Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/54—Interleukins [IL]
  • C07K14/5434—IL-12
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/67—General methods for enhancing the expression

Definitions

• the present invention relates to methods and polynucleotide compositions useful for enhancing the efficiency of expression of polynucleic acid sequences in a host cell by decreasing the expression of undesirable polynucleic acid sequences. More specifically, the invention relates to novel compositions and methods for preventing the expression of aberrant DNA and RNA sequences by use of such compositions.
• Polynucleotide compositions have been described for pharmaceutical uses, primarily for treatment or prophylaxis of disease in mammals, as well as in research in such fields. Specifically a great deal of activity presently surrounds the use of polynucleotide compositions in the treatment of pathogenic extracellular and intracellular infections, such as viral, bacterial, fungal infections, and the like.
• DNA vaccines are described to deliver to a mammalian cell in vivo an agent which will combat a pathogen by harnessing the mammalian immune system.
• such vaccines are designed to express, for example, a viral protein or polypeptide, and elicit a humoral or cellular immune response upon challenge by the infective agent.
• Gene therapy vectors are polynucleotide compositions generally designed to deliver to a mammalian cell a protein which is either not expressed, expressed improperly or under expressed in a mammal. Such vectors frequently must address species specific immune responses to the those polynucleotide sequences that are recognized as antigenic or which evoke an unwanted cellular immune response. Still other therapeutic uses of polynucleotide compositions are for the delivery of missing or under expressed proteins to a diseased mammalian patient.
• polynucleotides are useful themselves as in vivo reagents, in diagnostic/imaging protocols, as reagents in gene therapy, in antisense protocols, in vaccine applications or otherwise as pharmaceuticals used to treat or prevent a variety of ailments such as genetic defects, infectious diseases, cancer, and autoimmune diseases.
• Polynucleotides are also useful as in vitro reagents in assays such as biological research assays, medical, diagnostic, screening and contamination detection assays.
• Phenomena have been observed in plants, nematodes, and Drosophila that are mediated by polynucleotide compositions, and are referred to as post-transcriptional gene silencing and transcriptional silencing.
• This phenomenon demonstrates that transfection or infection of a plant, nematode or Drosophila with a virus, viroid, plasmid or RNA expressing a polynucleotide sequence having some homology to a regulatory element, such as a promoter or a native gene or a portion thereof already expressed in that cell, can result in the permanent inhibition of expression of both the endogenous regulatory element and/or gene and the exogenous sequence.
• This silencing effect was shown to be gene specific. See, for example, L. Timmons and A. Fire, Nature. 395:354 (Oct. 29, 1998); A. Fire et al, Nature. 391:806-810 (Feb. 19,
• a DNA plasmid encoding a full-length pro-alpha 1 collagen gene was transiently transfected into a rodent fibroblast tissue cell line and a "silencing" effect on the native collagen gene and the transiently expressed gene observed [Bahramian and Zarbl, Mol. Cell. Biol. 19(l):274-283 (Jan. 1999)].
• RNA or DNA is formed of aberrant RNA or DNA, instead of the gene-containing transcript intended.
• the formation of aberrant RNA or DNA has been postulated to occur in the use of any transfected plasmid or polynucleic acid molecule and to reduce the efficiency of the target gene expression.
• the formation of aberrant or abnormal RNA can be one cause of the silencing effect noted above.
• sequences which function to terminate transcription For example, a pause site for RNA polymerase II is associated with termination of transcription, particularly when the site is positioned immediately downstream of a strong polyadenylation signal in a transient expression system [P. Enriquez-Harris et al, EMBO J.. 9(7): 1833-1842 (1991); G. W. Hatfield et al, Mel Cell. Bio 3(10):1687-1693 (1983)].
• DNA molecules and increase expression of a selected polynucleotide sequence in a host cell.
• the invention provides a double stranded polynucleotide (preferably deoxyribonucleic acid) molecule comprising a first coding strand and a second transcription template strand.
• the first strand comprises (i) at least one expression cassette sequence which comprises, from 5' to 3', a promoter, a selected polynucleotide sequence the expression of which is controlled by the promoter, and a polyadenylation site, and (ii) at least one first strand terminator sequence.
