors such as gamma-interferon ( ⁇ -IFN), IL-2 or IL-5 for the targeted treatment of virus infected cell types.
  • ⁇ -IFN gamma-interferon
  • inverted repeats of the alphavirus vector may be selected to hybridize to any pathogen-specific RNA, for instance target cells infected by pathogens such as HIV, CMV, HBV, HPV and HSV.
  • pathogens such as HIV, CMV, HBV, HPV and HSV.
  • specific organ tissues may be targeted for the treatment of tissue-specific metabolic diseases utilizing gene replacement therapies.
  • liver is an important target tissue because it is responsible for many of the body's metabolic functions and is associated with many metabolic genetic disorders.
  • diseases include many of the glycogen storage diseases, phenylketonuria, Gaucher's disease and familial hypercholesterolemia.
  • liver-specific enzymes and markers which have been sequenced which may be used to engineer appropriate inverted repeats for alphavirus vectors.
  • liver-specific cDNAs include sequences encoding for S- adenosylmethione synthetase (Horikawa et al., Biochem. Int. 25:81, 1991); lecithin: cholesterolacyl transferase (Rogne et al., Biochem. Biophys. Res. Commun.
  • liver-specific alphavirus vector could be used to deliver the low density lipoprotein receptor (Yamamoto et al., Cell 39:27, 1984) to liver cells for the treatment of familial hypercholesterolemia (Wilson et al., Mol. Biol. Med. 7:223, 1990).
  • nucleotide sequences may be carried by the alphavirus vector constructs of the present invention.
  • the nucleotide sequences should be of a size sufficient to allow production of viable virus.
  • the production of any measurable titer for example, by plaque assay, luciferase assay, or ⁇ -galactosidase assay, of infectious virus on appropriate susceptible monolayers, is considered to be "production of viable virus.”
  • This may be, at a minimum, an alphavirus vector construct which does not contain any additional heterologous sequence.
  • the vector construct may contain additional heterologous or foreign sequences.
  • the heterologous sequence will comprise a heterologous sequence of at least about 100 bases, 2 kb, 3.5 kb, 5 kb, 7 kb, or even a heterologous sequence of at least about 8 kb.
  • the efficiency of packaging and hence, viral titer is to some degree dependent upon the size of the sequence to be packaged.
  • additional non-coding sequences may be added to the vector construct.
  • heterologous sequences may be included in the vector construct, including for example sequences which encode palliatives such as lymphokines, toxins, prodrugs, antigens which stimulate an immune response, ribozymes, and proteins which assist or inhibit an immune response, as well as antisense sequences (or sense sequences for "antisense applications").
  • palliatives such as lymphokines, toxins, prodrugs, antigens which stimulate an immune response, ribozymes, and proteins which assist or inhibit an immune response
  • antisense sequences or sense sequences for "antisense applications”
  • the alphavirus vector constructs provided herein may contain (and express, within certain embodiments) two or more heterologous sequences.
  • the heterologous sequence encodes a lymphokine.
  • lymphokines act to proliferate, activate, or differentiate immune effectors cells.
  • Representative examples of lymphokines include gamma interferon, tumor necrosis factor, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, GM-CSF, CSF-1 and G-CSF.
  • the heterologous sequence encodes an immunomodulatory cofactor.
  • immunomodulatory cofactor refers to factors which, when manufactured by one or more of the cells involved in an immune response, or 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 cofactor.
  • 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 5l Cr release) ⁇ see Wamer et al., AIDS Res. and Human Retroviruses 7:645-655, 1991).
  • in vitro assays which measure cellular proliferation ⁇ e.g., 3 H thymidine uptake
  • in vitro cytotoxic assays e.g., which measure 5l Cr release
  • immunomodulatory co-factors include alpha interferon (Finter et al.. Drugs 42 ⁇ 5): 749-765, 1991; U.S. Patent No. 4,892,743; U.S. Patent No. 4,966,843; WO 85/02862; Nagata et al.. Nature 284:316-320, 1980; Familletti et al., Methods in Era. 7 ⁇ .387-394, 1981 ; Twu et al., Proc. Natl. Acad. Sci. USA 86:2046-2050, 1989; Faktor et al., Oncogene 5:867-872, 1990), beta interferon (Seif et al., J. Virol.
  • GM- CSF WO 85/04188
  • TNFs Jayaraman et al., J. Immunology 144:942-951, 1990
  • Interleukin- 2 IL-2
  • IL-4 Interleukin- 2
  • ICAM-1 Altman et al., Nature 338:512- 514, 1989
  • ICAM-2 LFA-1
  • LFA-3 MHC class I molecules
  • MHC class II molecules MHC class II molecules
  • ⁇ 2 -microglobulin chaperones
  • CD3, B7/BB1 MHC linked transporter proteins or analogues thereof.
  • Peripheral blood lymphocytes are restimulated in vitro with autologous or HLA-matched cells ⁇ e.g., EBV transformed cells), and transduced with an alphavirus vector construct which directs the expression of an immunogenic portion of a hepatitis antigen and the immunomodulatory cofactor.
  • Stimulated PBLs are used as effectors in a CTL assay with the HLA-matched transduced cells as targets.
  • the immunomodulatory cofactor gamma interferon is particularly preferred.
  • Another example of an immunomodulatory cofactor is the B7/BB1 costimulatory factor.
  • 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 is delivered to the T cell by antigen-presenting cells.
  • the second signal is required for interleukin-2 (IL-2) production by T cells and appears to involve interaction of the B7/BB 1 molecule on antigen-presenting cells with CD28 and CTLA-4 receptors on T lymphocytes (Linsley et al., J. Exp. Med., 775:721-730, 1991a, and J.
  • B7/BB1 may be introduced into tumor cells in order to cause costimulation of CD8 + T cells, such that the CD8 + 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/BB1 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/BB 1.
  • the heterologous sequence encodes a toxin.
  • toxins act to directly inhibit the growth of a cell.
  • Representative examples of toxins include ricin (Lamb et al., Eur. J. Biochem. 148:265-270, 1985), abrin (Wood et al., Eur. J. Biochem. 198:723-732, 1991; Evensen et al., J. of Biol. Chem. 266:6848-6852, 1991; Collins et al., J. of Biol. Chem. 265:8665-8669, 1990; Chen et al., Fed. of Eur. Biochem Soc.
  • the heterologous sequence encodes a "pro-drugs".
  • pro-drugs refers to a gene product that activates a compound with little or no cytotoxicity into a toxic product.
  • Representative examples of such gene products include HSVTK and VZVTK (as well as analogues and derivatives thereof), which selectively monophosphorylate certain purine arabinosides and substituted pyrimidine compounds, converting them to cytotoxic or cytostatic metabolites.
  • ganciclovir acyclovir, or any of their analogues ⁇ e.g., FIAU, FIAC, DHPG
  • HSVTK phosphorylates the drug into its corresponding active nucleotide triphosphate form.
  • pro-drugs which may be utilized within the context of the present invention include: E. coli guanine phosphoribosyl transferase which converts thioxanthine into toxic thioxanthine monophosphate (Besnard et al., Mol. Cell. Biol.
  • alkaline phosphatase which will convert inactive phosphorylated compounds such as mitomycin phosphate and doxorubicin-phosphate to toxic dephosphorylated compounds
  • fungal ⁇ e.g., Fusarhtm oxysporum or bacterial cytosine deaminase, which will convert 5-fluorocytosine to the toxic compound 5-fluorouracil (Mullen, PNAS 89:33, 1992)
  • 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 ⁇ see generally, Vrudhula et al., J. of Med. Chem. 56(7): 919-923, 1993; Kern et
  • the heterologous sequence is 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 HER2 Coussens et al., Science 230: 1 132-1 139, 1985
  • antisense ABL Ferinstein et al., Oncogene 7: 1477-1481, 1989
  • antisense Myc Stanton et al.. Nature 570:423-425, 1984
  • antisense ras as well as antisense sequences which block any of the enzymes in the nucleotide biosynthetic pathway.
  • antisense sequences to interferon and 2 microglobulin may be utilized in order to decrease immune response.
  • antisense RNA may be utilized as an anti-tumor agent in order to induce a potent Class I restricted response.
  • high levels of specific antisense sequences are believed to induce the increased expression of interferons (including gamma-interferon) due to the formation of large quantities of double- stranded RNA.
  • the increased expression of gamma interferon boosts the expression of MHC Class I antigens.
  • Preferred antisense sequences for use in this regard include actin RNA, myosin RNA, and histone RNA. Antisense RNA which forms a mismatch with actin RNA is particularly preferred.
  • alphavirus vectors are provided which produce ribozymes upon infection of a host cell.
  • ribozymes are used to cleave specific RNAs and are designed such that it can only affect one specific RNA sequence.
  • the substrate binding sequence of a ribozyme is between 10 and 20 nucleotides long. The length of this sequence is sufficient to allow a hybridization with target RNA and disassociation of the ribozyme from the cleaved RNA.
  • Representative examples for creating ribozymes include those described in U.S. Patent Nos. 5, 1 16,742; 5,225,337 and 5,246,921.
  • Particularly preferred ribozymes for use within the present invention include those disclosed in more detail below in the Examples ⁇ e.g.. Examples 18 and 19).
  • proteins or other cellular constituents may be carried by the alphavirus vector construct.
  • Representative examples of such proteins include native or altered cellular components, as well as foreign proteins or cellular constituents, found in for example, viruses, bacteria, parasites or fungus.
  • alphavirus vector constructs which direct the expression of an immunogenic, non-tumorigenic, altered cellular component.
  • immunogenic refers to altered cellular components which are capable, under the appropriate conditions, of causing an immune response. This response must be cell-mediated, and may also include a humoral response.
  • non-tumorigenic refers to altered cellular components which will not cause cellular transformation or induce tumor formation in nude mice.
  • altered cellular component refers to proteins and other cellular constituents which are either associated with rendering a cell tumorigenic, or are associated with tumorigenic cells in general, but are not required or essential for rendering the cell tumorigenic.
  • the cellular components may be essential to normal cell growth and regulation and include, for example, proteins which regulate intracellular protein degradation, transcriptional regulation, cell-cycle control, and cell-cell interaction. After alteration, the cellular components no longer perform their regulatory functions and, hence, the cell may experience uncontrolled growth.
  • altered cellular components include ras*, p53*, Rb*, altered protein encoded by the Wilms' tumor gene, ubiquitin*, mucin*, protein encoded by the DCC, APC, and MCC genes, the breast cancer gene BRCAI *, as well as receptors or receptor-like structures such as neu, thyroid hormone receptor, platelet derived growth factor (PDGF) receptor, insulin receptor, epidermal growth factor (EGF) receptor, and the colony stimulating factor (CSF) receptor.
  • PDGF platelet derived growth factor
  • EGF epidermal growth factor
  • CSF colony stimulating factor
  • alphavirus vector constructs which direct the expression of a non-tumorigenic, altered ras (ras*) gene.
  • ras* a non-tumorigenic, altered ras
  • the ras* gene is an attractive target because it is causally linked to the neoplastic phenotype, and indeed may be necessary for the induction and maintenance of tumorigenesis in a wide variety of distinct cancers, such as pancreatic carcinoma, colon carcinoma and lung adenocarcinoma.
  • ras* genes are found in pre-neoplastic tumors and, therefore, immune intervention therapy may be applied prior to detection of a malignant tumor.
  • Normal ras genes are non-tumorigenic and ubiquitous in all mammals.
  • the normal ras protein is a G-protein which binds GTP and has GTPase activity, and is involved in transmitting signals from the external milieu to the inside of the cell, thereby allowing a cell to respond to its environment.
  • Ras* genes on the other hand alter the normal growth regulation of neoplastic cells by uncoupling cellular behavior from the environment, thus leading to the uncontrolled proliferation of neoplastic cells. Mutation of the ras gene is believed to be an early event in carcinogenesis (Kumar et al., Science 248.1 101 - 1104, 1990) which, if treated early, may prevent tumorigenesis.
  • Ras* genes occur in a wide variety of cancers, including for example, pancreatic, colon, and lung adenocarcinomas.
  • Table 1 summarizes known in vivo mutations (codons 12, 13 and 61) which activate human ras, as well as potential mutations which have in vitro transforming activity. Potential mutations with in vitro transforming activity were produced by the systematic substitution. of amino acids for the normal codon ⁇ e.g., other amino acids were substituted for the normal glycine at position 12). In vitro mutations, while not presently known to occur in humans or animals, may serve as the basis for an anti-cancer immunotherapeutic if they are eventually found to arise / " // vivo.
  • novel proteins encoded by these sequence(s) may be used as a marker of tumorigenic cells, and an immune response directed against these novel coding regions may be utilized to destroy tumorigenic cells containing the altered sequences (ras*).
  • alphavirus vector constructs which direct the expression of an altered p53 (p53*) gene.
  • p53 is a nuclear phosphoprotein which was originally discovered in extracts of transformed cells and thus was initially classified as an oncogene (Linzer and Levine, Cell 77:43-52, 1979; Lane and Crawford, Nature 278.261-263, 1979). It was later discovered that the original p53 cDNA clones were mutant forms of p53 ⁇ Hinds et al., J. Virol. 65:739-746, 1989). It now appears that p53 is a tumor suppressor gene which negatively regulates the cell cycle, and that mutation of this gene may lead to tumor formation. Of colon carcinomas that have been studied, 75%-80% show a loss of both p53 alleles, one through deletion and the other through point mutation. Similar mutations are found in lung cancer, and in brain and breast tumors.
  • Certain alterations of the p53 gene may be due to certain specific toxins.
  • Bressac et al. ⁇ Nature 350:429-431, 1991 describes specific G to T mutations in codon 249 in patients affected with hepatocellular carcinoma.
  • One suggested causative agent of this mutation is aflatoxin B j , a liver carcinogen which is known to be a food contaminant in Africa.
  • a sequence encoding the altered cellular component has been obtained, it is necessary to ensure that the sequence encodes a non-tumorigenic protein.
  • Various assays are known and may easily be accomplished which assess the tumorigenicity of a particular cellular component. Representative assays include a rat fibroblast assay, tumor formation in nude mice or rats, colony formation in soft agar, and preparation of transgenic animals, such as transgenic mice.
  • Tumor formation in nude mice or rats is a particularly important and sensitive method for determining the tumorigenicity of a particular cellular component.
  • Nude mice lack a functional cellular immune system ⁇ i.e., do not possess CTLs), and therefore provide a useful in vivo model in which to test the tumorigenic potential of cells.
  • Normal non-tumorigenic cells do not display uncontrolled growth properties if infected into nude mice. However, transformed cells will rapidly proliferate and generate tumors in nude mice.
  • the alphavirus vector construct is administered to syngeneic murine cells, followed by injection into nude mice. The mice are visually examined for a period of 2 to 8 weeks after injection in order to determine tumor growth.
  • mice may also be sacrificed and autopsied in order to determine whether tumors are present.
  • Giovanella et al. J. Natl. Cancer Inst. 75: 1531-1533, 1972; Furesz et al.. Abnormal Cells, New Products and Risk, Hopps and Petricciani (eds.). Tissue Culture Association, 1985; and Levenbook et al., J. Biol. Std. 73: 135-141, 1985.
  • Tumorigenicity may also be assessed by visualizing colony formation in soft agar (Macpherson and Montagnier, Vir. 23:291-294, 1964). Briefly, one property of normal non- tumorigenic cells is "contact inhibition" ⁇ i.e., cells will stop proliferating when they touch neighboring cells). If cells are plated in a semi-solid agar support medium, normal cells rapidly become contact inhibited and stop proliferating, whereas tumorigenic cells will continue to proliferate and form colonies in soft agar.
  • Transgenic animals such as transgenic mice, may also be utilized to assess the tumorigenicity of an altered cellular component.
  • the gene of interest may be expressed in all tissues of the animal. This dysregulated expression of the transgene may serve as a model for the tumorigenic potential of the newly introduced gene.
  • 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 downstream of the immunogenic region. Insertion of termination codons will prematurely terminate protein expression, thus preventing expression of the transforming portion of the protein.
  • the ras* gene is truncated in order to render the ras* protein non-tumorigenic.
  • the carboxy-terminal amino acids of ras* functionally allow the protein to attach to the cell membrane. Truncation of these sequences renders the altered cellular component non-tumorigenic.
  • the ras* gene is truncated in the purine ring binding site, for example around the sequence which encodes amino acid number 110.
  • the ras* gene sequence may be truncated such that as little as about 20 amino acids (including the altered amino acid(s)) are encoded by the alphavirus vector construct, although preferably, as many amino acids as possible should be expressed (while maintaining non- tumorigenicity).
  • the p53* protein is modified by truncation in order to render the cellular component non-tumorigenic. As noted above, not all mutations of the p53 protein are tumorigenic, and therefore, not all mutations would have to be truncated.
  • p53* is truncated to a sequence which encodes amino acids 100 to 300, thereby including all four major "hot spots.”
  • Other altered cellular components which are oncogenic may also be truncated in order to render them non-tumorigenic.
  • both neu and bcr/abl may be truncated in order to render them non-tumorigenic.
  • Non-tumorigenicity may be confirmed by assaying the truncated altered cellular component as described above.
  • altered cellular component is only associated with non-tumorigenic cells in general, and is not required or essential for making the cell tumorigenic, then it is not necessary to render the cellular component non-tumorigenic.
  • altered cellular components which are not tumorigenic include Rb*, ubiquitin*, and mucin*.
  • the altered cellular component in order to generate an appropriate immune response, the altered cellular component must also be immunogenic. Immunogenicity of a particular sequence is often difficult to predict, although T cell epitopes often possess an immunogenic amphipathic alpha-helix component. In general, however, it is preferable to determine immunogenicity in an assay.
  • Representative assays include an ELISA, which detects the presence of antibodies against the newly introduced vector, as well as assays which test for T helper cells such as gamma-interferon assays, IL-2 production assays, and proliferation assays.
  • the present invention may be co-expressed in order to form a general anti-cancer therapeutic.
  • this therapeutic may be targeted to a particular type of cancer.
  • An alphavirus vector construct which co-expresses a number of these altered cellular components may be administered to a patient with colon cancer in order to treat all possible mutations. This methodology may also be utilized to treat other cancers.
  • an alphavirus vector construct which co-expresses mucin*, ras*, neu, BRCA1* and p53* may be utilized to treat breast cancer.
  • alphavirus vector constructs which direct the expression of immunogenic portions of antigens from foreign organisms or other pathogens.
  • antigens include bacterial antigens ⁇ e.g., E.
  • influenza virus Human Immmunodeficiency Virus
  • HAV Human Immmunodeficiency Virus
  • HAV Hepatitis A, B and C Virus
  • HPV Human Papiloma Virus
  • EBV Epstein-Barr Virus
  • HSV Herpes Simplex Virus
  • HMV Hantavirus
  • TTLV I TTLV I
  • HTLV II Cytomegalovirus
  • CMV Cytomegalovirus
  • immunological portion refers to a portion of the respective antigen which is capable, under the appropriate conditions, of causing an immune response ⁇ i.e., cell-mediated or humoral).
  • "Portions” may be of variable size, but are preferably at least 9 amino acids long, and may include the entire antigen.
  • Cell-mediated immune responses may be mediated through Major Histocompatability Complex (“MHC”) class I presentation, MHC Class II presentation, or both.
  • MHC Major Histocompatability Complex
  • alphavirus vector constructs which direct the expression of immunogenic portions of Hepatitis B antigens.
  • the Hepatitis B genome is comprised of circular DNA of about 3.2 kilobases in length and has been well characterized (Tiollais et al., .Science 275:406-41 1, 1981; Tiollais et al.. Nature 577:489-495, 1985; and Ganem and Varmus, Ann. Rev. Biochem. 56:651-693, 1987; see also EP 0 278,940, EP 0 241,021, WO 88/10301, and U.S. Patent Nos. 4,696,898 and 5,024,938, which are hereby incorporated by reference).
  • the Hepatitis B virus presents several different antigens, including among others, three HB “S” antigens (HBsAgs), an HBc antigen (HBcAg), an HBe antigen (HBeAg), and an HBx antigen (HBxAg) ⁇ see Blum et al., TIG 5(5): 154- 158, 1 89).
  • HBeAg results from proteolytic cleavage of P22 pre- core intermediate and is secreted from the cell.
  • HBeAg is found in serum as a 17 kD protein.
  • the HBcAg is a protein of 183 amino acids
  • the HBxAg is a protein of 145 to 154 amino acids, depending on subtype.
  • the HBsAgs (designated “large,” “middle” and “small”) are encoded by three regions of the Hepatitis B genome: S, pre-S2 and pre-Sl .
  • the large protein which has a length varying from 389 to 400 amino acids, is encoded by pre-Sl, pre-S2, and S regions, and is found in glycosylated and non-glycosylated forms.
  • the middle protein is 281 amino acids long and is encoded by the pre-S2 and S regions.
  • the small protein is 226 amino acids long and is encoded by the S region. It exists in two forms, glycosylated (GP 27 s ) and non- glycosylated (P24 s ). If each of these regions are expressed separately, the pre-Sl region will code for a protein of approximately 1 19 amino acids, the pre-S2 region will code for a protein of approximately 55 amino acids, and the S region will code for a protein of approximately 226 amino acids.
  • the immunological variability is due to single nucleotide substitutions in two areas of the hepatitis B virus S open reading frame, resulting in the following amino acid changes: (1) exchange of lysine- 122 to arginine in the Hepatitis B virus S open reading frame causes a subtype shift from d to y, 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 adw 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 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 livers.
  • the polymerase protein consists of at least two domains: the amino terminal domain which 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 determined utilizing methods described herein ⁇ e.g., below and in Examples 12Aii and 13), utilizing alphavirus vector constructs described below, and administered in order to generate an immune response within a warm-blooded animal.
  • HBV antigens such as ORF 5 and ORF 6 (Miller et al., Hepalology 9:322-327, 1989) may be expressed utilizing alphavirus vector constructs as described herein.
  • ORF 5 and ORF 6 Representative examples of alphavirus vector constructs utilizing ORF 5 and ORF 6 are set forth below in the examples.
  • At least one immunogenic portion of a hepatitis B antigen is inco ⁇ orated into an alphavirus vector construct.
  • the immunogenic portion(s) which are inco ⁇ orated into the alphavirus vector construct may be of varying length, although it is generally preferred that the portions be at least 9 amino acids long and may include the entire antigen.