• the selected polynucleotide sequence of the expression cassette can be any polynucleotide sequence which is desired to be expressed in a cell to perform a biological function.
• the first strand terminator sequence is preferably located 5' to the promoter, located 3' to the polyadenylation site, located on the first strand outside of the expression cassette sequence, or located within the selected polynucleotide sequence of the expression cassette sequence.
• the first strand termination is located in a position which does not impede transcription from the sequence on the second strand which is complementary to the expression cassette sequence, nor effect the function of the polynucleotide sequence to be expressed.
• the second strand is complementary to the first strand. The portions of the second strand sequence complementary to the first strand terminator sequence do not impede transcription from the second strand sequence complementary to the first strand expression cassette. Further, the second strand comprises at least one second strand terminator sequence which terminates transcription initiated on the second strand.
• the second strand terminator sequence is preferably located on the second strand outside of the sequence complementary to the expression cassette of the first strand and in a position which does not impede transcription from the sequence complementary to the expression cassette sequence, nor impairs the biological function of the polynucleotide sequence, when expressed.
• the invention provides a pharmaceutical composition
• a pharmaceutical composition comprising the above-identified double-stranded polynucleotide molecule, an optional agent that facilitates polynucleotide (e.g., DNA) uptake in a cell, and a suitable pharmaceutically acceptable carrier.
• Such compositions are useful for treating intracellular pathogenic infections, such as viruses.
• Other such compositions are useful for treating certain cancers.
• Other such compositions are useful for treating certain extracellular pathogens.
• the invention provides a single stranded polynucleotide sequence selected from a first strand or second strand of a double stranded polynucleotide molecule described above.
• the invention provides a pharmaceutical composition
• a pharmaceutical composition comprising the above-identified single-stranded polynucleotide molecule, an optional agent that facilitates polynucleotide (e.g., DNA) uptake in a cell, and a suitable pharmaceutically acceptable carrier.
• the invention provides a method for enhancing the efficiency of expression of a selected polynucleotide sequence in a host cell, the method comprising the step of transfecting the host cell with a double stranded DNA molecule described above or a single strand thereof, and thereby inhibiting the formation of aberrant polynucleotide sequences transcribed from the polynucleotide molecule in the host cell.
• the invention provides a single stranded RNA molecule comprising a sequence of ribonucleic acids having a 5' end and a 3' end, modified to prevent the formation of double-stranded or partially double-stranded regions.
• this molecule comprises a 5' cap.
• this molecule does not have a cap.
• this molecule has a 3' polyA tail.
• this molecule has no polyA tail.
• the invention provides a pharmaceutical composition comprising the above-identified single-stranded RNA molecule, an optional agent that facilitates RNA uptake in a cell, and a suitable pharmaceutically acceptable carrier.
• the invention provides a method for enhancing the efficiency of expression of a selected polynucleotide sequence in a host cell, the method comprising the step of transfecting the host cell with the single-stranded RNA molecule described above, and thereby inhibiting the formation of aberrant RNA molecules in the host cell.
• the invention provides a method for treating a mammalian subject comprising administering an effective amount of a pharmaceutical composition comprising any of the polynucleotide molecules described above, an optional agent that facilitates DNA or RNA uptake in a cell, and a suitable pharmaceutically acceptable carrier.
• the invention provides a method for preventing the unintentional shutting off or down-regulation of a polynucleotide sequence in a host cell transfected with a polynucleotide molecule containing a selected polynucleotide sequence homologous to the polynucleotide sequence present in the host cell, the method comprising the steps of : administering an effective amount of a pharmaceutical composition comprising a polynucleotide molecule as described above, an optional agent that facilitates polynucleotide uptake in a cell, and a suitable pharmaceutically acceptable carrier.
• the polynucleotide compositions or molecules are made by enzymatic synthetic methods or chemical synthetic methods in vitro.
• compositions or molecules may be generated in a recombinant culture, e.g., bacterial cells, isolated therefrom, and used in the methods discussed below.
• composition generates the polynucleotide molecule in vivo after delivery to the host cell.
• compositions and molecules for use in research methods, such as a reagent for reducing or inhibiting undesired polynucleotide expression in host cells or tissue in vitro for use in diagnostic or other research assays, or ex vivo for return to the host subject for therapy or other medicinal uses.
• Fig. 1 A is an illustration of a double-stranded DNA molecule of this invention containing a first "sense" strand, with the 5' terminator sequence (T 1 ), the promoter (P), the selected polynucleotide sequence (PN), the polyadenylation sequence (pA), the 3 ' terminator sequence (T 2 ) and containing a second complementary strand containing the second strand terminator sequence (T 3 ), which in this case, is located in a position which is 3' relative to the polyA sequence on the sense strand.
• the first or second strand may be utilized as a single-stranded DNA molecule of this invention.
• IB is an illustration of another double-stranded DNA molecule of this invention containing a first "sense" strand, with two 5' terminator sequences (T 1 and T 2 ), the promoter (P), the selected polynucleotide sequence (PN), the polyadenylation sequence (pA), the 3' terminator sequence (T 3 ) and an RNA instability sequence (RIS); and containing a second complementary strand containing multiple second strand terminator sequences (T 4 through T 8 ) interspersed in the region which is not complementary to the expression cassette region on the first strand.
• terminators T 4 and T 5 are located in positions downstream relative to the polyA sequence on the sense strand; and terminators T 6 - T 8 are located in positions upstream relative to the promoter (P) on the sense strand.
• Either the first or second strand may be utilized as a single-stranded DNA molecule of this invention.
• Fig. 1C is an illustration of another double-stranded DNA molecule of this invention containing a first "sense" strand, with a padlock terminator (PT) and another terminator (T 2 ) located 5' to the promoter (P), the selected polynucleotide sequence (PN), the polyadenylation sequence (pA), the 3' terminator sequence (T 3 ); and containing a second complementary strand containing multiple second strand terminator sequences (T 4 through T 6 ) interspersed in the region which is not complementary to the expression cassette region on the first strand.
• PT padlock terminator
• T 2 another terminator located 5' to the promoter
• PN selected polynucleotide sequence
• pA polyadenylation sequence
• T 3 3' terminator sequence
• T 4 through T 6 containing a second complementary strand containing multiple second strand terminator sequences
• terminators T 4 and T 5 are located in positions downstream relative to the polyA sequence on the sense strand; and terminator T 6 is located in a position upstream relative to the promoter (P) on the sense strand.
• the first or second strand may be utilized as a single-stranded DNA molecule of this invention.
• Fig. ID is an illustration of another double-stranded DNA molecule of this invention having a number of terminators positioned as described in Fig. 1C, except that the padlock terminator (PT) is positioned on the sense strand within the selected polynucleotide (PN) sequence.
• Fig. 2A is a schematic demonstrating an RNA molecule made in vitro containing an inverted complementary repeat (Sequence B) of Sequence A, which can base pair and fold back upon itself to become a double stranded or partially double- stranded RNA.
• sequence B inverted complementary repeat
• the codon GCC codes for Ala
• AAG codes for Lys
• CUU and UUG both code for Leu
• GGC and GGA both code for Gly.
• Fig. 2B is a schematic demonstrating the altering of the wobble bases (i.e., those nucleotides in a codon which when altered change the codon but do not change the encoded amino acid) to remove the inverted complementary repeats.
• the codon GCC codes for Ala
• AAG codes for Lys, CUU and UUG both code for Leu
• GGC and GGA both code for Gly.
• Fig. 3 A is a schematic illustrating an RNA molecule with a small hairpin and the result of the action of endogenous RNA dependent RNA polymerases.
• Fig. 3B is a schematic illustrating the effect on the same molecule modified with a 3' chain terminator (*). No extension of the hairpin by endogenous RNA dependent RNA polymerases is possible.
• Fig. 4A is a schematic illustrating a DNA double-stranded plasmid.
• Fig. 4B is a schematic illustrating how transcripts are formed from the plasmid of Fig. 4 A in the absence of terminator sequences. Aberrant RNA strands are derived from both strands of the plasmid. Each arrow depicts the direction of transcription. Some of these transcripts can base pair with other transcripts to form double stranded
• Fig. 4C is a schematic illustrating the transcripts that occur from double- stranded DNA plasmids with terminators on both strands. Terminators on the coding strand are represented by *. Terminators on the non-coding strand are represented by 0. Arrows depict the direction of transcription.
• Fig. 5 is a schematic of an expanded region of the first (coding) strand of a plasmid. Terminators on the coding strand are represented by *.