  • Immunogenicity of a particular sequence is often difficult to predict, although T cell epitopes may be predicted utilizing computer algorithms such as TSITES (Medlmmune, Maryland), in order to scan coding regions for potential T-helper sites and CTL sites. From this analysis, peptides are synthesized and used as targets in an in vitro cytotoxic assay.
  • ELISA which detects the presence of antibodies against the newly introduced vector
  • assays which test for T helper cells such as gamma-interferon assays, IL-2 production assays and proliferation assays.
  • Immunogenic portions may also be selected by other methods.
  • the HLA A2.1 transgenic mouse has been shown to be useful as a model for human T-cell recognition of viral antigens. Briefly, in the influenza and hepatitis B viral systems, the murine T cell receptor repertoire recognizes the same antigenic determinants recognized by human T cells. In both systems, the CTL response generated in the HLA A2.1 transgenic mouse is directed toward virtually the same epitope as those recognized by human CTLs of the HLA A2.1 haplotype (Vitiello et al., J. Exp. Med. 775: 1007-1015, 1991; Vitiello et al., Abstract of Molecular Biology of Hepatitis B Virus Symposia, 1992). Particularly preferred immunogenic portions for incorporation into alphavirus vector constructs include HBeAg, HBcAg and HBsAgs, as described in greater detail below in Example 10.
  • Additional immunogenic portions of the hepatitis B virus may be obtained by truncating the coding sequence at various locations including, for example, the following sites: Bst UI, Ssp I, Ppu M l , and Msp I (Valenzuela et al., Nature 250:815-19, 1979; Valenzuela et al.. Animal Virus Genetics: ICWUCLA Sy p. Mol. Cell Biol., 1980, B. N. Fields and R. Jaenisch (eds ), pp. 57-70, New York: Academic). Further methods for determining suitable immunogenic portions as well as methods are also described below in the context of hepatitis C.
  • an immunogenic portion may be inco ⁇ orated into the alphavirus vector construct.
  • an alphavirus vector construct may express (either separately or as one construct) all or immunogenic portions of HBcAg, HBeAg, HBsAgs, HBxAg, as well as immunogenic portions of HCV antigens.
  • Sequences which encode the above-described proteins may be readily obtained from a variety of sources, including for example, depositories such as the American Type Culture Collection (ATCC, Rockville, MD), or from commercial sources such as British Bio- Technology Limited (Cowley, Oxford, England).
  • Representative examples include BBG 12 (containing the GM-CSF gene coding for the mature protein of 127 amino acids); BBG 6 (which contains sequences encoding gamma interferon), ATCC No. 39656 (which contains sequences encoding TNF), ATCC No. 20663 (which contain sequences encoding alpha interferon), ATCC Nos.
  • Sequences which encode altered cellular components as described above may be readily 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, MD), or from commercial sources such as Advanced Biotechnologies (Columbia, Maryland).
  • ATCC American Type Culture Collection
  • ATCC No. 41000 containing a G to T mutation in the 12th codon of ras
  • ATCC No. 41049 containing a G to A mutation in the 12th codon
  • plasmids which encode normal cellular components may also be obtained from depositories such as the ATCC ⁇ see, for example, ATCC No.
  • Sequences which encode the above-described viral antigens may likewise be obtained from a variety of sources.
  • molecularly cloned genomes which encode the hepatitis B virus may be obtained from sources such as the American Type Culture Collection (ATCC, Rockville, MD).
  • ATCC No. 45020 contains the total genomic DNA of hepatitis B (extracted from purified Dane particles) (.see Figure 3 of Blum et al., 77G 5(5): 154- 158, 1989) in the Bam HI site of pBR322 (Moriarty et al., Proc. Natl. Acad. Sci. USA 75:2606-2610, 1981 ).
  • cDNA sequences which encode the above-described heterologous sequences 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 oligonucleotide 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 PCR 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, dATP, dCTP, dGTP and dTTP. Double-stranded DNA is produced when synthesis is complete. This cycle may be repeated many times, resulting in a factorial amplification of the desired DNA. Sequences which encode the above-described proteins may also be synthesized, for example, on an Applied Biosystems Inc. DNA synthesizer ⁇ e.g., APB DNA synthesizer model 392 (Foster City, CA)).
  • DNA vectors (referred to as "Eukaryotic Layered Vector Initiation Systems") which are capable of directing the synthesis of viral RNA /// vivo.
  • eukaryotic layered vector initiation systems comprising a promoter which is capable of initiating the 5' synthesis of RNA from cDNA, a construct which is capable of autonomous replication in a cell, the construct also being capable of expressing a heterologous nucleic acid sequence, and a 3' sequence which controls transcription termination.
  • eukaryotic layered vector initiation systems provide a two-stage or "layered" mechanism which controls expression of heterologous nucleotide sequences.
  • the first layer initiates transcription of the second layer, and comprises a promoter which is capable of initiating the 5' synthesis of RNA from cDNA ⁇ e.g., a 5' promoter), a 3' transcription termination site, as well as one or more splice sites and/or a polyadenylation site, if desired.
  • a promoter which is capable of initiating the 5' synthesis of RNA from cDNA ⁇ e.g., a 5' promoter
  • a 3' transcription termination site as well as one or more splice sites and/or a polyadenylation site, if desired.
  • promoters suitable for use within the present invention include both eukaryotic ⁇ e.g., pol I, II, or III) and prokaryotic promoters, and inducible or non-inducible ⁇ i.e., constitutive) promoters, such as, for example, Murine Leukemia virus promoters (e.g., MoMLV), metallothionein promoters, the glucocorticoid promoter, Drosophila actin 5C distal promoter, SV 40 promoter, heat shock protein 65 promoter, heat shock protein 70 promoter, immunoglobulin promoters.
  • Murine Leukemia virus promoters e.g., MoMLV
  • metallothionein promoters metallothionein promoters
  • the glucocorticoid promoter e.g., Drosophila actin 5C distal promoter
  • SV 40 promoter e.g., heat shock protein 65 promoter
  • heat shock protein 70 promoter e
  • the second layer comprises a construct which is capable of expressing one or more heterologous nucleotide sequences, and of replication in a cell either autonomously or in response to one or more factors.
  • the second layer construct may be an alphavirus vector construct as described above.
  • vector systems may be utilized as the first layer of the eukaryotic layered vector initiation system, including for example, viral vector constructs developed from DNA viruses such as those classified in the Poxviridae, including for example canary pox virus or vaccinia virus (e.g., Fisher-Hoch et al., PNAS 56:317-321, 1989; Flexner et al., Ann. NY. Acad. Sci. 569:86-103, 1989; Flexner et al., Vaccine 5: 17-21, 1990; U.S. Patent Nos.
  • DNA viruses such as those classified in the Poxviridae
  • canary pox virus or vaccinia virus e.g., Fisher-Hoch et al., PNAS 56:317-321, 1989; Flexner et al., Ann. NY. Acad. Sci. 569:86-103, 1989; Flexner et al., Vaccine 5: 17-21, 1990; U.S. Patent Nos.
  • Papoviridae such as BKV, JCV or SV40 ⁇ e.g., Mulligan et al., Nature 277: 108-1 14, 1979); Adenoviridae such as adenovirus ⁇ e.g., Berkner, Biotechni ⁇ ies 6:616-627, 1988; Rosenfeld et al., Science 252:431-434, 1991); Parvoviridae such as adeno-associated virus ⁇ e.g., Samulski et al., J. Vir. 65:3822- 3828, 1989; Mendelson et al., Virol.
  • Retroviridae include leukemia in viruses such as MoMLV and immunodeficiency viruses such as HIV, e.g., Poznansky, J.
  • vector systems may be utilized as second layer of the eukaryotic layered vector initiation system, including for example, vector systems derived from viruses from the families: Picornaviridae (e.g., poliovirus, rhinovirus, coxsackieviruses), Caliciviridae, Togaviridae ⁇ e.g.
  • Picornaviridae e.g., poliovirus, rhinovirus, coxsackieviruses
  • Caliciviridae e.g., Togaviridae ⁇ e.
  • alphavirus, rubella Flaviviridae ⁇ e.g., yellow fever
  • Coronaviridae ⁇ e.g., HCV, TGEV, IBV, MHV, BCV
  • Rhabdoviridae Filoviridae
  • Paramyxoviridae ⁇ e.g., parainfluenza virus, mumps virus, measles virus, and respiratory syncytial virus
  • Orthomyxoviridae ⁇ e.g., influenza virus
  • Bunyaviridae Arenaviridae
  • Retroviridae ⁇ e.g., RSV, MoMLV, HIV, HTLV
  • Astrovirus hepatitis delta virus and Astrovirus.
  • RNA viruses may also be utilized, including for example, bacterial and bacteriophage replicases, as well as components derived from plant viruses such as Topamoviruses and Bromoviruses ⁇ see Strauss et al. Micro. Rev. 55:491-562, 1994).
  • the replication competency of the autocatalytic vector construct contained within the second layer of the eukaryotic vector initiation system, may be measured by a variety of assays known to one of skill in the art including, for example, ribonuclease protection assays which measure increases in both positive-sense and negative-sense RNA over time, in transfected cells, in the presence of an inhibitor of cellular RNA synthesis, such as dactinomycin, and assays which measure expression of a heterologous reporter gene in transfected cells.
  • assays known to one of skill in the art including, for example, ribonuclease protection assays which measure increases in both positive-sense and negative-sense RNA over time, in transfected cells, in the presence of an inhibitor of cellular RNA synthesis, such as dactinomycin, and assays which measure expression of a heterologous reporter gene in transfected cells.
  • eukaryotic layered vector initiation systems comprise a 5' promoter which is capable of initiating the synthesis of viral RNA from cDNA, followed by a 5' sequence which is capable of initiating transcription of an alphavirus, a nucleotide sequence encoding alphavirus non-structural proteins, a viral junction region which is either active or which has been inactivated such that viral transcription of the subgenomic fragment is prevented, an alphavirus RNA polymerase recognition sequence, and a 3' sequence which controls transcription termination.
  • the viral junction region may be modified, such that viral transcription of the subgenomic fragment is merely reduced, rather than inactivated.
  • a second viral junction region may be inserted following the first inactivated viral junction region, the second viral junction region being modified such that viral transcription of the subgenomic fragment is reduced.
  • the resulting alphavirus RNA vector molecule is comprised of a 5' sequence which is capable of initiating transcription of an alphavirus, a nucleotide sequence encoding alphavirus non-structural proteins, a viral junction region, a heterologous nucleotide sequence, an alphavirus RNA polymerase recognition sequence, and a polyadenylate sequence.
  • alphavirus cDNA vector constructs including the 5' sequence which is capable of initiating transcription of an alphavirus, the nucleotide sequence encoding alphavirus non-structural proteins, the viral junction region which has been inactivated such that viral transcription of the subgenomic fragment is prevented, and the alphavirus RNA polymerase recognition sequence.
  • modified junction regions and tandem junction regions have also been discussed above.
  • methods for delivering a heterologous nucleotide sequence to a warm-blooded animal, comprising the step of administering a eukaryotic layered vector initiation system as described above, to a warm ⁇ blooded animal.
  • Eukaryotic layered vector initiation systems may be administered to warm ⁇ blooded animals either directly ⁇ e.g., intravenously, intramuscularly, intraperitoneally, subcutaneously, orally, rectally, intraocularly, intranasally), or by various physical methods such as lipofection (Feigner et al., Proc. Natl. Acad. Sci.
  • eukaryotic layered vector initiation systems may either be administered directly ⁇ i.e., in vivo), or to cells which have been removed ⁇ ex vivo), and subsequently returned.
  • Eukaryotic layered vector initiation systems may be administered to a warm-blooded animal for any of the therapeutic uses described herein, including for example, for the pu ⁇ ose of stimulating a specific immune response; inhibiting the interaction of an agent with a host cell receptor; to express a toxic palliative, including for example, conditional toxic palliatives; to immunologically regulate the immune system; to express markers, and for replacement gene therapy.
  • a toxic palliative including for example, conditional toxic palliatives
  • immunologically regulate the immune system to express markers, and for replacement gene therapy.
  • ALPHA VIRI IS PACKAGING CELL LINES
  • alphavirus packaging cell lines are provided.
  • alphavirus packaging cell lines are provided wherein the viral structural proteins, supplied in trans from one or more stably integrated expression vectors, are able to encapsidate transfected, transduced, or intracellulariy produced vector RNA transcripts in the cytoplasm and release infectious packaged vector particles through the cell membrane, thus creating an alphavirus vector producing cell line.
  • Alphavirus RNA vector molecules capable of replicating in the cytoplasm of the packaging cell, can be produced initially utilizing, for example, a SP6 RNA polymerase system to transcribe /// vitro a cDNA vector clone encoding the gene of interest and the alphavirus nonstructural proteins (described previously).
  • Vector RNA transcripts are then transfected into the alphavirus packaging cell line, such that the vector RNA replicates to high levels, and is subsequently packaged by the viral structural proteins, yielding infectious vector particles. Because of the extended length of the alphavirus cDNA molecule, the in vitro transcription process is inefficient. Further, only a fraction of the cells contained in a monolayer are typically transfected by most procedures.
  • the vector may first be transfected into a primary alphavirus packaging cell line.
  • the transfected packaging cell line releases infectious vector particles into the culture supernatants and these vector-containing supernatants are subsequently used to transduce a fresh monolayer of alphavirus packaging cells.
  • Transduction into the final alphavirus vector producing cells is preferred over transfection because of its higher RNA transfer efficiency into cells, and optimized biological placement of the vector in the cell. This leads to higher expression and higher titer of packaged infectious recombinant alphavirus vector.
  • alphavirus vector particles may fail to transduce the same packaging cell line because the cell line produces extracellular envelope proteins which block cellular receptors for alphavirus vector particle attachment, a second type of alphavirus vector particle is generated which maintains the ability to transduce the alphavirus packaging cells.
  • This second type of viral particle is produced by a packaging cell line known as a "hopping cell line,” which produces transient vector particles as the result of being transfected with in vitro transcribed alphavirus RNA vector transcripts.
  • the hopping cell line is engineered to redirect the receptor tropism of the transiently produced vector particles by providing alternative viral envelope proteins which redirect alphavirus vectors to different cellular receptors, in a process termed pseudotyping.
  • the first approach consists of an alphavirus packaging cell line expressing the vesicular stomatitis virus G protein (VSV-G).
  • VSV-G vesicular stomatitis virus G protein
  • the second approach for producing a pseudotyped alphavirus vector particle is to use currently available retroviral packaging cell lines containing retroviral gaglpol and env sequences which would be capable of packaging an alphavirus RNA vector containing a retroviral packaging sequence ⁇ e.g., WO 92/05266).
  • a second approach has also been devised in which a stably integrated DNA expression vector is used to produce the alphavirus vector RNA molecule, which, as in the first approach, maintains the autocatalytic ability to self- replicate.
  • This approach allows for continued vector expression over extended periods of culturing because the integrated DNA vector expression system is maintained through a drug selection marker and the DNA system will constitutively express unaltered RNA vectors which cannot be diluted out by defective RNA copies.
  • the DNA-based alphavirus vector is introduced initially into the packaging cell line by transfection, since size restrictions could prevent packaging of the expression vector into a viral vector particle for transduction.
  • the SP6 RNA polymerase recognition site of the plasmid is replaced with another appropriate promoter sequence defined by the parent cell line used.
  • this plasmid sequence also contains a selection marker different from that used to create the packaging cell line.
  • alphavirus proteins and/or vector RNA above certain levels may result in cytotoxic effects in packaging cell lines. Therefore, within certain embodiments of the invention, it may be desirable for these elements to be expressed only after the packaging/producer cells have been propagated to a certain critical density. For this pu ⁇ ose, additional packaging or producer cell line modifications are made whereby the structural proteins necessary for packaging are synthesized only after induction by the RNA vector itself or some other stimulus. Also, other modifications allow for the individual expression of these proteins under the control of separate inducible elements, by utilizing expression vectors which unlink the genes encoding these proteins. In addition, expression of the integrated vector molecule itself, in some instances, is controlled by yet another inducible system. This configuration results in a cascade of events following induction, that ultimately leads to the production of packaged vector particles.
  • compositions and methods are provided for administering an alphavirus vector construct which is capable of preventing, inhibiting, stabilizing or reversing infectious, cancerous, auto-immune or immune diseases.
  • diseases include viral infections such as HTV, HBN HTLV I, HTLV II, CMV, EBV and HPV, melanomas, diabetes, graft vs. host disease, Alzheimer's disease and heart disease.
  • compositions and methods are provided for stimulating an immune response (either humoral or cell-mediated) to a pathogenic agent, such that the pathogenic agent is either killed or inhibited.
  • pathogenic agents include bacteria, fungi, parasites, viruses and cancer cells.
  • the pathogenic agent is a virus
  • methods are provided for stimulating a specific immune response and inhibiting viral spread by using recombinant alphavirus viral particles designed to deliver a vector construct that directs the expression of an antigen or modified form thereof to susceptible target cells capable of either (1) initiating an immune response to the viral antigen or (2) preventing the viral spread by occupying cellular receptors required for viral interactions.
  • Expression of the vector nucleic acid encoded protein may be transient or stable with time.
  • the recombinant alphavirus is preferably designed to express a modified form of the antigen which will stimulate an immune response and which has reduced pathogenicity relative to the native antigen.
  • alphavirus vectors can easily be thought of as safe viral vectors which can be used on healthy individuals for vaccine use.
  • This aspect of the invention has a further advantage over other systems that might be expected to function in a similar manner, in that the presenter cells are fully viable and healthy and low levels of viral antigens, relative to heterologous genes, are expressed.
  • Such an approach may be extended to the expression of a peptide having multiple epitopes, one or more of the epitopes being derived from different proteins.
  • this aspect of the invention allows efficient stimulation of cytotoxic T lymphocytes (CTL) directed against antigenic epitopes, and peptide fragments of antigens encoded by sub-fragments of genes, through intracellular synthesis and association of these peptide fragments with MHC Class I molecules.
  • CTL cytotoxic T lymphocytes
  • This approach may be utilized to map major immunodominant epitopes for CTL induction.
  • An immune response may also be achieved by transferring to an appropriate immune cell (such as a T lymphocyte) the gene for the specific T cell receptor which recognizes the antigen of interest (in the context of an appropriate MHC molecule if necessary), for an immunoglobulin which recognizes the antigen of interest, or 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
  • the gene for the specific T cell receptor which recognizes the antigen of interest in the context of an appropriate MHC molecule if necessary
  • an immunoglobulin which recognizes the antigen of interest
  • the recombinant alphavirus infected cells may be used as an immunostimulant, immunomodulator, or vaccine.
  • alphavirus vectors deliver and express defective interfering viral structural proteins, which inhibit viral assembly.
  • Such vectors may encode defective gag, pol, env or other viral particle proteins or peptides and these would inhibit in a dominant fashion the assembly of viral particles. This occurs because the interaction of normal subunits of the viral particle is disturbed by interaction with the defective subunits.
  • HIV protease cleaves the viral gag and gaglpol 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 HIV 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. Vectors to inhibit HIV will express one or multiple fused copies of such peptide inhibitors.
  • Another embodiment 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 by means of a viral vector leads to regression of the tumor phenotype in these cells.
  • cancers are retinoblastoma and Wilms Tumor. Since malignancy can be considered to be an inhibition of cellular terminal differentiation compared with cell growth, the alphavirus vector delivery and expression of gene products which lead to differentiation of a tumor should also, in general, lead to regression.
  • the alphavirus vector provides a therapeutic effect by encoding a ribozyme (an RNA enzyme) (Haseloff and Gerlach, Nature 557:585, 1989) which will cleave and hence inactivate RNA molecules corresponding to a pathogenic function. Since 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 species such as a RNA or a retroviral genome. Additional specificity may be achieved in some cases by making this a conditional toxic palliative (see below).
  • a ribozyme an RNA enzyme
  • autoimmune diseases involve the interaction of viral particles with cells, cells with cells, or cells with factors.
  • viral infections viruses commonly enter cells via receptors on the surface of susceptible cells.
  • cancers cells may respond inappropriately or not at all to signals from other cells or factors.
  • autoimmune disease there is inappropriate recognition of "self markers.
  • interactions may be blocked by producing, in vivo, an analogue to either of the partners in an interaction. This blocking action may occur intracellulariy, on the cell membrane, or extracellularly.
  • the blocking action of a viral or, in particular, an alphavirus vector carrying a gene for 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.
  • an appropriate blocker would be a vector construct expressing either an HIV 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 intracellulariy 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 an alphavirus vector encoding such a hybrid-soluble CD4 to a host results in a continuous supply of a stable hybrid molecule. Efficacy of treatment can be assayed by measuring the usual indicators of disease progression, including antibody level, viral antigen production, infectious HIV levels, or levels of nonspecific infections.
  • EXPRESSION OF PALLIATIVES Techniques similar to those described above can be used to produce recombinant alphavirus with vector constructs which direct the expression of an agent (or "palliative") which is capable of inhibiting a function of a pathogenic agent or gene.
  • "capable of inhibiting a function” means 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 adso ⁇ tion, replication, gene expression, assembly, and exit of the virus from infected cells.
  • functions for a cancerous cell or cancer-promoting growth factor include viability, cell replication, altered susceptibility to external signals ⁇ e.g., contact inhibition), and lack of production or production of mutated forms of anti-oncoge ⁇ e proteins.
  • the alphavirus vector construct directs the expression of a gene which can interfere with a function of a pathogenic agent, for instance in viral or malignant diseases.
  • a pathogenic agent for instance in viral or malignant diseases.
  • Such expression may either be essentially continuous or in response to the presence in the cell of another agent associated either with the pathogenic condition or with a specific cell type (an "identifying agent").
  • vector delivery may be controlled by targeting vector entry specifically to the desired cell type (for instance, a virally infected or malignant cell) as discussed above.
  • One method of administration is leukophoresis, in which about 20% of an individual's
  • PBLs are removed at any one time and manipulated /// vitro. Thus, approximately 2 x 10 9 cells may be treated and replaced. Repeat treatments may also be performed. Alternatively, bone marrow may be treated and allowed to amplify the effect as described above. In addition, packaging cell lines producing a vector may be directly injected into a subject, allowing continuous production of recombinant virions.
  • alphavirus vectors which express RNA complementary to key pathogenic gene transcripts can be used to inhibit translation of that transcript into protein, such as the inhibition of translation of the HIV tat protein. Since expression of this protein is essential for viral replication, cells containing the vector would be resistant to HIV replication.
  • RNA complementary to the viral packaging signal ⁇ e.g., an HIV packaging signal when the palliative is directed against HIV
  • an alphavirus vector may be introduced which expresses a palliative capable of selectively inhibiting the expression of a pathogenic gene, or a palliative capable of inhibiting the activity of a protein produced by the pathogenic agent.
  • a mutant tat protein which lacks the ability to transactivate expression from the HIV LTR and interferes (in a transdominant manner) with the normal functioning of tat protein.
  • Such a mutant has been identified for HTLV II tat protein ("XII Leu 5 " mutant; see Wachsman et al.. Science 255:674, 1987).
  • a mutant transrepressor tat should inhibit replication much as has been shown for an analogous mutant repressor in HSV-1 (Friedmann et al.. Nature 555:452, 1988).
  • Such a transcriptional repressor protein may be selected for in tissue culture using any viral-specific transcriptional promoter whose expression is stimulated by a virus-specific transactivating protein (as described above).
  • a virus-specific transactivating protein as described above.
  • HIV virus-specific transactivating protein
  • a cell line expressing HIV tat protein and the HSVTK gene driven by the HIV promoter will die in the presence of AC V.
  • a mutant with the appropriate properties ⁇ i.e., represses transcription from the HIV promoter in the presence of wild-type tat
  • the mutant gene can then be reisolated from these cells.
  • a cell line containing multiple copies of the conditionally lethal vector/tat system may be used to assure that surviving cell clones are not caused by endogenous mutations in these genes
  • a battery of randomly mutagenized tat genes are then introduced into these cells using a "rescuable" alphavirus vector ⁇ i.e., one that expresses the mutant tat protein and contains a bacterial origin of replication and drug resistance marker for growth and selection in bacteria). This allows a large number of random mutations to be evaluated and permits facile subsequent molecular cloning of the desired mutant cell line.
  • This procedure may be used to identify and utilize mutations in a variety of viral transcriptional activator/viral promoter systems for potential antiviral therapies.
  • Another approach for inhibiting a pathogenic agent is to express a palliative which is toxic for the cell expressing the pathogenic condition.
  • expression of the palliative from the vector should be limited by the presence of an entity associated with the pathogenic agent, such as a specific viral RNA sequence identifying the pathogenic state, in order to avoid destruction of nonpathogenic cells.
  • a recombinant alphavirus vector carries a vector construct containing a toxic gene (as discussed above) expressed from a cell-specific responsive vector.
  • a toxic gene as discussed above
  • rapidly replicating cells which contain the RNA sequences capable of activating the cell-specific responsive vectors, are preferentially destroyed by the cytotoxic agent produced by the alphavirus vector construct.
  • the alphavirus vector construct can carry a gene for phosphorylation, phosphoribosylation, ribosylation, or other metabolism of a purine- or pyrimidine-based drug.
  • This gene may have no equivalent in mammalian cells and might come from organisms such as a virus, bacterium, fungus, or protozoan.
  • An example of this would be the E. coli guanine phosphoribosyl transferase gene product, which is lethal in the presence of thioxanthine (.see Besnard et al., Mol. Cell. Biol. 7:4139-4141, 1987).
  • 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).
  • Mammalian viruses in general tend to have "immediate early" genes which are necessary for subsequent transcriptional activation from other viral promoter elements.
  • RNA sequences of this nature are excellent candidates for activating alphavirus vectors intracellular signals (or "identifying agents") of viral infection.
  • conditionally lethal genes expressed from alphavirus cell-specific vectors responsive to these viral "immediate early" gene products could specifically kill cells infected with any particular virus.
  • the human and interferon promoter elements are transcriptionally activated in response to infection by a wide variety of nonrelated viruses, the introduction of vectors expressing a conditionally lethal gene product like HSVTK, for example, in response to interferon production could result in the destruction of cells infected with a variety of different viruses.
  • the recombinant alphavirus viral vector carries a vector construct that directs the expression of a gene product capable of activating an otherwise inactive precursor into an active inhibitor of the pathogenic agent.
  • the HSVTK gene product may be used to more effectively metabolize potentially antiviral nucleoside analogues such as AZT or ddC.
  • the HSVTK gene may be expressed under the control of a cell-specific responsive vector and introduced into these cell types.
  • AZT and other nucleoside antivirals
  • HSVTK a nucleoside and nucleoside kinase with very broad substrate specificity
  • AZT or ddC therapy will thereby be more effective, allowing lower doses, less generalized toxicity, and higher potency against productive infection.
  • Additional nucleoside analogues whose nucleotide triphosphate forms show selectivity for retroviral reverse transcriptase but, as a result of the substrate specificity of cellular nucleoside and nucleotide kinases are not phosphorylated, will be made more efficacious.
  • the recombinant alphavirus vector carries a gene specifying a product which is not in itself toxic but, when processed or modified by a protein such as a protease specific to a viral or other pathogen, is converted into a toxic form.
  • the recombinant alphavirus could carry a gene encoding a proprotein for ricin A chain, which becomes toxic upon processing by the HIV protease.
  • a synthetic inactive proprotein form of the toxin ricin or diphtheria A chains could be cleaved to the active form by arranging for the HIV virally encoded protease to recognize and cleave off an appropriate "pro" element.
  • the alphavirus construct may express a "reporting product" on the surface of the target cells in response to the presence of an identifying agent in the cells (such as expression of a viral gene).
  • This surface protein can be recognized by a cytotoxic agent, such as antibodies for the reporting protein, or by cytotoxic T cells.
  • a cytotoxic agent such as antibodies for the reporting protein
  • cytotoxic T cells such as antibodies for the reporting protein
  • such a system can be used as a detection system ⁇ see below) to simply identify those cells having a particular gene which expresses an identifying protein.
  • a surface protein could be expressed which would itself be therapeutically beneficial.
  • expression of the human CD4 protein specifically in HIV-infected cells may be beneficial in two ways:
  • CD4 Binding of CD4 to HIV env intracellulariy 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
  • the alphavirus vector codes for a ribozyme which will cleave and inactivate RNA molecules essential for viability of the vector infected cell. By making ribozyme production dependent on a specific RNA sequence corresponding to the pathogenic state, such as HIV tat, toxicity is specific to the pathogenic state.
  • the above-described technique of expressing a palliative in a cell in response to a specific RNA sequence can also be modified to enable detection of a particular gene in a cell which expresses an identifying protein (for example, a gene carried by a particular virus), and hence enable detection of cells carrying that virus.
  • this technique enables the detection of viruses (such as HIV) in a clinical sample of cells carrying an identifying protein associated with the virus.
  • This modification can be accomplished by providing a genome coding for a product, the presence of which can be readily identified (the "marker product”), in an alphavirus vector which responds to the presence of the identifying protein in the infected cells.
  • the marker product For example, HIV, when it infects suitable cells, makes tat and rev.
  • the indicator cells can thus be provided with a genome (such as by infection with an appropriate recombinant alphavirus) which codes for a marker gene, such as the alkaline phosphatase gene, ⁇ -galactosidase gene, or the luciferase gene which is expressed by the recombinant alphavirus upon activation by the tat and/or rev RNA transcript.
  • ⁇ -galactosidase or alkaline phosphatase exposing the cells to substrate analogues results in a color or fluorescence change if the sample is positive for HIV.
  • luciferase exposing the sample to luciferin will result in luminescence if the sample is positive for HIV.
  • the viral titre can be measured directly by counting colored or fluorescent cells, or by making cell extracts and performing a suitable assay.
  • virus titre can also be measured by performing enzyme assays on the cell surface using a fluorescent substrate.
  • Further specificity can be incorporated into the preceding system by testing for the presence of the virus either with or without neutralizing antibodies to that virus. For example, in one portion of the clinical sample being tested, neutralizing antibodies to HIV may be present; whereas in another portion there would be no neutralizing antibodies. If the tests were negative in the system where there were antibodies and positive where there were no antibodies, this would assist in confirming the presence of HIV.
  • a particular gene such as a viral gene
  • the presence of a particular gene such as a viral gene
  • the cells of the sample are infected with a suitable alphavirus vector which carries the reporter gene which is only expressed in the presence of the appropriate viral RNA transcript.
  • the reporter gene after entering the sample cells, will express its reporting product (such as ⁇ -galactosidase or luciferase) only if the host cell expresses the appropriate viral proteins.
  • the present invention also provides recombinant alphavirus which carry a vector construct capable of suppressing one or more elements of the immune system in target cells infected with the alphavirus.
  • specific down-regulation of inappropriate or unwanted immune responses such as in chronic hepatitis or in transplants of heterologous tissue such as bone marrow, can be engineered using immune-suppressive viral gene products which suppress surface expression of transplantation (MHC) antigen.
  • MHC transplantation
  • Group C adenoviruses Ad2 and Ad5 possess a 19 kd glycoprotein (gp 19) encoded in the E3 region of the virus.
  • This gp 19 molecule binds to class I MHC molecules in the endoplasmic reticulum of cells, and prevents terminal glycosylation and translocation of class I MHC to the cell surface.
  • donor bone marrow cells may be infected with gp 19-encoding vector constructs which, upon expression of the gp 19, inhibit the surface expression of MHC class I transplantation antigens.
  • These donor cells may be transplanted with low risk of graft rejection and may require a minimal immunosuppressive regimen for the transplant patient. This may allow 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 lupus erythromiatis, multiple sclerosis, rheumatoid arthritis or chronic hepatitis B infection.
  • An alternative method involves the use of anti-sense message, ribozyme, or other specific 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 anti-sense, ribozyme, or other gene may be introduced using the viral vector delivery system.
  • One further aspect of the present invention relates to transforming cells of an animal with recombinant alphavirus vectors which serve as gene transfer vehicles to supply genetic sequences capable of expressing a therapeutic protein.
  • the viral vector construct is designed to express a therapeutic protein capable of preventing, inhibiting, stabilizing or reversing an inherited or noninherited genetic defect in metabolism, immune regulation, hormonal regulation, enzymatic or membrane associated structural function.
  • This embodiment also describes the viral vector capable of transducing individual cells, whereby the therapeutic protein is able to be expressed systemically or locally from a specific cell or tissue, whereby the therapeutic protein is capable of (a) the replacement of an absent or defective cellular protein or enzyme, or (b) supplement production of a defective of low expressed cellular protein or enzyme.
  • Such diseases may include cystic fibrosis, Parkinson's disease, hypercholesterolemia, adenosine deaminase deficiency, ⁇ -globin disorders. Hemophilia A & B, Gaucher's disease, diabetes and leukemia.
  • a recombinant alphavirus viral vector can be used to treat Gaucher disease.
  • Gaucher disease is a genetic disorder that is characterized by the deficiency of the enzyme glucocerebrosidase.
  • This type of therapy is an example of a single gene replacement therapy by providing a functional cellular enzyme.
  • This enzyme deficiency leads to the accumulation of glucocerebroside in the lysosomes of all cells in the body.
  • the disease phenotype is manifested only in the macrophages, except in the very rare neuronpathic forms of the disease. The disease usually leads to enlargement of the liver and spleen and lesions in the bones. (For a review, see Science 256:794, 1992, and The Metabolic Basis of Inherited Disease, 6th ed., Scriver et al., vol. 2, p. 1677).
  • LYMPHOKINES AND LYMI'HOKINE RECEPTORS As noted above, the present invention provides alphavirus particles which can, among other functions, direct the expression of one or more cytokines or cytokine receptors.
  • cytokines can have negative effects resulting in certain pathological conditions.
  • most resting T-cells, B cells, large granular lymphocytes and monocytes do not express IL-2R ⁇ .
  • IL-2R ⁇ is expressed by abnormal cells in patients with certain leukemias (ATL, Hairy-cell, Hodgkins, acute and chronic granulocytic), autoimmune diseases, and is associated with allograft rejection.
  • ATL IL-2R ⁇
  • IL-2R ⁇ is expressed by abnormal cells in patients with certain leukemias (ATL, Hairy-cell, Hodgkins, acute and chronic granulocytic), autoimmune diseases, and is associated with allograft rejection.
  • ATL Hairy-cell, Hodgkins, acute and chronic granulocytic
  • therapy may be effected by increasing the serum concentration of the soluble form of the cytokine receptor.
  • an alphavirus vector can be engineered to produce both soluble IL-2R ⁇ and EL-2R ⁇ , creating a high affinity soluble receptor.
  • serum IL-2 levels would decrease, inhibiting the paracrine loop.
  • This same strategy may also be effective against autoimmune diseases.
  • some autoimmune diseases ⁇ e.g.. Rheumatoid arthritis, SLE
  • SLE autoimmune diseases
  • blocking the action of IL-2 by increasing the serum level of receptor may also be utilized in order to treat such autoimmune diseases.
  • IL-1 consists of two polypeptides, IL-l ⁇ and IL-l ⁇ , each of which has plieotropic effects.
  • IL-1 is primarily synthesized by mononuclear phagocytes, in response to stimulation by microbial products or inflammation.
  • IL-I Ra IL-1 Receptor antagonist
  • This IL-IR antagonist has the same molecular size as mature IL-1 and is structurally related to it. However, binding of IL-IRa to the IL-IR does not initiate any receptor signaling.
  • this molecule has a different mechanism of action than a soluble receptor, which complexes with the cytokine and thus prevents interaction with the receptor.
  • IL-1 does not seem to play an important role in normal homeostasis.
  • antibodies to IL- 1 receptors reduce inflammation and anorexia due to endotoxins and other inflammation inducing agents.
  • IL-1 induces secondary compounds which are potent vasodilators.
  • exogenously supplied IL-1 decreases mean arterial pressure and induces leukopenia.
  • Neutralizing antibody to IL-1 reduced endotoxin-induced fever in animals.
  • the 28 day mortality was 16% compared to 44% in patients who received placebo infusions.
  • alphavirus vectors may be engineered to produce a soluble receptor or more specifically the IL-IRa molecule.
  • a single injection of IL-IRa producing vector particles could replace the current approach requiring a constant infusion of recombinant IL-IR.
  • Cytokine responses may also be involved in the failure to control or resolve infectious diseases. Perhaps the best studied example is non-healing forms of leishmaniasis in mice and humans which have strong, but counte ⁇ roductive TH2-dominated responses. Similarly, lepromotomatous leprosy is associated with a dominant, but inappropriate TH2 response. In these conditions, alphavirus-based gene therapy may be useful for increasing circulating levels of IFN gamma, as opposed to the site-directed approach proposed for solid tumor therapy. IFN gamma is produced by TH- I T-cells, and functions as a negative regulator of TH-2 subtype proliferation.
  • IFN gamma also antagonizes many of the IL-4 mediated effects on B-cells, including isotype switching to IgE.
  • IgE mast cells and eosinophils are involved in mediating allergic reaction.
  • IL-4 acts on differentiating T-cells to stimulate TH- development, while inhibiting Tpj-1 responses.
  • alphavirus-based gene therapy may also be accomplished in conjunction with traditional allergy therapeutics.
  • One possibility is to deliver alphavirus-IL4R with small amounts of the offending allergen ⁇ i.e., traditional allergy shots). Soluble IL-4R would prevent the activity of IL-4, and thus prevent the induction of a strong TH-2 response.
  • alphavirus suicide vectors to limit the spread of wild-type alphavirus in the packaging/producer cell lines.
  • the alphavirus suicide vector would be comprised of an antisense or ribozyme sequence, specific for the wild-type alphavirus sequence generated from an RNA recombination event between the 3' sequences of the junction region of the vector, and the 5' alphavirus structural sequences of the packaging cell line expression vector.
  • the antisense or ribozyme molecule would only be thermostable in the presence of the specific recombination sequence and would not have any other effect in the alphavirus packaging/producer cell line.
  • a toxic molecule (such as those disclosed below), may also be expressed in the context of a vector that would only express in the presence of wild-type alphaviais.
  • Ai.Pi IAVIRI is VECTORS TO PREVENT THE SPREAD OF METASTATIC TUMORS
  • One further aspect of the present invention relates to the use of alphavirus vectors for inhibiting or reducing the invasiveness of malignant neoplasms.
  • the extent of malignancy typically relates to vascularization of the tumor.
  • One cause for tumor vascularization is the production of soluble tumor angiogenesis factors (TAF) (Paweletz et al., C 7/7. Rev. Oncol. Hemalol. 9: 197, 1989) expressed by some tumors.
  • TAF tumor angiogenesis factors
  • tumor vascularization may be slowed by using alphavirus vectors to express antisense or ribozyme RNA molecules specific for TAF.
  • anti-angiogenesis factors may be expressed either alone or in combination with the above-described ribozymes or antisense sequences in order to slow or inhibit tumor vascularization.
  • alphavirus vectors can also be used to express an antibody specific for the TAF receptors on surrounding tissues.
  • recombinant alphavirus vectors which can be designed to be delivered by, for example, ( 1) direct injection into the blood stream; (2) direct injection into a specific tissue or tumor; (3) oral administration; (4) nasal inhalation; (5) direct application to mucosal tissues; or (6) ex vivo administration of transduced autologous cells into the animal.
  • the therapeutic alphavirus vector can be administered in such a fashion such that the vector can (a) transduce a normal healthy cell and transform the cell into a producer of a therapeutic protein or agent which is secreted systemically or locally, (b) transform an abnormal or defective cell, transforming the cell into a normal functioning phenotype, (c) transform an abnormal cell so that it is destroyed, and/or (d) transduce cells to manipulate the immune response.
  • alphavirus vectors may be utilized in order to regulate the growth control activity of transcription factors in the infected cell.
  • transcription factors directly influence the pattern of gene expression through sequence-specific tram-activation or repression (Karin, New Biologist 27: 126-131, 1990).
  • Alphavirus gene transfer therapy can be used, for example, to return control to tumor cells whose unregulated growth is activated by oncogenic transcription factors, and proteins which promote or inhibit the binding cooperatively in the formation of homo- and heterodimer tram-activating or repressing transcription factor complexes.
  • the nuclear oncogene c-myc is expressed by proliferating cells and can be activated by several distinct mechanisms, including retroviral insertion, amplification, and chromosomal translocation.
  • the Max protein is expressed in quiescent cells and, independently of c-myc, either alone or in conjunction with an unidentified factor, functions to repress expression of the same genes activated by the myc/Max heterodimer (Cole, Cell 65:715-716, 1991).
  • Inhibition of c- yc or c-m c/Max proliferation of tumor cells may be accomplished by the overexpression of Max in target cells controlled by alphavirus vectors.
  • the Max protein is only 160 amino acids (corresponding to 480 nucleotide RNA length) and is easily inco ⁇ orated into an alphavirus vector either independently, or in combination with other genes and/or antisense/ribozyme moieties targeted to factors which release growth control of the cell.
  • Modulation of homo/hetero-complex association is another approach to control transcription factor activated gene expression.
  • transport from the cytoplasm to the nucleus of the tra//.v-activating transcription factor NF-B is prevented while in a heterodimer complex with the inhibitor protein IB.
  • IB Upon induction by a variety of agents, including certain cytokines, IB becomes phosphorylated and NF-B is released and transported to the nucleus, where it can exert its sequence-specific traws-activating function (Baeuerle and Baltimore, Science 242:540-546, 1988).
  • the dissociation of the NF-B/IB complex can be prevented by masking with an antibody the phosphorylation site of IB. This approach would effectively inhibit the tra.
  • /.s-activation activity of the NF-IB transcription factor by preventing its transport to the nucleus.
  • Expression of the IB phosphorylation site specific antibody or protein in target cells may be accomplished with an alphavirus gene transfer vector.
  • An approach similar to the one described here could be used to prevent the formation of the tra//.v-activating transcription heterodimer factor AP-1 (Turner and Tijan, Science 275: 1689- 1694, 1989), by inhibiting the association between the jun and fos proteins.
  • the present invention also provides pharmaceutical compositions comprising a recombinant Sindbis particle or virus, or Sindbis vector construct, in combination with a pharmaceutically acceptable carrier, diluent, or recipient.
  • infectious recombinant virus may be preserved either in crude or purified forms.
  • virus- producing cells may first be cultivated in a bioreactor, wherein viral particles are released from the cells into the culture media.
  • Virus may then be preserved in crude form by first adding a sufficient amount of a formulation buffer to the culture media containing the recombinant virus to form an aqueous suspension.
  • 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 recombinant vi s can also be preserved in a purified form. More specifically, prior to the addition of the formulation buffer, the crude recombinant virus 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 in order to remove excess media components and to establish the recombinant virus in a more desirable buffered solution. The diafiltrate is then passed over a Sephadex S-500 gel column and a purified recombinant virus is eluted.
  • a cross flow concentrating system Frtron Technology Corp., Nortborough, MA.
  • DNase is added to the concentrate to digest exogenous DNA.
  • the digest is then diafiltrated in order to remove excess media components and to establish the recombinant virus in a more desirable buffered solution.
  • the formulation buffer may be 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.
  • Crude recombinant virus may also be purified by ion exchange column chromatography. Briefly, crude recombinant virus may be clarified by first passing it through a filter, followed by loading the filtrate onto a column containing a highly sulfonated cellulose matrix. The recombinant virus may then be eluted from the column in purified form by using a high salt buffer, and the high salt buffer exchanged for a more desirable buffer by passing the eluate over a molecular exclusion column. A sufficient amount of formulation buffer is then added, as discussed above, to the purified recombinant virus and the aqueous suspension is either dried immediately or stored, preferably at -70°C.
  • 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 virus.
  • aliquots of the formulated recombinant virus are placed into an Edwards Refrigerated Chamber (3 shelf RC3S unit) attached to a freeze dryer (Supermodulyo 12K).
  • a freeze dryer Supermodulyo 12K
  • ⁇ Cryobiology 75:414, 1981 is used to lyophilize the formulated recombinant virus, preferably from a temperature of -40°C to -45°C.
  • the resulting composition contains less than 10% water by weight of the lyophilized virus.
  • the recombinant viais is stable and may be stored at -20°C to 25°C, as discussed in more detail below.
  • water is removed from the aqueous suspension at ambient temperature by evaporation.
  • water is removed through spray-drying (EP 520,748).
  • spray-drying the aqueous suspension is delivered into a flow of preheated gas, usually air, whereupon water rapidly evaporates from droplets of the suspension.
  • Spray-drying apparatus are available from a number of manufacturers (e.g., Drytec, Ltd., Tonbridge, England; Lab-Plant, Ltd., Huddersfield, England). Once dehydrated, the recombinant virus is stable and may be stored at -20°C to 25°C.