• transcripts can still initiate at various cryptic sites on the plasmid DNA, the presence of terminators located throughout the plasmid prevents the initiated transcripts from extending past a terminator and thus prevent aberrant RNA formation from DNA double-stranded plasmids
• Fig. 6 illustrates the plasmid employed in Examples 1-4, which has an expression cassette containing a respiratory syncytial virus enhancer (RSVenh) and a human cytomegalovirus promoter (HCMV), a murine IL-12 p40 selected polynucleotide sequence, and an SV40 polyadenylation (polyA) site.
• the plasmid also contains a kanamycin resistance gene (Kan R ), and an origin of replication (ori).
• Kan R kanamycin resistance gene
• ori origin of replication
• the restriction endonuclease enzyme restriction sites are shown along with their respective nucleotide position numbers.
• the plasmid is 4709 bases in length for each strand.
• Fig. 7 illustrates a double-stranded plasmid used in Example 5, which contains two promoters: the simian cytomegalovirus (SCMV) promoter which directs transcription in one direction from the first strand and the HCMV promoter which directs transcription in the opposite direction from the second strand. Arrows denote direction of transcription. Terminator and RNA instability sequences are represented by •; a terminator on the second strand is •'; and a terminator on the first strand is 2 . Region A is located between ⁇ 1 and # 2 , as depicted.
• SCMV simian cytomegalovirus
• the present invention provides novel polynucleotide compositions and methods for therapy, prophylaxis, research and diagnostics in diseases and disorders which afflict invertebrate and vertebrate species, particularly mammalian species, in which the goal is to enhance the efficiency of expression of a selected polynucleotide sequence and reduce or inhibit the formation of undesired or aberrant polynucleotide species.
• These compositions and methods are also intended to prevent unintended shutting off or down-regulation of certain polynucleotide sequences which occur naturally in the host cell or may have been inserted therein.
• the terms "host cell” or "host” are intended to mean a vertebrate or invertebrate cell or living organisms made of those cells.
• this invention is intended for efficacy in mammals, preferably humans, domestic animals such as canines, felines, and equines, zoo animals, farmstock and laboratory animals, as well as in other vertebrates, such as avian species and fish, and their cells.
• This invention also has efficacy in eukaryotic cells, prokaryotic cells, other invertebrate cells, among others.
• the compositions and methods of this invention may also be useful in plant cells, and possibly in other organisms.
• compositions and methods have utility both in vitro, ex vivo and in vivo.
• cells in which the invention is particularly useful in vitro include stem cells, stable cell lines and primary cells.
• Cells in which the invention is particularly useful ex vivo include stem cells and primary cells. Any of the above cell types may be used for in vivo application of the methods and compositions herein. These compositions and methods further enable the harnessing of the molecular mechanisms of the cell to accomplish therapeutic, vaccine, diagnostic or research goals. /. DNA Molecules of the Invention
• polynucleotide molecule useful to accomplish the goals of this invention is a double stranded or partially double-stranded DNA molecule formed of a first "coding" strand and a complementary “transcription template” second strand, which are described in detail below.
• the term "complementary" has its traditional meaning in referring to double- stranded DNA, that is, for every purine in the first strand, i.e., adenine (A) and guanine (G), the second strand has in the corresponding position, a pyrimidine , i.e., either thymidine (T) or uridine (U) to hydrogen bond with every A in the first strand, or cytosine (C) to hydrogen bond with every G in the first strand.
• a pyrimidine i.e., either thymidine (T) or uridine (U) to hydrogen bond with every A in the first strand
• C cytosine
• This double-stranded or partially double-stranded molecule may be a DNA vector, a DNA plasmid or any double-stranded DNA construct designed to deliver a polynucleotide sequence to a cell for expression in the cell.
• This double-stranded molecule is linear in one embodiment; in another embodiment, this double stranded molecule is circular.
• the DNA molecule of this invention may be a double stranded plasmid or vector containing sequences under the control of RNA pol I, RNA pol II or RNA pol III, which can be transcribed into an RNA molecule in the cell according to this invention.
• the sequence encoding the RNA molecule has an open reading frame greater than about 300 nucleotides to avoid degradation in the nucleus via nonsense mRNA surveillance degradation mechanisms.