  • the resulting moisture content of the dried or lyophilized virus may be determined through use of a Karl-Fischer apparatus (EM Science Aquastar' V1B volumetric titrator. Cherry Hill, NJ), or through a gravimetric method.
  • aqueous solutions used for formulation are preferably 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 virus 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.
  • amino acids function to further preserve viral infectivity upon cooling and thawing of the aqueous suspension.
  • amino acids function to further preserve viral infectivity during sublimation of the cooled aqueous suspension and while in the lyophilized state.
  • a preferred amino acid is arginine, but other amino acids such as lysine, ornithine, serine, glycine, glutamine, asparagine, glutamic acid or aspartic acid can also be used.
  • a particularly preferred arginine concentration is 0.1% by weight.
  • the amino acid concentration ranges from 0.1% to 10% by weight.
  • the buffering component acts to buffer the solution by maintaining a relatively constant pH.
  • 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 virus 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 alphavirus 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.
  • the lyophilized or dehydrated viruses of the subject invention may be reconstituted using a variety of substances, but are preferably reconstituted using water.
  • dilute salt solutions which bring the final formulation to isotonicity may also be used.
  • aqueous solutions containing components known to enhance the activity of the reconstituted virus include cytokines, such as IL-2, polycations, such as protamine sulfate, or other components which enhance the transduction efficiency of the reconstituted virus.
  • Lyophilized or dehydrated recombinant virus 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.
  • viruses having an RNA genome with positive polarity is such that when introduced into a eukaryotic cell which serves as a permissive host, the purified genomic nucleic acid serves as a functional message RNA (mRNA) molecule. Therefore, this genomic RNA, purified from the virus, can initiate the same infection cycle which is characteristic of infection by the wild type virus from which the RNA was purified.
  • mRNA functional message RNA
  • Sindbis vi s strain Ar-339 (ATCC #VR-1248, Taylor et al.. Am. J. Trop. Med. Hyg. 7:844 1 55; isolated from the mosquito Culexus univittatus) may be propagated on baby hamster kidney (BHK) cells, infected at low multiplicity (0.1 PFU/cell).
  • Sindbis virus strain (Lee Biomolecular, San Diego, CA) may also be used and propagated by the same methods.
  • Sindbis virions can be precipitated from a clarified lysate at 48 hours post-infection with 10% (w/v) of polyethylene glycol (PEG-8000) at 0°C, as described previously. Sindbis virions which are contained in the PEG pellet may be lysed with 2% SDS, and the poly-adenylated mRNA isolated by chromatography utilizing commercially available oligo dT columns (Invitrogen).
  • this primer contains at its 5' end a five nucleotide 'buffer sequence' for efficient restriction endonuclease digestion, followed by the Xba I recognition sequence, 25 consecutive dT nucleotides and six nucleotides which are precisely complementary to the extreme Sindbis 3' end.
  • selection for first round cDNA synthesis occurs at two levels: (1) polyadenylated molecules, a prerequisite for functional mRNA, and (2) selective priming from Sindbis mRNA molecules, in a pool containing multiple mRNA species.
  • the reverse transcription is performed in the presence of 10 mM MeHgOH to mitigate the frequency of artificial stops during reverse transcription.
  • Sindbis cDNA Primary genomic length Sindbis cDNA is then amplified by PCR in six distinct segments using six pairs of overlapping primers.
  • the Sindbis 5' end forward primer is constructed to contain a 19 nucleotide sequence corresponding to the bacterial SP6 RNA polymerase promoter and the Apa I restriction endonuclease recognition sequence linked to its 5' end.
  • the bacterial SP6 RNA polymerase is poised such that transcription in vitro results in the inclusion of only a single non-viral G ribonucleotide linked to the A ribonucleotide, which corresponds to the authentic Sindbis 5' end.
  • Apa I recognition sequence facilitates insertion of the PCR amplicon into the plasmid vector (pKS II + , Stratagene) polylinker sequence.
  • a five nucleotide 'buffer sequence' is also inserted prior to the Apa I recognition sequence in order to permit efficient digestion.
  • the sequence of the SP6-5' Sindbis forward primer and all of the primer pairs necessary to amplify the entire Sindbis genome are shown below. (Note that "nt” and “nts” as utilized hereinafter refers to "nucleotide” and “nucleotides,” respectively).
  • the reference sequence (GenBank accession no. SI ⁇ CG) is from Strauss et al.. Virology 755:92-1 10.
  • reaction products are inserted first into the pCR II vector (Invitrogen), then using the appropriate enzymes shown above, are inserted, stepwise, into the pKS II + (Stratagene) vector, between the Apa I and Xba I sites.
  • pKS II + Stratagene
  • the Sindbis genomic cDNA clone pVGSP ⁇ GEN is linearized by digestion with Xba I, which cuts pVGSP ⁇ GEN once, immediately adjacent and downstream of the 25 nucleotide long poly dA:dT stretch.
  • the linearized pVGSP ⁇ GEN clone is purified with "GENECLEAN" (BIO 101 , La Jolla, CA), and adjusted to a concentration of 0.5 mg/ml.
  • the in vitro transcription reaction products can be digested with DNase I (Promega) and purified by sequential phenol/CHCl3 and ether extraction, followed by ethanol precipitation, or alternatively, can be used directly for transfection.
  • the / ' // vitro transcription reaction products or purified RNA are complexed with a commercial cationic lipid compound ("LIPOFECTIN,” GIBCO-BRL, Gaithersburg, MD), and applied to Baby Hamster Kidney- 21 (BHK-21) cells maintained in a 60 mM petri dish at 75% confluency. The transfected cells are incubated at 30°C. After 94 hours post-transfection, extensive cytopathologic effects (CPE) are observed.
  • CPE cytopathologic effects
  • virion RNA is amplified by RT-PCR as described above, and sequence relating to the nucleotides in question is determined by direct sequencing of the RT-PCR amplicon product, using a commercially available kit (Promega, Madison WI), and compared to the corresponding pVGSP ⁇ GEN sequence.
  • Table 2 The results of this study are given in Table 2. Briefly, three non conservative amino acid changes, Gly ⁇ Glu, Asp - Gly, and Tyr ⁇ Cys, which are a result of cloning artifact are observed respectively at viral nucleotides 2245, 6193, and 6730.
  • NSP viral non-structural protein
  • the RT-PCR amplicon product is digested with Eco 47III and Bgl II, and the 882 bp fragment is purified by 1% agarose/TBE gel electrophoresis, and exchanged into the corresponding region of the pVGSP ⁇ GEN clone, prepared by digestion with Eco 47III and Bgl II, and treatment with CIAP.
  • the 3A/7349R primer pair described above is used to repair the nt 6193 and nt 6730 changes.
  • the RT-PCR amplicon product is digested with Eco RI and Hpa I, and the 1,050 bp fragment is purified by 1% agaroseTBE gel electrophoresis, and exchanged into the corresponding region of the pVGSP ⁇ GEN clone.
  • This clone is designated pVGSP ⁇ GENrep.
  • Noncoding Region
  • Nonstmctural Proteins 2032 Glu ⁇ Gly +*
  • the present invention provides eukaryotic layered vector initiation systems which generally comprise a promoter which is capable of initiating the 5' synthesis of RNA from cDNA, a construct which is capable of autonomous or autocatalytic replication in a cell, the constmct also being capable of expressing a heterologous nucleic acid sequence, and a 3' sequence which controls transcription termination.
  • constmcts may be constmcted of the following ordered elements: a 5' eukaryotic promoter capable of initiating the synthesis of viral RNA at the authentic alphavirus 5' end, a 5' sequence which is capable of initiating transcription of an alphavirus, a nucleotide sequence encoding alphavims non-structural proteins, a viral junction region, a heterologous sequence, an alphavims RNA polymerase recognition sequence, and a 3' transcription termination polyadenylation signal sequence.
  • a 5' eukaryotic promoter capable of initiating the synthesis of viral RNA at the authentic alphavirus 5' end
  • a 5' sequence which is capable of initiating transcription of an alphavirus a nucleotide sequence encoding alphavims non-structural proteins
  • a viral junction region a heterologous sequence
  • an alphavims RNA polymerase recognition sequence an alphavims RNA polymerase recognition sequence
  • Such alphavims cDNA expression vectors may also include intervening sequences (introns), which are spliced from the pre-RNA in the nucleus prior to transport to the cytoplasm, and which may improve the overall efficiency of the system, in terms of molecules of functional mRNA transported to the cytoplasm/nuclear DNA template.
  • the intron splicing signals are located, for example, between Sindbis and heterologous gene regions as described in Example 3. Constmction of a eukaryotic layered vector initiation system utilizing the Sindbis clone pVGSP ⁇ GENrep and mammalian RNA polymerase II promoters may be accomplished essentially as follows.
  • plasmid pVGSP ⁇ GENrep is digested with Bgl II and Xba I, and the reaction products are electrophoresed on a 0.8% agarose/TBE gel.
  • the resulting 9,438 bp fragment is excised, purified with "GENECLEAN" (BIO 101, Vista, CA), and ligated into the 4,475 bp vector fragment resulting from treatment of pCDNA3 (Invitrogen, San Diego, CA) with Bgl II, Xba I, and CIAP. This constmction is designated as pcDNASINbgl/xba.
  • the U3 region of the long terminal repeat (LTR) from Moloney murine leukemia vims (Mo-MLV) is positioned at the 5' viral end such that the first transcribed nucleotide is a single G residue, which is capped in vivo, followed by the Sindbis 5' end. Juxtaposition of the Mo-MLV LTR and the Sindbis 5' end is accomplished by overlapping PCR as described below. Amplification of the Mo-MLV LTR in the first primary PCR reaction is accomplished in a reaction containing the BAG vector (Price et al., PNAS 57:156-160, 1987) and the following primer pair
  • Reverse primer B AGwt441 R2 (SIN nts 5- 1 /Mo-MLV LTR nts 441 -406):
  • PCR amplification of the Mo-MLV LTR with the primer pair shown above is performed using the Thermalase thermostable DNA polymerase (Amresco Inc., Solon, Ohio) and the buffer containing 1.5 mM MgCl2, provided by the supplier. Additionally, the reaction contains 5% DMSO, and the "HOT START WAX" beads (Perkin-Elmer), using the following PCR amplification protocol shown below:
  • Amplification of the Sindbis 5' end in the second primary PCR reaction is accomplished in a reaction containing the pVGSP ⁇ GENrep clone and the following primer pair:
  • PCR amplification of the Mo-MLV LTR is accomplished with the primer pair and amplification reaction conditions described above, utilizing the PCR amplification protocol shown below:
  • PCR amplification of the primer PCR amplicon products is accomplished utilizing the primer pair and amplification reaction conditions shown above, and using the following PCR amplification protocol:
  • the 25 3' terminal bases of the first primary PCR amplicon product overlaps with the
  • pVGELVIS plasmid DNA is complexed with LIPOFECTAMINE (GIBCO-BRL, Gaithersburg, MD) according to the conditions suggested by the supplier (ca. 5 ⁇ g DNA/8 ⁇ g lipid reagent) and added to 35 mm wells containing BHK -21 cells at approximately 75% confluency. Cytopathic effects (CPE), characteristic of wild type Sindbis vims infection are observed within 48 hours post-infection. Addition of 1 ml of transfection supernatant to fresh BHK-21 monolayers results in CPE within 16 hrs. This data demonstrates the correct juxtaposition of viral cDNA and RNA polymerase II expression cassette signals in the pVGELVIS construct, resulting in the de novo initiation of an RNA vims from a DNA expression module.
  • LIPOFECTAMINE GEBCO-BRL, Gaithersburg, MD
  • an infectious centers assay is performed. Briefly, 5 ⁇ g of pVGELVIS plasmid DNA is transfected onto BHK cells in 35 mm wells as described above, and at 1.5 hours post transfection the cells are trypsinized and serially diluted 10,000-fold, over 10-fold increments, into 5 x 10 3 untreated BHK cells. This transfected and untreated BHK cell mixture is then added to 35 mm wells The cells are allowed to attach to the plate, and subsequently overlayed with media containing 1.0% Noble Agar.
  • plaques due to cell lysis may be visualized either directly or after overlaying with a second layer containing Neutral Red Stain. This experiment reveals that the efficiency of the pVGELVIS plasmid in generating wild type Sindbis vims after transfection onto BHK cells is approximately 1 x 10 3 PFU/ mg of plasmid DNA.
  • a first step in the construction of the Sindbis Basic Vector is the generation of two plasmid subclones containing separate elements from the viral 5' and 3' ends. These elements may then be utilized in order to subsequently assemble a basic gene transfer vector.
  • the first plasmid subclone is constmcted to contain the 40 terminal nucleotides of the viral 3' end and a 25 base pair stretch of consecutive dA:dT nucleotides.
  • the following oligonucleotide pairs are first synthesized:
  • SIN 1 1664F (buffer sequence/Not I site/ SIN nts 1 1664-1 1698):
  • oligonucleotides are then mixed together at equal molar concentrations in the presence of 10 mM MgC , heated to 100°C for 5 minutes and cooled slowly to room temperature.
  • the partially double-stranded molecule is then filled in using Klenow DNA polymerase and 50 uM dNTPs.
  • the resultant 89 bp molecule is then digested with Not I and Sac I, purified on a 2% NuSieve/1% agarose gel, and ligated into pKS 11+ plasmid (Stratagene, La Jolla, CA), prepared by digestion with Not I and Sac I and treatment with CIAP, at a 10: 1 molar excess of insert:vector ratio. This constmction is designated pKSID'SIN.
  • the second plasmid subclone is constmcted to contain the first 5' 7,643 nucleotides of Sindbis, and a bacteriophage RNA polymerase promoter is positioned at the viral 5' end such that only a single non-viral nucleotide is added to the authentic viral 5' end after in vitro transcription.
  • a bacteriophage RNA polymerase promoter is positioned at the viral 5' end such that only a single non-viral nucleotide is added to the authentic viral 5' end after in vitro transcription.
  • the 3' end of this clone is derived by a standard three temperature PCR amplification with a reverse primer having the sequence shown below.
  • the reverse primer maps to viral nucleotides 7643-7621 and is 41 bp downstream from the junction core element 3' end. Additionally, viral nucleotide 7643 is 4 nucleotides upstream from the stmctural protein gene translation initiation codon. The first five 5' nucleotides in this primer are included to serve as a 'buffer sequence' for the efficient digestion of the PCR amplicon products, and are followed by 6 nucleotides comprising the Xho I recognition sequence.
  • primer 2A (described in Example 1), having the following sequence:
  • ATACTAGCCACGGCCGGTATC SEQ. ID NO. 6
  • the 4510 bp amplicon product resulting from the PCR amplification shown above with pVGSP ⁇ GENrep plasmid (described in Example 1) as template, is digested with the enzymes Sfi I and Xho I. The resultant 2526 bp fragment is gel purified. Sindbis cDNA clone pVGSP ⁇ GENrep is also digested with Apa I and Sfi I, and the resultant 5144 bp fragment which includes the SP6 RNA polymerase promoter at its 5' end is gel purified.
  • the 5144 bp fragment is ligated together with the 2526 bp fragment from above, along with Apa I and the Xho I digested CIAP treated pKS 11+ plasmid.
  • a clone is isolated having the Sindbis nucleotides 1 - 7643 including the RNA polymerase promoter at its 5' end contained in the pKSII+ plasmid vector. This constmction is designated pKSII5'SIN.
  • Assembly of the complete basic vector is accomplished by digesting pKSII5'SIN with Xho I and Sac I, treating with CIAP, and gel purifying of a large 10,533 bp fragment.
  • 10,533 bp fragment is then ligated together with a 168 bp small fragment resulting from digestion of pKSII3'SIN with Xho I and Sac I. This resultant constmction is designated pKSSINBV ⁇ see Figure 4).
  • the firefly luciferase reporter gene is inserted into the Sindbis Basic Vector in order to demonstrate the expression of a heterologous gene in cells transfected with RNA that is transcribed in vitro from the Sindbis vector clone, and to demonstrate the overall functionality of the Sindbis basic vector.
  • Constmction of the Sindbis luciferase vector is performed by assembling together components of 3 independent plasmids: pKSIIS'SIN, pKSII3'SIN, and pGL2-basic vector.
  • the pGL2 -basic vector plasmid contains the entire firefly luciferase gene.
  • the luciferase gene is first inserted into the pKSII3'SIN plasmid. This is accomplished by digesting pGL2 with Bam HI and Hind III, and gel purifying a 2689 bp containing fragment. This fragment is ligated with a gel purified 3008 bp large fragment resulting from digestion of pKSIM'SIN with Bam HI and Hind III and treatment with CIAP. The resultant constmction is designated pKSI13'SIN-luc.
  • pKSII5'SIN with Xho I and Sac I
  • CIAP treating with CIAP
  • gel purifying the large 10,533 bp fragment The pKS 5'SIN 10,533 bp fragment is ligated together with the 2854 bp small fragment resulting from digestion of pKSII3'S_N-luc with Xho I and Sac I.
  • This constmction contains the entire Sindbis nonstmctural gene coding region and 3' viral elements necessary for genome replication, as well as the firefly luciferase gene positioned between these two viral 5' and 3' elements.
  • This vector is designated pKSSINBV-luc, and is shown schematically in Figure 4.
  • a complementary packaging vector which is deleted of most of the non stmctural gene region, is constmcted by digestion of pVGSP ⁇ GENrep with Bsp El and re- ligation under dilute conditions.
  • This constmction designated pVGSP6GENdlRsp, is deleted of nonstmctural gene sequences between bases 422-7,054, and is shown schematically in Figure 5.
  • Transcription in vitro of Xba I-linearized pVGSP6GENdl-5-»p is as described in Example 1. Transfections and co-transfections are performed by complexing in vitro transcription products with LIPOFECTIN (Gibco-BRL, Gaithersburg, MD) and applying to BHK cells.
  • luciferase in transfected cells is tested 18 hours after transfection. Additionally, 1 ml of the transfection supernatant is used to infect a confluent monolayer of BHK cells and the expression of luciferase is tested at 24 hours post- infection.
  • a fragment is PCR amplified from the pKSSINBV clone under nonstringent reaction cycle conditions utilizing a reverse primer having the following sequence:
  • the underlined bases in the reverse primer relate to nucleotide changes which can be made in the junction region without affecting the coded amino acid (see below). All of the nucleotide changes are transversions.
  • ser leu arg trp ser stop (SEQ. ID NO. 25) G C A T (resulting nt changes from reverse primer)
  • the reverse primer is complementary to Sindbis nts 7597-7566 (except at nucleotides, as shown, where junction region changes were made), and includes at its 5' end the 6 nucleotide Apa I recognition sequence following a 5' terminal TAT AT tail 'buffer sequence' for efficient enzyme digestion.
  • primer 2A (described in Example 1), having the following sequence:
  • ATACTAGCCACGGCCGGTATC SEQ. ID NO. 6
  • the 4,464 bp amplicon resulting from a PCR reaction with pKSSINBV template and using the primer pair described above is digested with Sfi I and Apa I and the gel purified 2,480 bp fragment is ligated together with the gel purified 5,142 bp fragment resulting from the digestion of pKSSINBV with Apa I and Sfi 1, and with the gel purified 2,961 bp fragment resulting from the digestion of pKSII+ with Apa I and from the treatment with CIAP.
  • This constmction comprised of Sindbis nucleotides 1 - 7597, including the changes in the junction region described above, and including the bacterial SP6 promoter attached to Sindbis nt 1 is referred to as pKS5'SINdlJR.
  • Final constmction of the inactivated junction region vector is accomplished by ligation of the 7,622 bp large Sindbis fragment resulting from digestion of pKS5'SINdlJR with Apa I, with the 3,038 bp fragment resulting from digestion of pKSI 'SIN with Apa I and treatment with CIAP.
  • the positive orientation of the 5' Sindbis element, relative to the 3' Sindbis element, is confirmed by restriction endonuclease analysis. This constmction is referred to as pKSSINBVdIJR.
  • the RNase protection assay demonstrates that cells transfected with pKSSINBV have two fragments, of genomic and subgenomic specificity, while cells transfected with pKSSINBVdIJR have only a single fragment of genomic specificity. These results prove that the junction region in the pKSSINBVdIJR vector is indeed inactivated.
  • the luciferase reporter gene is inserted into the inactivated junction region vector pKSSINBVdIJR described above. This constmction is accomplished by digesting the pKSSINBVdIJR with Xho I and Sac I, treating with CIAP, and gel purifying the resulting 10, 197 bp fragment. The pKSSINBVdIJR fragment is ligated together with the 2854 bp small fragment resulting from digestion of pKSII3'SIN-luc with Xho I and Sac I.
  • This constmction contains the entire Sindbis nonstructural gene coding region terminating in an inactivated junction region at Sindbis nt 7597, and 3' viral elements necessary for genome replication; the firefly luciferase gene is placed between these two viral 5' and 3' elements.
  • This vector is known as pKSSINBVdlJR-luc.
  • the expression of the reporter gene from the pKSSINBVdlJR-luc vector is tested in transfected BHK-21 cells. Translation of functional luciferase protein is determined by the luciferin luminescent assay, using a luminometer for detection. The sensitivity in this assay is 1 x 10"20 moles of luciferase.
  • the minimal -19-. +5 junction region core oligonucleotide pair comprised of Sindbis nts 7579-7602, is synthesized ... vitro, and flanked with Apa I and Xho I recognition sequences as shown:
  • the oligonucleotides above are mixed together in the presence of 10 mM Mg 2+ , heated to 100°C for 5 minutes and cooled slowly to room temperature.
  • the annealed oligonucleotides are ligated at a 25: 1 molar ratio of insert to the pKSSINBVdIJR vector, prepared accordingly: complete digestion with Xho 1, followed by digestion with Apa I under partial conditions, resulting in one Apa I induced cleavage per molecule (of two cleavages possible), gel purification of the 10,655 bp fragment, and treatment with CIAP.
  • pKSSINdlJRsjrc In order to regulate the level of subgenomic mRNA synthesis, further modifications of the tandemly inserted synthetic junction region core in plasmid pKSSINdlJRsjrc are performed. These modifications of the junction region core may be accomplished by at least two approaches: nucleotide changes within the junction region core; or extension at the 5' and 3' junction region core termini of flanking Sindbis nucleotides, according to the authentic viral sequence.
  • the minimal junction region core, spanning viral nts 7579 - 7602 is shown below:
  • These changes observed in the junction region between alphavimses at the level of NSP 4 coding potential and at the level of junction region cis activity may represent either, or both, permissive changes in NSP 4 and the junction region which do not affect functionality, or on the other hand, simply different viruses.
  • junction region changes at Sindbis nts 7600 and 7602 are downstream of the NSP 4 termination codon and upstream of the stmctural proteins initiation codon.
  • junction region nucleotides corresponding to permissive changes are given in the table above.
  • Fourteen junction region nucleotides for which no changes are observed among the eight alphavimses sequenced are given below:
  • Changes within the junction region observed among alphavimses may reflect a specific interaction between a given alphaviral RNA polymerase and its cognate junction region.
  • changes among the "permissive" nucleotides may result in as marked a decrease in the subgenomic mRNA synthesis levels as changes among the "nonpermissive" nucleotides of the junction region.
  • these may indeed be sites of permissive change within the junction region core.
  • the single authentic nonpermissive change within the junction region core is likely Sindbis nt 7598, corresponding to the subgenomic mRNA initiation point. Changes of this nucleotide in the tandemly inserted junction region core of plasmid pKSSINdlJRsjrc are not described here.
  • the oligonucleotides above are mixed together in the presence of 10 mM Mg, heated to 100 °C for 5 minutes and cooled slowly to room temperature.
  • the annealed oligonucleotides are ligated at a 25 1 molar ratio of insert to the pKSSINBVdIJR vector, prepared accordingly: complete digestion with Xho I, followed by digestion with Apa I under partial conditions, resulting in one Apa I induced cleavage per molecule (of two cleavages possible), gel purification of the 10,655 bp fragment, and treatment with CIAP.
  • This vector is known as pKSSINdlJRsjrPc.
  • nt 7582 is changed from T ⁇ G, using the following oligonucleotide pair, synthesized /// vitro, and flanked with Apa I and Xho I recognition sequences as shown:
  • the oligonucleotides above are mixed together in the presence of 10 mM Mg 2+ , heated to 100°C for 5 minutes and cooled slowly to room temperature.
  • the annealed oligonucleotides are ligated at a 25: 1 molar ratio of insert to the pKSSINBVdIJR vector, prepared accordingly: complete digestion with Xho I, followed by digestion with Apa I under partial conditions, resulting in one Apa I induced cleavage per molecule (of two cleavages possible), gel purification of the 10,655 bp fragment, and treatment with CIAP.
  • This vector is known ⁇ KSSINdlJRsjrNP7582.
  • the luciferase reporter gene is inserted into the modified tandem junction region vectors. This constmction is accomplished by digesting with Xho I and Sac I and treating with CIAP the tandemly inserted synthetic junction region core vectors and gel purifying the resulting approximate 10,200 bp fragment. The treated vector fragment is then ligated together with the 2854 bp small fragment resulting from digestion of pKSID'SIN-luc.with Xho I and Sac I.
  • constmctions contain the entire Sindbis nonstmctural gene coding region terminating in an inactivated junction region at Sindbis nt 7597, the tandemly inserted synthetic junction region core (modified or unmodified), the firefly luciferase gene, and 3' viral elements necessary for genome replication.
  • the names of these vectors follows:
  • the relative levels of subgenomic synthesis are determined by comparing the levels of luciferase production at 16 hours post-transfection of BHK-21 cells.
  • the relative levels of subgenomic transcription are determined by comparing luciferase production by the vectors pKSSINBV-luc and pKSSINdlJRsjrc-luc with all of the modified junction region luciferase vectors shown above.
  • pKSSINdlJRsjrc should have a lower level of subgenomic mRNA expression, relative to the pKSSINBV constmct. Therefore, in certain embodiments, it may be necessary to increase the level of subgenomic mRNA expression observed from the pKSSINdlJRsjrc vector. This may be accomplished by extension at the 5' and 3' synthetic junction region core termini with 1 1 additional flanking Sindbis nucleotides, according to the authentic viral sequence.
  • the synthetic oligonucleotide pair shown below is synthesized in vitro, and contains 46 Sindbis nts, including all 24 nts (shown in boldface type) of the minimal junction region core.
  • the Sindbis nts are flanked with the Apa I and Xho I recognition sequences as shown:
  • the oligonucleotides above are mixed together in the presence of 10 mM Mg, heated to 100 °C for 5 minutes and cooled slowly to room temperature.
  • the annealed oligonucleotides are ligated at a 25: 1 molar ratio of insert to the pKSSINBVdIJR vector, prepared accordingly: complete digestion with Xho I, followed by digestion with Apa I under partial conditions, resulting in one Apa I induced cleavage per molecule (of two cleavages possible), gel purification of the 10,655 bp fragment, and treatment with CIAP.
  • This vector containing the entire nonstmctural protein coding region which terminates in an inactivated junction region core, attached to an extended synthetic junction region, and followed by 3' viral elements required for replication, and contained in the pKSII+ plasmid, is known pKSSINdlJRsexjr.
  • the luciferase reporter gene is inserted into the extended tandem junction region pKSSINdlJRsexjr vector. This constmction is accomplished by digesting the pKSSINdlJRsexjr plasmid with Xho I and Sac I, treating with CIAP, and gel purifying the resulting approximate 10,200 bp fragment. The thus-treated vector fragment is ligated together with the 2854 bp small fragment resulting from digestion of pKSI13'S_N-luc with Xho I and Sac I.
  • This constmction contains the entire Sindbis nonstmctural gene coding region terminating in an inactivated junction region at Sindbis nt 7597, the tandemly inserted extended synthetic junction region, the firefly luciferase gene, and 3' viral elements necessary for genome replication.
  • the name of this vector is pKSSrNdlJRsexjr-luc.
  • the relative levels of subgenomic transcription are determined by comparing luciferase production by the pKSSINdlJRsexjr-luc vector with the pKSSINBV-luc and pKSSINdlJRsjrc-luc vectors.
  • the SIN BV and SIN-BV-luciferase constmcts described in sections A and B of Example 3, above, are inserted into the pVGELVIS vector configurations described in Example 2 such that expression of the heterologous gene from Sindbis vectors occurs after direct introduction of the plasmid DNA into cells.
  • the ability to transfect alphavims-based vector plasmid DNA directly onto cells resulting in expression levels of heterologous genes typical of transfection of RNA-based alphavims vectors, without a primary step consisting of/ ' // vitro transcription of linearized template vector DNA enhances greatly the utility and efficiency of certain embodiments of the alphavims-based expression vector system.
  • Figure 8 is a schematic representation of one mechanism of expression of heterologous genes from a plasmid DNA alphavims expression (ELVIS) vectors.
  • ELVIS plasmid DNA alphavims expression
  • the ELVIS vectors may be introduced into the target cells directly by physical means as a DNA molecule, as a complex with various liposome formulations, or as a DNA ligand complex including the alphavims DNA vector molecule, a polycation compound such as polylysine, a receptor specific ligand, and, optionally, a psoralen inactivated vims such as Sendai or Adenovirus.
  • a DNA ligand complex including the alphavims DNA vector molecule, a polycation compound such as polylysine, a receptor specific ligand, and, optionally, a psoralen inactivated vims such as Sendai or Adenovirus.
  • the first step of constructing one representative plasmid DNA Sindbis expression vector consists of digesting pKSSINBV with Sac I, blunting with T4 polymerase, digesting with Sfi I, isolating the 2,689 bp fragment, and ligating into the pVGELVIS 10,053 bp vector fragment prepared by digestion with Xha ⁇ , blunting with T4 polymerase, digesting with Sfi I, treatment with CIAP, and 1% agarose/TBE gel electrophoresis. This constmction is known as pVGELVIS-SINBV.
  • the SV40 intron and transcription termination sequences at the 3' end of luciferase must be removed so that when the pre-RNA, transcribed from the plasmid DNA luciferase vector after transfection into cells, is processed the 3' end of the reporter gene is not separated from the Sindbis vector 3' end.
  • the Sindbis 5' and 3' ends contained within the pVGELVIS-SINBV vector are required in tv ' .v for the autocatalytic replication activity of the vector.
  • the Sindbis vector 3' end is required for initiation of synthesis of the antigenomic strand, which is the template for the subgenomic RNA encoding the heterologous or reporter protein.
  • the SV40 RNA processing signals positioned at the 3' end of the luciferase gene are removed from the SIN-BV-luc constmction described in section B above.
  • the modified luciferase fragment is then placed in the pVGELVIS-SINBV constmction described above via unique restriction sites.
  • the alteration of the luciferase gene is accomplished with the primer pair shown below:
  • Reverse primer LucStop (buffer sequence/M?t I. Xba I recognition sequences/pGL-2 nts 1725-1703):
  • the primers shown above are used in a PCR reaction with a three temperature cycling program using a 3 minute extension period.
  • the amplification products are purified with GENECLEAN (Bio 101 , Vista, C A), digested with Xho I and Xba I, purified again with GENECLEAN, and the 2,037 bp fragment is ligated into the 13,799 bp fragment of pVGELVIS-SINBV resulting from digestion with Xho I and Xba I, and treatment with CIAP.
  • This constmction is known as pVGELVIS-SINBV-luc (abbreviated as ELVIS-luc).
  • luciferase in BHK cells transfected with pVGELVIS-SINBV- luc DNA is measured in order to demonstrate that the Sindbis physical gene transfer vector is functional.
  • 5 ⁇ g of pVGELVIS-SINBV-luc DNA or 5 ⁇ g of in vitro transcribed RNA from linearized SINBV-luc template as described in section B, above are complexed with 10 ⁇ l of lipofectamine or lipofectin (GIBCO-BRL, Gaithersburg, MD), respectively, and transfected into 5 x 10 5 BHK cells contained in 35 mM petri plates.
  • the luciferase activity is determined from each of three samples at 2, 4, 8, 16, 20, 28, 48, 72, 96, and 120 hrs.
  • Sindbis NSPs are first deleted from the pVGELVIS-SINBV-luc vector in order to demonstrate the requirement for the viral enzymatic proteins for high levels of reporter gene expression. This is accomplished by digestion of pVGELVIS-SINBV-luc DNA with Bsp El, purification with GENECLEAN, and ligation under dilute conditions. This constmction is deleted of nonstmctural gene sequences between bases 422-7,054 and is analogous to the pVGSP6GENdl#.s/.
  • the constmction described here is known as pVGELVIS-SINBVdl/.sp-luc (abbreviated as dlNSP ELVIS-luc).
  • dlNSP ELVIS-luc pVGELVIS-SINBVdl/.sp-luc
  • the reporter is first inserted into the pCDNA3 vector (Invitrogen, San Diego, CA) between the Bam HI and Hind III sites.
  • the luciferase fragment is derived from pGL2 plasmid exactly as described in Example 3 section B, above, and inserted into the 5428x bp fragment of pCDNA3 prepared by digestion with Hind III and Bam HI, treatment with CIAP, and purification on a 1% agarose/TBE gel. This constmction is known as pCDNA3-luc.
  • the U3 region of the MoMuLV LTR is amplified from the BAG vector using the PCR primers shown below as described in Example 2.
  • BAGwt441R2 (SIN nts 5-1 /Mo-MLV LTR nts 441-406):
  • the amplification products are purified with GENECLEAN and the ends are first blunted with T4 DNA polymerase, then digested with Bgl II, purified with GENECLEAN and ligated into the pCDNA3-luc plasmid prepared by digestion with Hind III, blunting with the Klenow enzyme and 50 ⁇ M dNTPs, digestion with Bgl II, and purification by 1% agarose/TBE gel electrophoresis. This constmction is known as LTR-luc.
  • the plasmids ELVIS-luc, dlNSP ELVIS-luc, LTR-luc, and pCDNA3 are each complexed with 10 ⁇ l of lipofectamine and transfected into 5 x 10 5 BHK cells contained in 35 mM petri plates.
  • the luciferase activity is determined from each of three samples at 48 hrs. post transfection.
  • the results of this study, given in Figure 10, demonstrate that the level of heterologous gene expression enhancement provided by the ELVIS system, compared to the same promoter linked directly to the heterologous gene is at least 100-fold.
  • the comparatively low level of luciferase expression in cells transfected with the dlNSP ELVIS-luc constmction demonstrates that the expression enhancement is a direct result of functional Sindbis NSPs.
  • the autocatalytic amplification of the reporter gene mRNA as diagrammed in Figure 8 provides a significant advantage in terms of levels of gene expression, compared to primary transcription from simple promoter-heterologous gene constmctions.
  • the overall efficiency of the ELVIS vector is enhanced by several modifications to the pVGELVIS- SINBV-luc vector. These modifications include alternate RNA polymerase II promoters and transcription termination signals, additions of intron sequences in the vector constmct, and substitution with a smaller plasmid vector. The constmction of these modified ELVIS vectors is detailed below.
  • the modified ELVIS vector is assembled on the plasmid vector pBGS131 (ATCC # 37443) which is a kanamycin resistant analogue of pUC 9 (Spratt et al., Gene 77:337-342, 1986). Propagation of pBGS131 is in LB medium with 10 ⁇ g/ml kanamycin.
  • the transcription termination signals from the SV40 early region or Bovine growth hormone are inserted between the Sac I and Eco RI sites of pBGS131.
  • the SV40 nts between viral nts 2643 to 2563 containing the early region transcription termination sequences are isolated by PCR amplification using the primer pair shown below and the pBR322/SV40 plasmid (ATCC # 45019) as template.
  • Reverse primer RSVTT2563R (buffer sequence/ / w RI site/SV40 nts 2563-2588):
  • the primers shown above are used in a PCR reaction with a three temperature cycling program as described throughout this example, using a 30 second extension period.
  • the amplification products are purified with GENECLEAN (Bio 101, Vista, CA), digested with Sac I and Eco RI, purified again with GENECLEAN, and the 90 bp fragment is ligated into the 3,655 bp fragment of pBGS131 resulting from digestion with Sac I and Eco RI, and treatment with CIAP. This constmction is known as pBGS131-3'SV40TT
  • Bovine growth hormone transcription termination sequences are isolated by PCR amplification using the primer pair shown below and the pCDNA3 plasmid (Invitrogen, San Diego, CA) as template.
  • Reverse primer BGHTTR buffer sequence/Eco RI site/pCDNA3 nts 1 180-1 154):
  • the primers shown above are used in a PCR reaction with a three temperature cycling program, using a 30 sec. extension period.
  • the amplification products are purified with GENECLEAN (Bio 101, Vista, CA), digested with Sere I and Eco RI, purified again with GENECLEAN, and the 58 bp fragment is ligated into the 3,655 bp fragment of pBGS131 resulting from digestion with Sac I and Eco RI, and treatment with CIAP. This constmction is known as pBGS 13 1 -3'BGHTT.
  • the transcription termination signals are fused directly to the 3' end of the ELVIS vector and the poly-adenylate tract is deleted, or alternatively the antigenomic ribozyme sequence of hepatitis delta virus (HDV) is placed between the poly-adenylate tract at the 3' end of the ELVIS-luc vector and the transcription termination signals.
  • HDV hepatitis delta virus
  • the HDV ribozyme-containing constmct is generated with PCR techniques and overlapping oligonucleotide primers which contain the minimal 84 nucleotide antigenomic ribozyme sequence (Perotta and Been, Nature 350:434-6, 19 1).
  • the primers contain flanking Sac I recognition sites for insertion at the 3' end of the
  • the HDV ribozyme sequence is generated with the three overlapping primers shown below.
  • Nested primer HDV 17-68 5'-TATATGAGCTCGGGTCGGCATGGCATCTCCACCTCCTCGCGGTCCG (SEQ. ID NO. 39) Nested primer HDV 17-68:
  • the primers shown above are used in a PCR reaction with a three temperature cycling program as described throughout this example, using a 30 sec. extension period.
  • the amplification products are purified with GENECLEAN (Bio 101, Vista CA), digested with Sac I, purified again with GENECLEAN, and the 94 bp fragment is ligated into Sac I linearized and CIAP treated pBGS 131-3'SV40TT or pBGS131-3 ⁇ GHTT. These const ctions are known as pBGS 131/HDV/3'SV40TT and pBGS131/HDV/3'BGHTT. Insertion of the HDV ribozyme in the correct orientation in the Sac I site is determined by sequencing.
  • the SV40 or BGH transcription termination signals are fused directly to the 3' end of the ELVIS vector corresponding to Sindbis nt 1 1,700 and the poly-adenylate tract is deleted. This constmction is accomplished according to the steps outlined above in Example 3, sections A and B for the assembly of the pKSSINBV and pKSSINBV-luc vectors.
  • the vector 3' end primer does not contain a 25 poly-adenylate tract.
  • the 3' end of the vector is synthesized with the primer pair shown below:
  • SIN 1 1664F (buffer sequence/Not I site/ SIN nts 1 1664-1 1698):
  • the ELVIS expression vectors are assembled further onto the various 3' end processing plasmid constructions described above.
  • the Sindbis vectors containing a polyadenylate tract are combined with the plasmid constmctions containing the HDV ribozyme sequence and the SV40 or BGH transcription termination signals.
  • This constmction corresponds to the insertion of pKSSINBV and pKSSINBV-luc vector sequences into the pBGS 131/HDV/3'SV40TT and pBGS131/HDV/3'BGHTT plasmids.
  • the Sindbis vectors terminating precisely at the viral 3' end corresponding to viral nt 11,700 are linked directly to the SV40 or BGH transcription termination signals.
  • This constmction corresponds to the insertion of pKSSINBVdlA and pKSSINBVdlA-luc vector sequences into the pBGS 13 1/HDV/3'SV40TT and pBGS131/HDV/3'BGHTT plasmids.
  • Sindbis vectors pKSSINBV and pKSSINBV-luc are digested with Sac I and Bgl II, and the 5,522 bp (pKSSINBV) or 821 1 bp (pKSSINBV-luc) fragments are purified by 1% agarose/TBE gel electrophoresis and inserted into the linearized pBGS131/HDV/3'SV40TT and pBGS131/HDV/3'BGHTT plasmids prepared by digestion with Sac I and Bgl II and treatment with CIAP. These constmctions are known as:
  • Sindbis vectors pKSSINBVdlA and pKSSINBVdlA-luc are digested with Sac I and Bgl II, and the 5,497 bp (pKSSINBVdlA) or 8186 bp (pKSSINBVdlA-luc) fragments are purified by 1% agaroseTBE gel electrophoresis and inserted into the linearized pBGS 131/3'SV40TT and pBGS131/3'BGHTT plasmids prepared by digestion with Sac I and Bgl II and treatment with CIAP.
  • These constmctions are known as:
  • RNA polymerase II promoter and Sindbis nucleotides 1-2289 is the last step required to complete the constmction of the modified ELVIS expression vectors of the four constmctions shown below: pBGS 13 l/dlproSINBV/HDV/3'SV40TT pBGS13 l/dlproSINBV-luc/HDV/3'BGHTT pBGS13 l/dlproSINBV/3'SV40TT pBGS131/dlproSINBV-luc/3'BGHTT
  • RNA polymerase II promoter and Sindbis nucleotides 1-2289 are inserted into these constmctions by the overlapping PCR technique described for the pVGELVIS constmction in Example 2.
  • the four constmctions shown above are digested with Bgl II and treated with CIAP.
  • the U3 region of the long terminal repeat (LTR) from Moloney murine leukemia vims (Mo-MLV) is positioned at the 5' viral end such that the first transcribed nucleotide is a single G residue, which is capped /// vivo, followed by the Sindbis 5' end.
  • Amplification of the Mo-MLV LTR in the first primary PCR reaction is accomplished in a reaction containing the BAG vector (Price et al., PNAS 84: 156-160, 1987) and the following primer pair:
  • BAGwt441 R2 (SIN nts 5- 1 /Mo-MLV LTR nts 441-406):
  • the primers shown above are used in a PCR reaction with a three temperature cycling program using a 30 second extension period.
  • Amplification of the Sindbis 5' end in the second primary PCR reaction is accomplished in a reaction containing the pVGSP ⁇ GENrep clone and the following primer pair:
  • the primers shown above are used in a PCR reaction with a three temperature cycling program using a 3 minute extension period.
  • the 457 bp and 3202 bp products from the primary PCR reactions are purified with
  • the primers shown above are used in a PCR reaction with a three temperature cycling program using a 3 minute extension period.
  • the 25 3' terminal bases of the first primary PCR amplicon product overlaps with the
  • the CMV promoter is positioned at the 5' viral end such that transcription initiation results in the addition of a single non-viral nucleotide at the Sindbis 5' end.
  • Amplification of the CMV promoter in the first primary PCR reaction is accomplished in a reaction containing the pCDNA3 plasmid (Invitrogen, San Diego, CA) and the following primer pair:
  • the primers shown above are used in a PCR reaction with a three temperature cycling program using a 1 minute extension period.
  • Amplification of the Sindbis 5' end in the second primary PCR reaction is accomplished in a reaction containing the pVGSP ⁇ GENrep clone and the following primer pair:
  • the primers shown above are used in a PCR reaction with a three temperature cycling program using a 3 minute extension period.
  • the 600 bp and 3200 bp products from the primary PCR reactions are purified with GENECLEAN, and used together in a PCR reaction with the following primer pair: Forward primer: pC7?y/233F (buffer sequence/ ⁇ / II recognition sequence/CMV promoter nts 1-22):
  • the primers shown above are used in a PCR reaction with a three temperature cycling program using a 3 minute extension period.
  • the 26 3' terminal bases of the first primary PCR amplicon product overlaps with the 26 5' terminal bases of the second primary PCR amplicon product; the resultant 2,875 bp overlapping secondary PCR amplicon product is purified by 1% agarose/TBE electrophoresis, digested with Bgl II, and ligated into the four ELVIS constmctions described above. These constmctions are named as shown below:
  • the SV40 late region promoter is positioned at the 5' viral end such that the major cap site of transcription initiation results in the addition of a single non-viral nucleotide at the Sindbis 5' end.
  • Amplification of the SV40 promoter in the first primary PCR reaction is accomplished in a reaction containing the pBR322/SV40 plasmid (ATCC # 45019) and the following primer pair:
  • B2SVpr250F buffer sequence//. ⁇ / II recognition sequence/SV40 nts 250- 231):
  • the primers shown above are used in a PCR reaction with a three temperature cycling program using a 30 second extension period.