• plasmids or vectors can include sequences from bacteria, viruses or phages.
• vectors include chromosomal, episomal and virus-derived vectors e.g., vectors derived from bacterial plasmids, bacteriophages, yeast episomes, yeast chromosomal elements, and viruses, vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, cosmids and phagemids.
• one exemplary vector is a double-stranded DNA phage vector.
• Another exemplary vector is double-stranded DNA viral vector.
• Still another embodiment of a DNA molecule of this invention is a single- stranded DNA sequence which comprises, for example, one of the first or second strands of the double-stranded DNA molecules. These strands are described in detail below.
• the single stranded DNA molecule may also be synthesized and is designed so that the single strand encodes all of the information for the first coding strand, described in detail below, including the sequences which form the complements of any terminator sequences present on the second, complementary strand.
• the single stranded DNA molecule may also be synthesized as the non-coding or transcription template second strand.
• This second strand is designed so that the single strand encodes all of the complementary information for the first (coding) strand, described in detail below, including the complements to the terminator sequences which form part of the first strand, as well as the sequences forming the terminator sequences present on this non-coding strand.
• the single-stranded molecule may be a
• DNA vector a circular single stranded DNA such as that isolated from a bacteriophage M13, or any single-stranded DNA construct designed to deliver a polynucleotide sequence to a cell for expression in the cell.
• the single-stranded plasmids or vectors can include sequences from bacteria, viruses or phages.
• one exemplary molecule is a single-stranded DNA phage vector.
• Another exemplary molecule is a single-stranded DNA viral vector. This single-stranded molecule is linear in one embodiment; in another embodiment, this single-stranded molecule is circular.
• DNA molecules can be employed for in vivo, ex vivo and in vitro expression of the selected polynucleotide sequence in an efficient manner, while reducing the expression of undesired sequences from the constructs.
• the double-stranded, partially double-stranded or single-stranded molecule of this invention comprises a first strand (also referred to as a sense or coding strand) comprising at least one expression cassette sequence. If the first strand is bicistronic, it contains more than one expression cassette sequence.
• an expression cassette sequence includes a selected polynucleotide sequence operatively linked to regulatory components in a manner which permits transcription thereof in a host cell.
• "operably linked" sequences include both expression control sequences that are contiguous with the selected polynucleotide sequence and expression control sequences that act in trans or at a distance to control the polynucleotide sequence.
• the expression cassette contains at a minimum, from 5' to 3', a promoter, a selected polynucleotide sequence, and a polyadenylation (polyA) site.
• Other expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product.
• the first strand of the DNA construct is further provided with a nuclear localization signal, which targets the selected polynucleotide sequences to the nucleus of the cell transfected with the molecule, where transcription occurs.
• a nuclear localization signal which targets the selected polynucleotide sequences to the nucleus of the cell transfected with the molecule, where transcription occurs.
• Suitable nuclear localization signals are known to those of skill in the art and are not a limitation of the present invention [see, e.g., D. A. Dean, Exp. Cell Res.. 230(2):293-302 (Feb. 1, 1997)].
• suitable promoters are selected from among the constitutive, inducible and/or tissue-specific promoters known in the art for use in this invention.
• the promoter used in the expression cassette is desirably a weak promoter, e.g., a promoter that initiates slowly in the selected host cell; as one example, a weak promoter in some circumstances in the adeno-associated virus ITR.
• Examples of useful constitutive promoters include, without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al, Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the ⁇ -actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFl ⁇ promoter [Invitrogen].
• RSV Rous sarcoma virus
• CMV cytomegalovirus
• PGK phosphoglycerol kinase
• Inducible promoters are regulated by exogenously supplied compounds, including, the zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)- inducible mouse mammary tumor virus (MMTV) promoter, the T 7 polymerase promoter system [International Patent Application No. WO 98/10088]; the ecdysone insect promoter [No et al, Proc Natl Acad Sci USA. 93 3346-3351 (1996)], the tetracycline-repressible system [Gossen et al, Proc Natl Acad Sci USA. 89 5547- 5551 (1992)], the tetracycline-inducible system [Gossen et al, Science.