  • Amplification of the Sindbis 5' end in the second primary PCR reaction is accomplished in a reaction containing the pVGSP ⁇ GENrep clone and the following primer pair:
  • the primers shown above are used in a PCR reaction with a three temperature cycling program using a 3 minute extension period.
  • the 280 bp and 3, 194 bp products from the primary PCR reactions are purified with GENECLEAN, and used together in a PCR reaction with the following primer pair:
  • B2SVpr250F buffer sequence/ ⁇ / II recognition sequence/ SV40 nts 250- 231):
  • the 25 3' terminal bases of the first primary PCR amplicon product overlaps with the
  • the luciferase expression levels after transfection of BHK cells are determined with each of the reporter gene containing complete modified ELVIS constmctions detailed above in order to determine the desired configuration.
  • the heterologous gene is inserted into the multiple cloning site of the ELVIS vector, as described for the insertion of the luciferase gene in Example 3, section B.
  • the SV40 small t antigen intron can be inserted into the ELVIS expression vectors. Insertion of the SV40 small t antigen intron sequences is at the vector unique Xho I site immediately downstream of the 5' Sindbis sequences, or alternatively at the Not I site immediately upstream of the 3' Sindbis sequences. For insertion into the Xho I site of the ELVIS vectors, amplification of the SV40 small t antigen intron sequences is accomplished in a reaction containing the pBR322/SV40 plasmid (ATCC # 4501 ) and the following primer pair:
  • Reverse primer 5'-TATATATCTCGAGAAGCTCTAAGGTAAATATAAAATTTACC (SEQ. ID NO. 50)
  • Reverse primer XSVSA4562R (buffer seq ⁇ ence/ ⁇ 77r. I recognition sequence/SV40 nts 4562-4537):
  • the primers shown above are used in a PCR reaction with a three temperature cycling program using a 30 second extension period.
  • the amplification products are purified with GENECLEAN, digested with Xho I, re-purified with GENECLEAN and inserted into Xho I linearized and CIAP treated complete modified ELVIS vectors described above. Insertion of the SV40 small t antigen intron in the correct orientation in the ELVIS vector is determined by sequencing.
  • amplification of the SV40 small t antigen intron sequences is accomplished in a reaction containing the pBR322/SV40 plasmid (ATCC #) and the following primer pair:
  • NSVSD4647F buffer sequence/Not I recognition sequence/SV40 nts 4647-4675
  • Reverse primer XSVSA4562R (buffer sequence/Not I recognition sequence/SV40 nts 4562-4537):
  • the primers shown above are used in a PCR reaction with a three temperature cycling program using a 30 second extension period.
  • the amplification products are purified with GENECLEAN, digested with Not I, re-purified with GENECLEAN and inserted into Not I linearized and CIAP treated complete modified ELVIS vectors described above. Insertion of the SV40 small t antigen intron in the correct orientation in the ELVIS vector is determined by sequencing. Alternatively, the SV40 small t antigen may be inserted at other sites within the ELVIS vector, which do not impair function of the vector, using the disclosure provided herein.
  • the luciferase expression levels after transfection of BHK cells with the SV40 small t antigen intron containing ELVIS vectors are assayed in order to determine the desired configuration.
  • the heterologous gene is inserted into the multiple cloning site of the ELVIS vector, as described for the insertion of the luciferase gene in Example 3, section B.
  • a linker sequence is inserted into the pKSSINBV and into the pVGELVIS-SINBV constmcts to facilitate the insertion of heterologous sequences.
  • the linker is constmcted using two complementary 35nt oligonucleotides that form a duplex with ⁇ Tr ⁇ l and Xba l compatible sticky ends when hybridized.
  • SINBVLinkF 5TCGAGCACGTGGCGCCTGATCACGCGTAGGCCT (SEQ. ID NO. 54)
  • SINBVLinkR 5'CTAGAGGCCTACGCGTGATCAGGCGCGCCACGTGC (SEQ. ID NO. 55)
  • the oligonucleotides are phosphorylated with T4 polynucleotide kinase, heated to 90°C, and slow cooled to allow hybridization to occur.
  • the hybrid is then ligated to the 10.6kb fragment of pKSSINBV-Luc obtained after digestion with Xho I and Xba I, followed by treatment with alkaline phosphatase and agarose gel purification.
  • the resulting constmct contains Xho I, Pml I, Asc I, Bel I, Mlu I, Slit I, Xba I, and Not I as unique sites between the Sindbis junction region and the Sindbis 3' end. This constmct is known as pKSSINBV- Linker.
  • This linker also is cloned into the pVGELVIS-SINBV constmcts.
  • the linker is inserted by digestion of pVGELVIS-SINBV-luc with Sfi I and Not I.
  • the 10. lkb fragment is agarose gel purified, and this fragment was ligated to the gel purified 2.6kb fragment from a Sfi l/Nol l digest of pKSSI ⁇ BV-Linker.
  • the resulting constmct contains Xho I, Pml I, Asc I, Mlu I, and Not I as unique sites between the Sindbis junction region and the Sindbis 3' end. This constmct is known as pVGELVIS-SI ⁇ BV-Linker.
  • Ad 2 Adenovims type 2 (Ad 2) E3/19K gene ATCC No. VR-846 is cloned into the pKSSINdlJRsjrc plasmid, immediately downstream from the junction region core.
  • Ad 2 is propagated in a permissive cell line, for example HeLa or Vero cells, and after evidence of cytopathologic effects, virions are purified from the cell lysate, and the Ad 2 DNA is purified from the vims.
  • Ad 2 DNA E3/19K gene including the amino terminal signal sequence, followed by the intraluminal domain and carboxy terminal cytoplasmic tail which allows the E3 19K protein to embed itself in the endoplasmic reticulum, is located between viral nucleotides
  • Ad 2 E3 Forward primer (Ad 2 nucleotides 28,812-28,835):
  • Ad 2 E3 Reverse primer (Ad 2 nucleotides 29,241-29,213):
  • both primers contain a five nucleotide 'buffer sequence 1 at their 5' ends for efficient enzyme digestion of the PCR amplicon products.
  • This sequence in the forward primer is followed by the Xho I recognition site, and in the reverse primer this sequence is followed by the Cla I recognition site.
  • the E3/19K gene is flanked by Xho I and Cla I recognition sites.
  • Amplification of the E3/19K gene from Ad 2 DNA is accomplished with the following PCR cycle protocol:
  • the 451 bp amplicon is purified on a 1.5% agarose gel, and subsequently digested with the Xho I and Cla I enzymes and ligated into the CIAP treated pKSSINdlJRsjrc plasmid, previously digested with Xho I and Cla I.
  • This clone is designated pKSSINdlJRsjrcAdE3.
  • the Ad 2 E3/19K gene is inserted into all of the modified synthetic junction region vectors described in Example 2.
  • HCMV human cytomegalovirus
  • the HCMV H301 gene is located between viral nucleotides 23,637 and 24,742. Isolation of the HCMV H301 gene from the viral genomic DNA is accomplished by PCR amplification, with the primer pair shown below:
  • HCMV H301 Forward primer buffer sequence/ ⁇ T/o I site/ HCMV nucleotides
  • HCMV H301 Reverse primer buffer sequence/(7 ⁇ I site/HCMV nucleotides 24,744- 24,722):
  • both primers contain a five nucleotide 'buffer sequence' at their 5' ends for efficient enzyme digestion of the PCR amplicon products. This sequence in the forward primer is followed by the Xho I recognition site, and in the reverse primer this sequence is followed by the Cla I recognition site. Thus, in the 5' to 3' direction, the HCMV H301 gene is flanked by Xho I and Cla I recognition sites. Amplification of the HCMV H301 gene from HCMV DNA is accomplished with the following PCR cycle protocol:
  • the 1,129 bp amplicon product is purified on a 1.0% agarose gel, and subsequently digested with the Xho I and Cla I enzymes and ligated into the CIAP treated pKSSINdlJRsjrc plasmid, previously digested with Xho I and Cla I.
  • This clone is designated pKSSINdlJRsjrcH30 l .
  • the HCMV H301 gene is inserted into all of the modified synthetic junction region vectors described in Example 3.
  • the plasmid pBS-ECAT (Jang et al., ./. Virol 63: 1651, 1989) includes the 5' nontranslated region of Encephalomycarditis vims (EMCV) from nts 260-848 of the viral genome, which contains the internal ribosome entry site (IRES).
  • EMCV nucleotides 260- 827 are amplified from pBS-ECAT by PCR, using the following primer pair:
  • the amplicon resulting from amplification with the forward primer B and the reverse primer is flanked by Cla I and Neo I recognition sites, inside a 5 bp 'buffer sequence'.
  • Amplification of the EMCV IRES sequence from the pBS-ECAT plasmid is accomplished with the following PCR cycle protocol:
  • the 589 bp amplicon is digested with Apa I and Neo I, purified on a 1% agarose gel, and ligated into the CIAP treated vector digested with Apa 1 and Neo I.
  • the ATG corresponding to the start codon of the heterologous gene to be inserted immediately downstream of the EMCV IRES insert is modified to contain an Neo I site (CCATGG).
  • the 589 bp amplicon is digested with Cla I and Neo I, purified on a 1% agarose gel, and ligated into the bicistronic heterologous gene vector digested with Cla I and Neo I and treated with CIAP.
  • the 3' end of the upstream heterologous gene is modified to terminate in a Cla I recognition site.
  • the ATG corresponding to the start codon of the second downstream heterologous gene to be inserted immediately downstream of the EMCV IRES insert is modified to contain an Neo I site (CCATGG).
  • the order of components is: pKSSINBV or pKSSINBVdIJRsjrc-gene #l-Cla/Nco EMCV IRES gene #2-3' SIN. Insertion into all of the modified junction region vectors described in Example 2 follows the strategy given here for the pKSSINBV or pKSSINBVdlJRsjrc vectors.
  • the pKSSINBVdIJR vector containing a bicistronic heterologous configuration is constmcted with each of the EMCV IRES amplicons described above.
  • the first EMCV IRES amplicon is flanked by Apa I and Neo I sites and is inserted immediately downstream of the disabled junction region at the Apa I site, as described above.
  • This EMCV IRES sequence is followed by the first heterologous gene, which terminates in a Cla I recognition site.
  • the first heterologous gene is followed by the second EMCV IRES sequence, using the amplicon flanked by Cla I and Neo I recognition sites.
  • the second heterologous gene follows the second EMCV IRES sequence.
  • the order of components is: SINBVdlJR-/_/>-7/Nco EMCV IRES gene #l-Cla Nco EMCV IRES gene #2-3' SIN.
  • the plasmid pP2-5' (Pelletier et al., Mol. Cell Biol. 8: ⁇ ⁇ 03, 1988) includes the 5' nontranslated region of the poliovims P2/Lansing strain from nucleotides 1-1,872 of the viral genome, which contains the polio IRES. Poliovims nucleotides 320-631 are amplified from pP2-5' by PCR, using the following primer pair:
  • Polio IRES Forward primer A (For insertion next to disabled junction region in vector pKSSINBVdIJR at Apa I site):
  • Polio IRES Forward primer B (For insertion between heterologous genes terminating with Cla I sites and initiating with Neo I sites):
  • Polio IRES Reverse Primer (To be used with either primers A or B):
  • the amplicon resulting from PCR with the Polio IRES forward primer A/reverse primer pair shown above is flanked by Apa I and Neo I recognition sites, inside a 5 bp 'buffer sequence'.
  • the amplicon resulting from PCR with the Polio IRES forward primer B/reverse primer pair is shown above is flanked by Cla I and N o I recognition sites, inside a 5 bp 'buffer sequence'.
  • Amplification of the polio IRES sequence from the pP2-5' plasmid is accomplished with the PCR protocol shown in Example 5.
  • the 333 bp amplicon is digested with
  • Apa I and Neo 1 purified on a 1.5% agarose gel, and ligated into the vector digested with Apa I and Neo 1 and treated with CIAP.
  • the ATG corresponding to the start codon of the heterologous gene to be inserted immediately downstream of the polio IRES insert is modified to contain an Neo I site (CCATGG).
  • the 333 bp amplicon is digested with Cla I and Neo I, purified on a 1.5% agarose gel, and ligated into the bicistronic heterologous gene vector digested with Cla I and Neo I and treated with CIAP.
  • the 3' end of the upstream heterologous gene is modified to terminate in a Cla I recognition site.
  • the ATG corresponding to the start codon of the second downstream heterologous gene to be inserted immediately downstream of the polio IRES insert is modified to contain an Neo I site (CCATGG).
  • the order of components is: pKSSINBV or pKSSINBVdIJRsjrc-gene #l-Cla/Nco polio IRES gene #2-3' SIN. Insertion into all of the modified junction region vectors described in Example 3 follows the strategy given here for the pKSSINBV or pKSSINBVdlJRsjrc vectors.
  • the pKSSINBVdIJR vector containing a bicistronic heterologous configuration is constmcted with each of the polio IRES amplicons described above.
  • the first polio IRES amplicon is flanked by Apa I and Neo I sites and is inserted immediately downstream of the disabled junction region at the Apa 1 site, as described above.
  • This polio IRES sequence is followed by the first heterologous gene, which terminates in a Cla I recognition site.
  • the first heterologous gene is followed by the second polio IRES sequence, using the amplicon flanked by Cla I and Neo I recognition sites.
  • the second heterologous gene follows the second polio IRES sequence.
  • the order of components is: SINBVdlJR- _/r ⁇ /Nco polio IRES gene # l -Cla/Nco EMCV IRES gene #2-3' SIN.
  • the 220 bp BiP cDNA corresponding to the 5' leader region of the human immunoglobulin heavy-chain binding protein mRNA, is amplified from the clone pGEM5ZBiP5', using PCR.
  • the sequence corresponding to BiP cDNA was determined originally in the bacteriophage lambda hu28- 1 clone of the human GRP78 gene (Ting and Lee, DNA 7:275-286, 1988).
  • the forward primer to be used in the PCR reaction varies, depending on the Sindbis vector into which the BiP cDNA is inserted.
  • the reverse primer for the PCR reaction is the same for all Sindbis vectors.
  • Amplification of the BiP cDNA sequence from pGEM5ZBiP5' from the plasmid for insertion into the Sindbis vector pKSSINBVdIJR, immediately downstream of the disabled junction region, is accomplished by amplification with the following forward primer:
  • the primer contains a five nucleotide 'buffer sequence' at its 5' end for efficient enzyme digestion of the PCR amplicon products. This sequence is followed by the Apa I recognition site.
  • Amplification of the BiP cDNA sequence from the pGEM5ZBiP5' plasmid for insertion into the Sindbis vectors pKSSINBV, or pKSSINBVdlJRsjrc, is accomplished by amplification with the following forward primer shown below.
  • the BiP cDNA is inserted between two heterologous genes, which are placed in the region corresponding to the Sindbis stmctural genes.
  • the primer contains a five nucleotide 'buffer sequence' at its 5' end for efficient enzyme digestion of the PCR amplicon products. This sequence is followed by the Cla I recognition site.
  • the reverse primer for amplification of the BiP cDNA sequence from the ⁇ GEM5ZBiP5' plasmid for insertion into the Sindbis vectors pKSSINBVdIJR, pKSSINBV, or pKSSINBVdlJRsjrc, is:
  • the reverse primer contains a five nucleotide 'buffer sequence' at its 5' end for efficient enzyme digestion of the PCR amplicon products. This sequence is followed by the Neo I recognition site. Amplification of the BiP cDNA from the pGEM5ZBiP5' is accomplished with PCR protocol that are described above.
  • the 242 bp amplicon is digested with Apa I and Neo I, purified on a 2% agarose gel, and ligated into the vector digested with Apa
  • Neo I and Neo I and treated with CIAP The ATG corresponding to the start codon of the heterologous gene to be inserted immediately downstream of the BiP cDNA insert is modified to contain an Neo I site (CCATGG).
  • the 242 bp amplicon is digested with Cla I and Neo I, purified on a 2% agarose gel, and ligated into the bicistronic heterologous gene vector digested with Cla I and
  • Neo I and treated with CIAP In a biscistronic heterologous gene configuration, the 3' end of the upstream heterologous gene is modified to terminate in a Cla I recognition site.
  • the ATG corresponding to the start codon of the second downstream heterologous gene to be inserted immediately downstream of the BiP cDNA insert is modified to contain an Neo I site (CCATGG).
  • CCATGG Neo I site
  • the pKSSINBVdIJR vector containing a bicistronic heterologous configuration is constmcted with each of the BiP cDNA amplicons described above.
  • the first BiP cDNA amplicon is flanked by Apa I and Neo I sites and is inserted immediately downstream of the disabled junction region at the Apa I site, as described above.
  • This BiP sequence is followed by the first heterologous gene, which terminates in a Cla I recognition site.
  • the first heterologous gene is followed by the second BiP cDNA sequence, using the amplicon flanked by Cla I and Neo I recognition sites.
  • the second heterologous gene follows the second BiP sequence.
  • the order of components is: SINBVdlJR- Apa/Nco BiP-gene # I -Cla/Nco BiP-gene #2-3' SIN.
  • Sequences which promote ribosomal readthrough are placed immediately downstream of the disabled junction region in the pKSSINBVdIJR vector, which allows ribosomal scanning in genomic mRNA from non-stmctural gene termination to the heterologous genes.
  • the heterologous proteins are expressed from genomic length mRNA by ribosomal scanning. This extends the life of the infected target cell because no subgenomic transcription occurs in cells infected with this vector.
  • these same ribosomal scanning sequences are placed between heterologous genes contained in polycistronic subgenomic mRNAs.
  • the ribosomal spanning sequence to be used in the pKSDINBVdlJR vector and between heterologous genes in the polycistronic mRNA region is:
  • the boldfaced codons refer to the ochre stop codon and AUG start codon, respectively.
  • the bases underlined surrounding the stop codon refer to the Pac I recognition site and the bases underlined surrounding the start codon refer to the Neo I recognition site.
  • the intercistronic distance of 15 bp between the start and stop codons allows efficient ribosomal readthrough, as shown previously (Levine et al.. Gene 108: 167-174, 1991).
  • the sequences surrounding the ATG start codon from bases -9 to +1 conform to the Kozak consensus sequence for efficient translational initiation (Kozak, Cell 77:283-292, 1986).
  • the 3' terminal nucleotide corresponding to the carboxy terminal amino acid is changed to T, by site-directed mutagenesis.
  • the 5' terminal nucleotide corresponding to the amino terminal amino acid in the downstream cistron is changed to G, by site-directed mutagenesis.
  • Insertion of the intercistronic sequence between heterologous genes, or downstream of the disabled junction region in vector pKSDINBVdlJR, modified as described above, is accomplished by insertion of the double-stranded oligonucleotide pair shown below, into compatible Pac l/Nco I ends:
  • oligonucleotides above are mixed in equal molar quantities in the presence of 10 mM MgC ⁇ , heated at 95°C for 5 min, then allowed to cool slowly to room temperature, yielding the desired intercistronic sequence flanked by Pac I and Neo I sites.
  • the intercistronic sequence is then ligated into the appropriate vector containing Pac I and Neo I compatible sites.
  • RNA molecules in the same alphavims vector particle can be useful for the expression of multiple heterologous gene products from a single alphaviais vector particle.
  • this concept can also be adapted in order to allow very large genes to be carried on RNA molecules separate from the alphavims vector
  • RNA containing the nonstmctural genes thus avoiding the need to package very long vector
  • RNA molecules In order to accomplish such copackaging, all RNA fragments must contain a
  • RNA polymerase recognition sequence for minus-strand synthesis, and at least one copy of the RNA packaging sequence. At least one of the RNA fragments also must contain sequences which code for the alphavims non-stmctural proteins. Within preferred embodiments of the invention, one or more of the RNA fragments to be copackaged also will contain a viral junction region followed by a heterologous gene.
  • A. CONSTRUCTION OF COPACKAGED EXPRESSION CASSETTES FOR EXPRESSION OF MI JLTII'I ,E HI .TEI .( )i.( x . )i is GENES
  • the first constmct consists of a 5' sequence that is capable of initiating transcription of Sindbis vims RNA, Sindbis RNA sequences required for packaging, sequences encoding the synthesis of nonstmctural proteins 1-4, a Sindbis junction region, the luciferase gene, and Sindbis 3' sequences required for synthesis of the minus strand RNA.
  • the second constmct consists of a 5' sequence that is capable of initiating transcription of a Sindbis vims, a Sindbis Junction region, Sindbis sequences required for packaging, Sequences encoding the LacZ gene, and Sindbis 3' sequences required for synthesis of the minus strand RNA.
  • RNA transcripts of these constmcts transfected into a packaging cell line are copackaged to produce a vector particle capable of transferring expression of both luciferase and ⁇ -galactosidase into the same eukaryotic cell.
  • the ⁇ -galactosidase reporter gene is inserted into the Sindbis Basic Vector
  • RNA from this constmct is cotransfected with RNA from Sindbis Luciferase Vector (pKSSINBV-luc) and is copackaged by one of the methods described in Example 7. Infection of fresh BHK-21 cells with vector particles containing the copackaged RNA expression cassettes should result in the expression of both luciferase and ⁇ -galactosidase in the same cell.
  • pKSSINBV-Linker- Pmel The Pme I recognition site is substituted for the Sac I site in order to create a site for linearization of the plasmid prior to SP6 transcription.
  • the lacZ gene contains several Sac I sites.
  • pKSSINBV-Linker-/ '/WI is digested with Pml I and Bel I followed by purification with GENECLEAN.
  • the lacZ gene is obtained by digestion of pSV ⁇ -galactosidase vector DNA (Promega Corp., Madison, WI) with the enzyme Hindlll.
  • the digest is blunt-ended with Klenow DNA polymerase and dNTPs.
  • the Klenow is heat killed and the plasmid is fiirther digested with Bam HI and Xmn I.
  • Xmn I reduces the size of the remaining vector fragment to simplify gel purification of the lacZ fragment.
  • the 3.7kbp lacZ fragment is purified from a 1% agarose gel and ligated into the Pml UBcl I digested pKSSINBV-Linker- Pmel fragment. This constmct is known as pKSSINBV-lacZ.
  • pKSSINBV-lacZ is digested with Bsp El and religated under dilute conditions.
  • Sindbis constmct is known as pKSSINBVdlNSP-lacZ.
  • pKSSINBVdlNSP-lacZ and pKSSINBV-luc are linearized with Pme I and Sac I, respectively, and SP6 transcripts are prepared as described in Example 3. These RNA transcripts are cotransfected into packaging cells that express the Sindbis stmctural proteins by one of the mechanisms described in Example 7.
  • Each RNA transcript contains a 5' sequence that is capable of initiating transcription of a Sindbis vims, RNA sequences required for packaging, a Sindbis junction region, a reporter gene, and Sindbis 3' sequences required for synthesis of the minus strand RNA.
  • the pKSSINBV-luc transcript also contains the Sindbis non-stmctural proteins.
  • both RNA transcripts are replicated and some viral particles will contain both RNA transcripts copackaged into the same particle. Infection of fresh cells with the copackaged RNA particles will result in cell that express both luciferase and ⁇ -galactosidase.
  • RNA transcript approaching 16 kb in length. Because of the increased length, this RNA cannot be replicated or packaged efficiently.
  • Sindbis nonstmctural proteins and the Factor VIII gene could be divided onto separate RNA molecules of approximately 8 kb and 9 kb in length, and copackaged into the same particles.
  • the pKSSINBV-Linker- I constmct is digested with the enzyme Bsp El and religated under dilute conditions. This results in the removal of the Sindbis nonstmctural proteins between nt# 422-7054. This constmct is known as pKSSINBVdlNSP-Linker- Pme .
  • the pKSSINBVdlNSP-Linker-/...-'l constmct is digested with the enzymes Pml I and Slit I and purified by using Geneclean.
  • the source of Factor VIII cDNA is clone pSP64- VIII, an ATCC clone under the accession number 39812 having a cDNA encoding the fiill- length human protein.
  • pSP64-VIII is digested with Sal I, the ends are blunted with T4 DNA polymerase and 50 uM of each dNTP, and the ca. 7700 bp. fragment is electrophoresed on a 0.7% agarose/TBE gel and purified with Geneclean.
  • the 7.7 kb fragment encoding Factor VIII is purified in a 0.7% agarose gel and subsequently ligated to the Pml l/Slu I digested pKSSINBVdlNSP-Linker- /wI fragment.
  • This constmct is known as pKSSINBVdlNSP- Factor VIII.
  • pKSSINBVdINSP-Factor VIII and pKSSINBV constmcts are linearized with
  • RNA transcripts are prepared as described in Example 3. These RNA transcripts are cotransfected into packaging cells that express the Sindbis stmctural proteins by one of the mechanisms described in Example 7. Both RNA transcripts contain a 5' sequence that is capable of initiating transcription of Sindbis RNA, sequences required for RNA packaging, a Sindbis Junction region, and the Sindbis 3' sequences required for synthesis of the minus strand RNA.
  • the pKSSINBV transcript contains the Sindbis nonstmctural protein genes
  • the pKSSINBVdINSP-Factor VIII constmct contains the Factor VIII gene, but not the Sindbis nonstmctural protein genes.
  • both RNA transcripts are replicated and some viral particles will contain both RNA transcripts copackaged into the same vector particle. Infection of fresh BHK-21 cells with the copackaged RNA will result in Factor VIII expression only if both RNA molecules are present in the same cell.
  • Sindbis standard techniques known in the art ⁇ e.g., Sambrook et al.. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989), as well as specific approaches described herein, will be utilized for constmctions.
  • Vims obtained from the ATCC, is propagated on cultured cells, its virion RNA extracted, and cDNA spanning the entire genome synthesized and cloned using conventional techniques.
  • This cDNA is then used to constmct gene transfer vector systems similar in principal to those described above, including, but not limited to, a replicon capable of carrying the heterologous gene(s), packaging cell lines that express the stmctural protein genes, and unique to this system, a separate packaging-competent subgenomic vector capable of carrying the additional heterologous gene(s). Since Aura vims subgenomic RNA contains a packaging signal, preliminary experiments are performed to identify this sequence, in order to prevent its inactivation during replacements with heterologous the gene(s). After identification of the packaging sequence, the individual elements of this Aura-based system are generated.
  • a basic replicon vector is constmcted to contain the following minimum elements: Aura 5' sequences necessary for replication, nonstmctural protein coding regions, a modified or unmodified junction region for subgenomic mRNA synthesis, a multiple cloning site for insertion of heterologous gene(s), one or more copies of the packaging signal, and 3' Aura sequences necessary for replication, including a polyadenylate sequence.
  • An upstream bacteriophage RNA polymerase promoter will be utilized for in vitro transcription of replicon RNA; alternatively, a eukaryotic RNA polymerase promoter will be utilized for transcription directly from cDNA.
  • a packaging-competent subgenomic vector is also constmcted to contain the following minimum elements: a modified or unmodified junction region, a multiple cloning site for insertion of heterologous gene(s), one or more copies of the packaging signal, and 3' Aura sequences necessary for replication/minus-strand synthesis, including a polyadenylate sequence.
  • the subgenomic vector may, in some cases, be constmcted with the Aura 5' replication sequences positioned upstream of the junction region, such that the vector will function as an amplicon.
  • RNA Transcription of subgenomic vector RNA can be accomplished in vitro using a bacteriophage RNA polymerase promoter, or cDNA in vivo using a eukaryotic RNA polymerase promoter. Further, the initial transcript may be of the sense-configuration or of the antisense-configuration.
  • Packaging cell lines are also constmcted as described previously for Sindbis vectors, such that mRNA for one or more of the stmctural proteins will be transcribed from the junction region and be inducible by the Aura replicon.
  • one or more of the stmctural proteins can be expressed under the control of an inducible or constitutive eukaryotic promoter.
  • specific inactivating mutations are made in any packaging sequences present in the stmctural protein genes, in order to prevent encapsidation of these sequences with the replicon. These mutations should be silent changes, usually at the third position of the codon, which do not affect the amino acid encoded.
  • heterologous genes can be exploited for many therapeutic applications, which include, but are not limited to, expression of multiple cytokines, multiple CTL epitopes, combinations of cytokines and CTL epitopes to enhance immune presentation, multiple subunits of a therapeutic protein, combinations of therapeutic proteins and antisense RNAs, etc.
  • the packaging of subgenomic mRNAs into virions also enables this vector system for the transfer of extremely long heterologous sequences.
  • alphavims packaging cell lines An important criteria in selecting potential parent cell lines for the creation of alphavims packaging cell lines, is the choice of cell lines that exhibit little or no cytopathological effects, prior to the appropriate production of alphavims vector particles. This criteria is essential for the development of an alphavims vector producer cell line which can be propagated for long periods of time and used as a stable source of vector. It is known that alphavims infection of most mammalian cells results in cytopathology and lysis of the cell. However, the derivation of packaging cells from various insect cell lines may circumvent this problem.
  • insect cell lines such as Aedes albopictus, Aedes aegypli, Spodoplera fiugiperda, and Drosophi/a me/anogaster cells
  • alphavims packaging cell lines are provided using an configuration uses an insect parent cell line, such as the Aedes albopictus, containing a stably transfected expression cassette vector which allows for expression of alphavims stmctural proteins under the control of inducible or non-inducible promoters active in these cell types, and co-expressing a selectable marker.
  • Sindbis virus-induced protein of cellular origin which has been associated with the down-regulation of Sindbis vims production in some infected Aedes albopictus cells, has been identified and purified ⁇ Virology 194:44).
  • the protein is a small hydrophobic peptide of approximately 3200 Da., which can induce an antiviral state and inhibit both 49S and 26S viral RNA synthesis.
  • Cells treated with the antiviral peptide usually demonstrate quiescent arrest of cellular division for 96 hours in uninfected cells, and then normal growth rates are restored. Cells that have been exposed to this peptide prior to infection are unable to replicate Sindbis vims and appear to maintain this phenotype by constitutively producing the antiviral protein through 10 months of continuous passage.
  • the first method entails purification of this cellular protein described above, and determination of a portion of the primary amino acid sequence using established techniques known in the art. The resulting amino acid sequence is then used to derive possible corresponding genomic sequences, enabling one to design a degenerate PCR primer pair which can be used to amplify the specific cellular sequence.
  • This amplified sequence is then cloned using standard techniques known in the art, to obtain a discreet region of the gene encoding this inhibitory protein. Determination of the nucleotide sequence of this clone then enables one to design a vector which will integrate specifically within this Sindbis inhibitory gene by homologous recombination, and "knock out" its capacity to express a functional protein. Cell clones which contain the knock out sequence are identified by insertion of a selectable marker into the discreet cloned region of the inhibitory protein, prior to transfecting cells with the vector.
  • a second method for disabling this Sindbis vims inhibitory protein involves the treatment of Aedes a/bopictus- ⁇ e ⁇ ved packaging cells with a mutagen, for example, BUDR (5-bromodeoxyuridine).
  • BUDR 5-bromodeoxyuridine
  • the tnutagenized packaging cell line population is then transfected or transduced with a Sindbis vector, which is able to express the neomycin resistance marker.
  • BUDR 5-bromodeoxyuridine
  • Packaging cell lines may also be modified by overexpressing the bcl-2 gene product in potential parent cell lines, such as canine D-17 and Cf2; human HT1080 and 293; quail QT-6; baby hamster kidney BHK-21; mouse neuroblastoma N18; and rat prostatic adenocarcinoma AT-3.
  • potential parent cell lines such as canine D-17 and Cf2; human HT1080 and 293; quail QT-6; baby hamster kidney BHK-21; mouse neuroblastoma N18; and rat prostatic adenocarcinoma AT-3.
  • a bcl-2 expression vector is constmcted by using standard recombinant DNA techniques in order to insert the 910 base pair Eco RI cDNA fragment derived from the plasmid p84 ⁇ Nature 336:259) into any commercially available expression vector containing a constitutive promoter and encoding a selectable marker, for example, pCDNA3 (Invitrogen, San Diego, CA). Careful consideration must be taken to avoid any type of homology between alphavims nucleic acid sequences and other transduced vectors.
  • Sindbis infection is tested, followed by its use as a parent cell line for alphavims packaging cell line development.
  • Other gene products in addition to the bcl-2 oncogene, which suppress apoptosis may likewise be expressed in an alphavims packaging or producer cell line.
  • Three viral genes which are particularly preferred include: the adenovims E1B gene encoding the 19-kD protein (Rao et al., PNAS 89: 7742-7746, 1992), the he ⁇ es simplex vims type 1 ⁇ 34.5 gene (Chou and Roizman, PNAS ⁇ ".
  • each gene product inhibits apoptosis by its own unique mechanism. Therefore, the genes may also be introduced into packaging or producer cell lines in various combinations in order to obtain a stronger suppressive effect. Finally, other gene products having similar effects on apoptosis can also be readily incorporated into packaging cell lines as they are identified.
  • alphavims vector packaging and producer cell lines many approaches are outlined to control the expression of viral genes, such that producer cell lines stably transformed with both vector and vector packaging cassettes, can be derived. These approaches include inducible and/or cellular differentiation sensitive promoters, antisense stmctural genes, heterologous control systems, and mosquito or other cells in which viral persistent infections are established. Regardless of the final configuration for the alphavims vector producer cell line, the ability to establish persistent infection, or at least delay cell death as a result of viral gene expression, may be enhanced by inhibiting apoptosis.
  • the DNA tumor viruses including adenovims, HPV, SV40, and mouse polyomavims (Py), transform cells in part, by binding to, and inactivating, the retinoblastoma (Rb) gene product pi 05 and its closely related gene product, pi 07, and other gene products involved in the control of the cell cycle including cyclin A, ⁇ 33 cdk2 and p34 cdc2 . All of these vimses, except for Py, encode gene products which bind to and inactivate p53.
  • adenovims including adenovims, HPV, SV40, and mouse polyomavims (Py)
  • Rb retinoblastoma
  • pi 07 closely related gene product
  • other gene products involved in the control of the cell cycle including cyclin A, ⁇ 33 cdk2 and p34 cdc2 . All of these vimses, except for Py, encode gene products which bind to and inactivate p53
  • packaging and producer cells are transformed with viral genomic DNA from Py or SV40
  • SV40 and Py transformed cell lines are established, and the kinetics and level of Sindbis production and cytopathology after viral infection determined. If apoptic events characteristic of Sindbis proliferation in hamster cells are diminished, each prototype alphavims packaging and producer cell line subsequently is transformed with Py or SV40, in order to increase the yield of packaged vector from these cells.
  • the Sindbis E2 glycoprotein is synthesized as a precursor, PE2.
  • This PE2 precursor along with the second viral glycoprotein. El, associate in the endoplasmic reticulum and are processed and transported to the infected cell membrane as a heterodimer for virion incorporation.
  • PE2 is cleaved into E3 and the mature virion glycoprotein E2.
  • E3 is the 64 amino-terminal residues of PE2 and is lost in the extracellular void during maturation.
  • the larger cleavage product, E2 is associated with El and anchored in what becomes the viral envelope.
  • Host cell protease(s) is responsible for processing of the PE2 precursor, cleaving at a site that immediately follows a highly conserved canonical four amino acid (aa) residue motif, basic-X-basic-basic aa's.
  • a mutant cell line derived from the CHO-K 1 strain, designated RPE.40 (Watson et al., J. Virol 65:2332-2339, 1991 ), is defective in the production of Sindbis vims strain AR339, through its inability to process the PE2 precursor into the E3 and mature E2 forms.
  • the envelopes of Sindbis virions produced in the RPE.40 cell line therefore contain a PE2/E1 heterodimer.
  • RPE.40 cells are at least 100-fold more resistant to Sindbis vims infection than the parental CHO-K1 cells, suggesting an inefficiency in the ability of PE2 containing virions to infect these cells.
  • the defective virions produced by the RPE.40 cell line can be converted into a fully infectious form by treatment with trypsin.
  • any wild-type alphavims that is produced by recombination between vector and stmctural protein gene RNAs will re-infect cells and be rapidly amplified; thus, significantly contaminating and decreasing the titer of packaged vector preparations.
  • Packaging and producer cells developed from the RPE.40 line are an alternative to other cell lines permissive for alphavims infection due to the inefficient amplification of any wild-type vims generated during vector production and packaging. Thus, vector preparations are not significantly contaminated with wild-type vims.
  • the benefits of this system are extended to other packaging and producer cell lines by developing "knock-out" mutants in their analogous cellular protease(s), using techniques known in the art.
  • Alphavims hopping cell lines are used transiently to produce infectious RNA vector particles which have been pseudotyped for a different cellular receptor tropism. Once the hopping cell line produces vector particles, it is no longer required because only the infectious culture supernatants are needed to transduce the original alphavims packaging cell lines discussed above. Therefore, the hopping cell line need not exhibit persistent infection by alphavims in order to transiently produce vector particles.
  • the parent cell line can be either an insect cell line that exhibits persistent infection, or a mammalian cell line which is likely to lyse within 24-72 hours after a productive alphavims infection.
  • the alphavims hopping cell line can be any of the aforementioned parent cell lines able to support either alphavims or retroviral replication, without the additional cell modifications discussed previously, such as bcl-2 oncogene expression.
  • VSV-G pseudotyped alphavims vector particles can be accomplished by at least three alternative approaches, two of which are dependent on the stable integration of a VSV-G expression cassette into cells.
  • VSV-G protein is known to be highly cytotoxic when expressed in cells. Therefore, synthesis of this protein by the expression cassette is controlled by an inducible promoter.
  • a DNA fragment containing the VSV-G protein gene is isolated from plasmid pLGRNL (Emi et al., J. Virol. 65: 1202-1207, 1991 ) by digestion with Bam HI, the termini made blunt using Klenow fragment enzyme and dNTPs, and the 1.7 kb fragment purified from a 1% agarose gel.
  • Plasmid vector pVGELVIS-SINBV-linker (from Example 3), is digested with the enzyme Bsp El to remove Sindbis nonstmctural protein coding sequences nts. 422-7054, and the remaining vector is re-ligated to itself to generate plasmid pVGELVISdlNSP-BV-linker. This plasmid is then digested with Xho 1 and the termini made blunt using Klenow fragment enzyme and dNTPs. The previously purified VSV-G fragment is subsequently ligated with this vector DNA, and resulting clones are screened for proper VSV-G insert orientation.
  • This pVGELVIS-based VSV-G expression constmct in which VSV-G synthesis is controlled by a Sindbis replicon-inducible junction region, is designated pVGELVISdl-G.
  • pVGELVISdl-G a similar Sindbis replicon-inducible VSV-G expression cassette may be generated in the antisense configuration.
  • plasmid vector pKSSINBV-linker (described in Example 3) is digested with the enzymes Apa I and Bam HI to most of the Sindbis nonstmctural protein coding region, and the resulting 3309 bp vector fragment is purified from a 1% agarose gel.
  • Plasmid pd5'-BVlinker is subsequently digested with Xho I, the termini made blunt using Klenow fragment enzyme and dNTPs, and ligated with the previously purified VSV-G fragment.
  • the resulting constmct containing the expression cassette elements HDV antigenomic ribozyme/Sindbis 5'-end 299 nts./Sindbis junction region/VSV-G protein gene/Sindbis 3'-end untranslated region, is designated as plasmid pd5'-BV-G. Insertion of this VSV-G gene cassette into the pcDNA3 vector is as follows. Plasmid ⁇ d5'-BV-G is digested with the enzymes Pme I and Apa I, and the termini are made blunt by the addition of T4 DNA polymerase and dNTPs. The entire 2.5 kb VSV- G protein gene cassette is purified in a 1% agarose gel.
  • Plasmid pcDNA3 is digested with the enzymes Hindlll and Apa I and the termini are made blunt by the addition of T4 DNA polymerase and dNTPs, and the 5342 bp vector is purified in a 1% agarose gel. The two purified, blunt-end DNA fragments are subsequently ligated, and the resulting VSV-G protein gene expression cassette vector is known as plasmid pCMV/d5NSV-G. Further modifications of the VSV-G expression cassettes pVGELVISdl-G and pCMV/d5NSV-G to substitute other selectable markers, for example hygromycin resistance or E.
  • coli gpt for the current neomycin resistance, or other promoter elements, for example Drosophilia metallothionein or hsp 70, for the current CMV, MuLV, and SV40 promoters, may be readily accomplished given the disclosure provided herein.
  • promoter elements for example Drosophilia metallothionein or hsp 70, for the current CMV, MuLV, and SV40 promoters, may be readily accomplished given the disclosure provided herein.
  • VSV-G expression cassette plasmid D ⁇ A (pVGELVISdl-G or pCMV/d5NSV-G, or modified versions thereof) is transfected into the appropriate cell type (for example, BHK-21 cells) and selection for G418 resistance is applied using media containing 400 ⁇ g/ml of G418 as described elsewhere in this example.
  • G418-resistant cells are cloned by limiting dilution and the individual cell lines expanded for screening.
  • VSV-G expressing cell lines are detected by transfection with any nonstmctural protein gene-containing R ⁇ A vector (see Example 3) to induce the VSV- G expression cassette, followed by immunofluorescence using polyclonal rabbit anti-VSV antibody as described (Rose and Bergmann, Cell 37:513-524, 1983).
  • the stably transfected VSV-G expressing cell line in some cases, is subsequently transfected with plasmid expression cassette(s) which express one or more Sindbis stmctural proteins (described elsewhere in this example).
  • the appropriate vector RNA is transfected into the VSV-G hopping cell line, and vector particle-containing supernatants are recovered at least 24 hours post-transfection.
  • VSV-G expression cassette DNA (pVGELVISdl-G or pCMV/d5'VSV-G, or modified versions thereof) is transfected into previously derived alphavims packaging cell lines (described elsewhere in this example) and the appropriate selection is applied as described previously. The selected cells are cloned by limiting dilution and the individual cell lines expanded for screening.
  • VSV-G expressing cell lines are detected by transfection with any nonstmctural protein gene-containing RNA vector (see Example 3) to induce the VSV-G expression cassette, followed by immunofluorescence using polyclonal rabbit anti-VSV antibody as described (Rose and Bergmann, Cell 57:513-524, 1983).
  • the appropriate vector RNA is transfected into the VSV-G hopping cell line, and vector particle-containing supernatants are recovered at least 24 hours post- transfection.
  • VSV-G expression cassette DNA is co-transfected with the appropriate vector RNA into previously derived alphavims packaging cell lines (described elsewhere in this example). Supernatants containing pseudotyped vector particles are recovered at least 24 hours post-transfection.
  • any cell line referenced in the literature which expresses retroviral gag-pol and env sequences, may be used to package alphavims RNA vector that has been engineered to contain a retroviral packaging sequence.
  • the retrovims psi packaging sequence is inserted between the inactivated junction region and a synthetic junction region tandem repeat, such that only genomic-length vector, and not subgenomic RNA, is packaged by the retroviral envelope proteins.
  • Retroviral-based particles containing alphavirus vector RNA are produced by transfecting / ' // vitro transcribed alphavims vector RNA using procedures that have been described previously. Supernatants with pseudotyped retroviral particles containing alphavims RNA vector are harvested at 24 hours post-transfection, and these supernatants are then used to transduce an alphavims packaging cell line.
  • non-heat inactivated semm, medium, and heat- inactivated semm are placed in separate 1.5 ml tubes (Fisher Scientific, Pittsburgh, PA) and mixed with 10 ⁇ Plaque Forming units (PFU) of Sindbis vims and incubated at 37°C for 1 hour. After incubation the tubes are placed on ice.
  • PFU Plaque Forming units
  • the non-heat inactivated semm, medium, and heat-inactivated semm vims preparations are titered by plaque assay on BHK cells. Equivalent vims titers regardless of incubation with non-heat inactivated semm, medium, or heat-inactivated semm, are indicative of parent cell line hosts from which Sindbis vims is resistant to human complement inactivation
  • alphavims packaging cell lines The development of alphavims packaging cell lines is dependent on the ability to synthesize high intracellular levels of the necessary stmctural proteins: capsid, pE2 and/or
  • alphavims stmctural proteins is under control of the RSV LTR, in conjunction with the inducible lac operon sequences. This is achieved by insertion of alphavirus cDNA corresponding to the viral stmctural protein genes into the pOP13 and pOPRSVl vectors (Stratagene). These vectors, used separately, are co ⁇ transfected with the p3'SS vector (Stratagene), which expresses the lac repressor "i" protein.
  • IPTG Isopropyl-B-D-thiogalactopyranoside
  • Sindbis stmctural protein gene (SP) cDNA is inserted into the pOP13 and pOPRSVl vectors as follows.
  • the SP coding region is amplified in toto with a primer pair whose 5' ends map, respectively, to the authentic AUG translational start and UGA translational stop sites, including the surrounding nucleotides corresponding to the Kozak consensus sequence for efficient translational initiation at Sindbis nt 7638.
  • the forward primer is complementary to Sindbis nts 7638-7661
  • the reverse primer is complementary to Sindbis nts 1 1,384-1 1,364.
  • PCR amplification of Sindbis cDNA corresponding to the stmctural protein genes is accomplished by a standard three- temperature cycling protocol, using the following oligonucleotide pair:
  • a 5 nucleotide "buffer sequence" followed by the Not I recognition sequence is attached to the 5' ends of each primer.
  • the 3,763 bp fragment is purified in a 1% agarose gel, then subsequently digested with the Not I enzyme.
  • the resulting 3,749 bp fragment is then ligated, separately, into the pOP13 and pOPRSVl vectors, which are digested with Not 1 and treated with calf intestine alkaline phosphatase.
  • These expression cassette vectors which contain the entire coding capacity of the Sindbis stmctural proteins are known as pO 13-SINSP and pOPRSV l -SINSP. Variations of the lac operon-Sindbis stmctural protein gene expression cassettes also can be constmcted using other viral, cellular or insect-based promoters.
  • the lac operon and the RSV LTR promoter, or just the RSV LTR promoter, sequences can be switched out of the Stratagene pOP13 and pOPRSVl vectors and replaced by other promoter sequences, such as the cytomegalovirus major immediate promoter (pOPCMV-SINSP); the adenovims major late promoter (pOPAMLP-SINSP); the SV40 promoter (pOPSV-SINSP): or insect promoter sequences, which include the Drosophila metallothionein inducible promoter (pMET-SINSP), Drosophila actin 5C distal promoter (pOPA5C-SINSP), heat shock promoters HSP65 or HSP70 (pHSP-SINSP), or the baculovims polyhedrin promoter (pPHED-SINSP).
  • pMET-SINSP Drosophila metallothionein inducible promoter
  • pOPA5C-SINSP Drosophil
  • Alphavirus stmctural protein expression can be increased if the level of mRNA transcripts is increased.
  • Increasing the level of mRNA transcripts can be accomplished by modifying the expression cassette such that alphavims nonstmctural proteins recognize these transcripts, and in turn, replicate the message to higher levels. This modification is performed by adding the wild-type minimal junction region core (nucleotides 7579 to 7602) to the extreme 5'-end of the Sindbis stmctural protein coding region, prior to the first authentic ATG start site for translation and inverting the expression cassette in the vector, so as to produce antisense stmctural protein gene transcripts.
  • the resulting 3,787 bp fragment is purified in a 1% agarose gel, then subsequently digested with the Not I enzyme.
  • the resulting 3,773 bp fragment is then ligated, separately, into the pOP13 and pOPRSVl vectors which are digested with Not I and treated with calf intestine alkaline phosphatase.
  • the resulting expression cassette vectors are known as pOP13-JUNSINSP and pOPRSVl-JUNSINSP.
  • structural protein expression cassettes will introduce sequences which may possibly lead to undesirable recombination events, leading to the generation of wild-type vims.
  • packaging cell line expression cassettes are constmcted which contain regulatory elements for the high level induction of stmctural protein synthesis via nonstmctural proteins supplied in trans by the alphavims vector, but with no basal level of synthesis until appropriately stimulated.
  • a structural protein gene cassette is constmcted, whereby transcription of the stmctural protein genes occurs from an adjacent alphavims junction region sequence.
  • the primary features of this cassette are: an RNA polymerase II promoter positioned immediately adjacent to alphavims nucleotide 1, such that transcription initiation begins with authentic alphavirus nucleotide 1, the 5'-end alphavims sequences required for transcriptase recognition, the alphavims junction region sequence for expression of the stmctural protein gene mRNA, the alphavims stmctural protein gene sequences, the 3'-end alphavims sequences required for replication, and a transcription termination polyadenylation sequence.
  • the constmction of a positive-sense, vector-inducible Sindbis packaging cassette is accomplished as follows. Briefly, the pVGELVIS vector described previously is digested with the enzyme Bsp El to remove nucleotides 422 to 7054, including most of the nonstmctural gene coding sequences, and the remaining 9925 bp fragment is purified in a 0.8% agarose gel, and subsequently re-ligated to itself to generate the constmct known as pLTR/Sindl_?.spE ( Figure 1 1 ).
  • This deletion leaves the 5'-end authentic translation start codon at nts 60-62 intact, and creates in-frame downstream UAA and UGA stop codons at nts 7130-7132 and 7190-7192 (original numbering), respectively, thus preventing translation of the downstream stmctural protein gene open-reading frame.
  • the pLTR/Sindli?.spE packaging cassette constmct is subsequently transfected into BHK cells (ATCC #CCL 10) and transfectants are selected using the G418 dmg at 400 ug/ml and cloned by limiting dilution.
  • a similar packaging constmct can also be made using the pVG-ELVISd clone (described previously) as initial material for creation of the Bsp El deletion.
  • the Sindbis 3'-end sequence is followed by a catalytic ribozyme sequence to allow more precise processing of the primary transcript adjacent to the 3'-end sequences of Sindbis.
  • these packaging cassette constmctions can be made given the disclosure provided herein, including for example, the substitution of other RNA polymerase promoters for the current MuLV LTR, the addition of 1 or more nucleotides between the RNA polymerase promoter and the first Sindbis nucleotide, the substitution of other ribozyme processing sequences, or the substitution of a non-Sindbis-encoded open reading frame upstream of the stmctural protein gene sequences, which may or may not retain the 5'- end Sindbis sequences required for transcriptase recognition.
  • constmcts can be transfected into other cell lines, as discussed previously
  • expression cassettes contain a cDNA copy of the alphavirus structural protein gene sequences flanked by their natural junction and 3'-untranslated regions, and are inserted into an expression vector in an orientation, such that primary transcription from the promoter produces antisense stmctural protein gene RNA molecules.
  • these constmcts contain, adjacent to the junction region, alphavims 5'-end sequences necessary for recognition by the viral transcriptase, and a catalytic ribozyme sequence positioned immediately adjacent to alphavims nucleotide 1 of the 5'-end sequence.
  • this ribozyme cleaves the primary RNA transcript precisely after the first alphavims nucleotide.
  • the stmctural protein genes cannot be translated, and are dependent entirely on the presence of alphavims vims nonstmctural proteins for transcription into positive-strand mRNA, prior to their expression. These nonstmctural proteins again are provided by the alphavims vector itself.
  • this configuration contains the precise alphavims genome 5'- and 3'-end sequences, the stmctural protein gene transcripts undergo amplification by utilizing the same nonstmctural proteins provided by the alphavims vector.
  • Sindbis stmctural protein gene cDNA is removed from the genomic clone pVGSP ⁇ GEN and inserted into the pcDNA3 (Invitrogen Corp., San Diego, CA) expression vector as follows.
  • plasmid pVGSP ⁇ GEN is digested with the enzymes Apa I and Bam HI to remove all Sindbis sequences through nucleotide 7335, including the genes encoding nonstmctural proteins 1, 2, 3, and most of 4.
  • the remaining 7285 bp vector fragment which contains the Sindbis stmctural protein genes, is purified in a 0.8% agarose gel, and subsequently ligated with a polylinker sequence, called SinMCS, that is obtained by annealing two synthetic oligonucleotides.
  • SinMCSI and SinMCSII contain the recognition sites for Cla I, Bgl II, and Spe I, and have Apa I and Bam HI ends after annealing. Their sequences are as follows:
  • the resulting constmct is then modified to contain the 5'-end 299 nucleotides of Sindbis, fused to an 84 nucleotide ribozyme sequence from the antigenomic strand of hepatitis delta vims (HDV) ⁇ Nature 350:434), using overlapping PCR amplification.
  • Two primer pairs are used initially in separate reactions, followed by their overlapping synthesis in a second round of PCR.
  • the forward primer (HDV49-XC) is complementary to HDV genome nucleotides 823-859
  • the reverse primer (HDV 17-68) is complementary to HDV genome nucleotides 839-887, with sequences as follows:
  • primer HDV49-XC contains flanking Xba I and Cla I recognition sequences at the 5'-end.
  • PCR amplification of HDV sequences is accomplished by a standard three-temperature cycling protocol with these primers and Vent polymerase.
  • the forward primer (SIN-HDV), which joins precisely the HDV and Sindbis sequences, is complementary to nucleotides 1-21 of Sindbis, and genomic nucleotides 871 -903 of HDV, and overlaps the sequence of primer HDV 17-68 (from above) by 20 nucleotides
  • the reverse primer (SIN276-SPE) is complementary to Sindbis nucleotides 299-276, with sequences as follows:
  • primer SIN276-SPE contains a flanking UAA translation termination codon and Spel recognition sequence at its 5' end.
  • PCR amplification of the fragment containing Sindbis 5'-end sequences fused to HDV ribozyme sequences is accomplished by a standard three-temperature cycling protocol, using Vent polymerase, these primers, and pVGSP ⁇ GEN plasmid as template. After the first round of PCR amplification, I /20th of the total amounts from each of reaction #1 and reaction #2 is combined and used as template in a second round of PCR amplification with additional input of primers HDV49-XC and SIN276-SPE and a standard three-temperature cycling protocol.
  • the 414 bp amplicon is purified with the MERMAID KIT (Bio 101, La Jolla, CA), and digested with the enzymes Clal and Spel.
  • the digested amplicon is purified in a 1% agarose gel, and subsequently ligated into plasmid pMCS-26s, which also is digested with Clal and Spel and purified in a 1% agarose gel.
  • the resulting constmct containing the expression cassette elements HDV antigenomic ribozyme/Sindbis 5'-end 299 nts/Sindbis junction region/Sindbis stmctural protein genes/Sindbis 3'-end untranslated region, is known as pd5'26s.
  • Insertion of the stmctural protein gene cassette from pd5'26s into the pcDNA3 vector is performed as follows. Plasmid pd5'26s is digested with the enzyme Xba I and the 3'- recessed ends are made blunt by the addition of Klenow enzyme and dNTPs. The entire 4798 bp stmctural protein gene cassette is purified in a 1% agarose gel. Plasmid pcDNA3 is digested with the enzymes H//.dIII and Apa I and the ends are made blunt by the addition of T4 DNA polymerase enzyme and dNTPs, and the 5342 bp vector is purified in a 1% agarose gel.
  • the two purified, blunt-end DNA fragments are subsequently ligated, and the resulting stmctural protein gene expression cassette vector is known as pCMV-d5'26s ⁇ see Figure 11). Transfection of this DNA into cells and selection for G418 resistance is performed as previously described.
  • Modifications of the CMV promoter/antisense-Sindbis stmctural protein vector also can be constmcted using other viral, cellular, or insect-based promoters.
  • the CMV promoter can be switched out of the Invitrogen pcDNA3 vector and replaced by promoters such as those listed previously.
  • this antisense packaging cassette may include, but are not limited to: the addition of 1 or more nucleotides between the first Sindbis nucleotide and the catalytic ribozyme, the use of longer or shorter HDV or other catalytic ribozyme sequences for transcript processing, the substitution of a precise transcription termination signal for the catalytic ribozyme sequence, or the antisense expression of stmctural protein gene cassettes using any downstream sequence recognized by an RNA polymerase which results in transcription of a stmctural protein gene mRNA.
  • each of the vector-inducible constmcts described contains sequences homologous to the Sindbis vector itself. Therefore, the potential exists for the generation of wild-type vims by recombination between the two RNA molecules. Additional modifications may be made to eliminate this possibility as described below.
  • Packaging cell lines may also be generated which segregate the integration and expression of the stmctural protein genes, allowing for their transcription as non- overlapping, independent RNA molecules. For example, the expression of capsid protein independently of glycoproteins E2 and El , or each of the three proteins independent of each other, eliminates the possibility of recombination with vector RNA and subsequent generation of contaminating wild-type vims.
  • capsid protein is expressed independently from an inducible expression vector, such that sequences which might result in recombination with vector RNA are eliminated.
  • the capsid protein gene is amplified from plasmid pVGSP ⁇ GEN with a primer pair complementary to nucleotides 7632-7655 (forward primer) and 8415- 8439 (reverse primer), with sequences as follows.
  • Reverse primer (Sin8439R): 5'-CAGTCTCGAGTTACTACCACTCTTCTGTCCCTTCCGGGGT-3' (SEQ. ID NO. 84)
  • the forward primer contains Nhe I and Hindlll recognition sequences at its 5'-end
  • the reverse primer contains both UAG and UAA translation stop codons and a Xho I recognition sequence at its 5'-end.
  • Amplification is accomplished using a standard three-temperature cycling protocol, and the resulting amplicon is digested with the enzymes Nhe I and )Cho I, and purified in a 1% agarose gel.
  • Expression plasmid pMAM (Clontech), which contains a dexamethasone-inducible MMTV LTR promoter sequence, is digested with the enzymes Nhe I and Xho I and the plasmid DNA purified in a 1% agarose gel.
  • the capsid protein gene fragment is ligated into the pMAM vector, and the resulting constmct is known as pMAM-SinC.
  • Plasmid pMAM-SinC is transfected into the appropriate cell line as described previously and selection for stable transfectants is accomplished by using HAT (hypoxanthine, aminopterin, thymidine) media, supplemented with dialyzed fetal calf semm, mycophenolic acid and xanthine, as described by Mulligan and Berg ⁇ PNAS 78: 2072-2076, 1981 ).
  • HAT hypoxanthine, aminopterin, thymidine
  • Cell lines expressing capsid protein are identified following dexamethasone induction by immunofluorescence using polyclonal rabbit anti-Sindbis antibody.
  • capsid protein is expressed using the lac-inducible vectors (Stratagene) described previously.
  • the Sindbis capsid protein gene is amplified by PCR using primers Sin7632F and Sin8439R (described previously), and ligated with TA vector DNA (Stratagene).
  • the resulting plasmid, designated TA/SinC is digested with Eco RI, the termini are made blunt by the addition of Klenow fragment enzyme and dNTPs, and the capsid protein gene purified from a 1% agarose gel.
  • Plasmid vectors pOP13 and pORSVl are digested with Nol 1, their termini made blunt by the addition of Klenow fragment enzyme and dNTPs, and subsequently treated with calf intestinal alkaline phosphatase.
  • the capsid protein gene is ligated with both pOP13 and pORSVl vector DNA to generate the expression constmcts designated pOP13CAP and pORSVl CAP, respectively.
  • Each plasmid is co-transfected with p3'SS into the appropriate cell line as described previously, and selection for stable transfectants is accomplished using G418 and hygromycin selection.
  • Cell lines expressing capsid protein are identified following IPTG induction by immunofluorescence using polyclonal rabbit anti-Sindbis antibody.
  • the glycoprotein genes. El and E2 are expressed together using one of the inducible systems previously described.
  • the Sindbis El and E2 genes are amplified from plasmid pVGSP ⁇ GEN using a primer pair complementary to Sindbis nucleotides 8440-8459 (forward primer) and Sindbis nts 1 1,384-1 1,364 (reverse primer).
  • PCR amplification is performed using a standard three-temperature cycling protocol and the following oligonucleotide pair: Reverse primer ( 1 1384R):
  • the forward primer contains an "in-frame" AUG translation initiation codon, and both primers contain a NotI recognition sequence at their 5'-ends.
  • the amplicon is digested with the NotI enzyme and purified in a 1% agarose gel. The resulting fragment is then ligated separately into the pOP13 and pOPRSV l vectors (Stratagene), digested with Not I and treated with calf intestinal alkaline phosphatase, as described previously.
  • These glycoprotein expression vectors are used to transfect cells that have been previously transfected with a capsid protein expression constmct, and stable glycoprotein gene transfectants are identified by selection for G418 and hygromycin resistance.
  • the E 1 and E2 glycoproteins are expressed under the control of the replicon-inducible junction region promoter, described previously.
  • the ELVIS expression plasmid pVGELVISOSI ⁇ BV-linker (Example 3) is digested with the enzyme Not I, and treated with calf intestinal alkaline phosphatase. PCR amplified Sindbis El and E2 glycoprotein genes digested with Not 1 (previous paragraph) are then ligated to the ELVIS vector to generate a constmct designated ⁇ VGELVIS-El/E2.
  • Plasmid pVGELVIS-El/E2 subsequently is digested with the enzyme Bsp El, removing most of the nonstmctural protein gene coding region, and the remaining El- and E2-containing vector DNA is re-ligated to itself, creating an inducible expression cassette identified as pVGELVdl-El/E2.
  • This glycoprotein expression vector is used to transfect cells that have been previously transfected with a capsid protein expression constmct, and stable glycoprotein gene transfectants are identified by selection for G418 resistance.
  • BHK-21 cell line and replicon-inducible packaging expression cassette are used to demonstrate assembly of the components.
  • other possible parent cell lines can be used to create alphavims packaging cell lines and have been discussed previously.
  • BHK-21 cells CCL 10 are grown at 37°C in 5% CO 2 in Dulbecco's modified Eagle's Media (DMEM), 2 mM L-glutamine, and 10% fetal bovine semm (optimal media).
  • Transfectam Promega
  • any method of transfection is rapidly substituted, i.e., by electroporation, calcium phosphate precipitation, or by using any of the readily available cationic liposome formulations and procedures commonly known in the art.
  • the cells are trypsinized and reseeded in 100 mm dishes in 10 ml of optimal media, as described above, supplemented with 400 ug/ml of G418 (Gibco/BRL) and selected over a period of 5 to 7 days. Colonies displaying resistance to the G418 dmg are then pooled, dilution cloned, and propagated. Individual clones are screened for high levels of Sindbis stmctural protein expression and functional packaging after transfection with Sindbis-luciferase vector RNA transcribed /// vitro from Sad linearized plasmid pKSSINBV-luc (see Example 3).
  • LTR/Sindl/ty/E transfected BHK-21 cells grown in 60 mm petri dishes are transfected with 2 ug of Sindbis-luciferase vector RNA and overlayed with 3 ml of optimal media (see above).
  • the supernatants are removed, and clarified by centrifugation for 30 min. at 3000 rpm in a Sorvall RT6000B tabletop centrifuge.
  • the transfected cell monolayer is lysed in reporter lysis buffer (Promega) as described by the manufacturer, and assayed for luciferase expression as described previously.
  • luciferase activity (and thus functional packaging) is tested by using 1 ml of the above supernatants to infect fresh monolayers of BHK-21 cells in 60 mm dishes. At 20 hours post-infection, the cell monolayers are lysed as described above, and tested for luciferase expression. As shown in Figure 12, three clones (#13, 18, and 40) produce packaged Sindbis-luciferase vector and are the first examples of alphavims packaging cell lines. In addition, transfected clone # 18 cells are tested for increased vector packaging over a timecourse following transfection.
  • a cell whose differentiation state can be controlled is likely an ideal host in which to derive an alphavims vector producer cell line.
  • the vector expression cassette and, in some instances, stmctural components are coupled to terminal differentiation state-inducible promoters, according to the strategy described for ELVIS, and used to transform stably an undifferentiated host cell. Terminal differentiation of the host producer cell after induction with the appropriate stimuli coincidentally results in induction of the alphavims replication cycle and production of packaged vector.
  • the block to viral propagation occurs at the level of transcription and replication, and maps to the enhancers, contained within the viral non-coding control regions (Linney et al.. Nature 308:470-472, 1984; Fujimura et al., Cell 25:809-814, 1981 ; Katinka and Yaniv, Cell 20:393-399, 1980).
  • M-MuLV infects undifferentiated EC cells
  • the viral DNA integrates into the genome.
  • expression of viral genes or of heterologous genes is blocked. This block of viral expression is released upon terminal differentiation of EC cells by addition of retinoic acid to the growth medium.
  • plasmid DNA is complexed with LIPOFECTAMINE (GIBCO-BRL, Gaithersburg, MD) according to the conditions suggested by the supplier (ca. 5 ⁇ g DNA/8 ⁇ g lipid reagent) and added to 35 mm wells containing undifferentiated PCC4 or F9 cells (Fujimura et al., 1981, Cell 23:809-814) at approximately 75% confluency.
  • LIPOFECTAMINE GIP-BRL, Gaithersburg, MD
  • CPE cytopathic effects
  • undifferentiated F9 or PCC4 cells are transfected with ⁇ LTR/SINdl/_.s/ E and G418 selected as described above. Differentiation state-sensitive clones are then selected by infection at high multiplicity with packaged SIN-luc vector. Clones which are resistant to cell lysis or do not produce packaged SIN-luc vector particles, are candidate vector packaging clones. These candidate clones are tested for SIN-luc vector particle production following terminal differentiation with retinoic acid, as described.
  • the murine wild type polyomavims is unable to replicate in the teratocarcinoma cell lines PCC4 or F9.
  • This block of replication in undifferentiated cells occurs at the level of transcription of early region ⁇ i.e., T antigen) genes, and is released by induction of terminal differentiation with vitamin A.
  • Py mutants which are able to establish productive infection in undifferentiated PCC4 and F9 cells map to the viral enhancer region. The genesis of an embryonic tissue specific transcriptional enhancer element has resulted in these mutants.
  • the viral regulatory non-coding region including the enhancer, is coupled to the genomic cDNA of Sindbis vims, according to the ELVIS strategy.
  • the precise transcriptional start site of the Py early region has been determined (see Tooze, DNA Tumor Vimses).
  • the PCC4 and F9 cell lines are stably transformed with the Py-Sindbis vectors.
  • Sindbis productive infection occurs after addition of retinoic acid to the culture medium and induction of terminal differentiation.
  • the Py non-coding region from bases 5021-152 which includes the sequences corresponding to the viral enhancers, 21 bp repeats, replication origin, CAAT and TATA boxes, and the early mRNA transcription 5' cap site, is positioned at the 5' viral end such that n vivo, only a single capped C residue is added to the Sindbis 5' end. Juxtaposition of the Py non-coding region and the Sindbis 5' end is accomplished by overlapping PCR as described in the following detail. Amplification of the Py non-coding region in the first primary PCR reaction is accomplished in a reaction containing the pBR322/Py, strain A2 plasmid (ATCC number 45017-p53.A6.6 (pPy- 1 )) and the following primer pair:

Abstract

L'invention concerne une composition et des procédés utilisant des vecteurs d'alphavirus de recombinaison.
PCT/US1995/015490 1994-11-30 1995-11-30 Vecteurs d'alphavirus de recombinaison WO1996017072A2 (fr)

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