• MT zinc-inducible sheep metallothionine
• Dex dexamethasone- inducible mouse mammary tumor virus
• T 7 polymerase promoter system International Patent Application No. WO 98/10088
• Any component of the expression cassette may be readily selected from among conventionally employed promoters and regulatory sequences used in the genetic engineering field See, e g , Sambrook et al, "Molecular Cloning A
• the selected polynucleotide sequence can be any polynucleotide sequence which encodes a polypeptide, protein, or other product of interest or performs a biological function of interest and is desired to be preferentially and efficiently expressed in a cell in vitro, ex vivo or in vivo for therapeutic use, prophylactic use, gene therapy use, or research or diagnostic use
• a host of such selected polynucleotide sequences may be selected by one of skill in the art depending upon which condition is being treated or prevented
• the selection of the polynucleotide sequence will depend upon the use to which the resulting molecule is put
• one type of selected polynucleotide sequence includes a reporter sequence, which upon expression produces a detectable signal
• reporter sequences include without limitation, DNA sequences encoding ⁇ -lactamase, ⁇ - galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein
• GFP chloramphenicol acetyltransferase
• CAT chloramphenicol acetyltransferase
• membrane bound proteins including, for example, CD2, CD4, CD8, the influenza hemagglutinin protein, and others well known in the art, to which high affinity antibodies directed thereto exist or can be produced by conventional means, and fusion proteins comprising a membrane bound protein appropriately fused to an antigen tag domain from, among others, hemagglutinin or Myc.
• sequences when associated with regulatory elements which drive their expression, provide signals detectable by conventional means, including enzymatic, radiographic, colorimetric, fluorescence or other spectrographic assays, fluorescent activating cell sorting assays and immunological assays, including enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and immunohistochemistry.
• ELISA enzyme linked immunosorbent assay
• RIA radioimmunoassay
• immunohistochemistry for example, where the marker sequence is the LacZ gene, the presence of helper virus is detected by assays for beta-galactosidase activity. Where the selected polynucleotide is luciferase, the helper virus may be measured by light production in a luminometer.
• the selected polynucleotide is a non-marker sequence encoding a product which is useful in biology and medicine, such as proteins, peptides, anti-sense nucleic acids (e.g., RNAs), enzymes, or catalytic RNAs.
• the selected polynucleotide may be used to correct or ameliorate gene deficiencies, which may include deficiencies in which normal genes are expressed at less than normal levels or deficiencies in which the functional gene product is not expressed.
• a preferred type of selected polynucleotide sequence encodes a therapeutic protein or polypeptide which is expressed in a host cell.
• the invention further includes using multiple selected polynucleotides, e.g., to correct or ameliorate a gene defect caused by a multi-subunit protein.
• a different selected polynucleotide may be used to encode each subunit of a protein, or to encode different peptides or proteins. This is desirable when the size of the DNA encoding the protein subunit is large, e.g., for an immunoglobulin, the platelet-derived growth factor, or a dystrophin protein.
• a cell is infected with the recombinant virus containing each of the different subunits.
• different subunits of a protein may be encoded by the same selected polynucleotide.
• a single selected polynucleotide includes the DNA encoding each of the subunits, with the DNA for each subunit separated by an internal ribozyme entry site (IRES).
• IRES internal ribozyme entry site
• the selected polynucleotide may encode any product desirable for study. The selection of the selected polynucleotide sequence is not a limitation of this invention.
• hormones, growth and differentiation factors including, without limitation, insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormone releasing factor (GRF), follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietins, angiostatin, granulocyte colony stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), transforming growth factor ⁇ (TGF ⁇ ), platelet-derived growth factor (PDGF), insulin growth factors I and II (IGF-I and IGF-II), any one of the transforming growth factor ⁇ superfamily, including TGF ⁇ , activins, inhibins, or any of the bone
• cytokines and lymphokines such as thrombopoietin (TPO), interleukins (DL) IL-1 through IL-17, monocyte chemoattractant protein, leukemia inhibitory factor, granulocyte-macrophage colony stimulating factor, Fas ligand, tumor necrosis factors ⁇ and ⁇ , interferons ⁇ , ⁇ , and ⁇ , stem cell factor, flk-2/flt3 ligand.
• immunoglobulins IgG, IgM, IgA, IgD and IgE include, without limitations, immunoglobulins IgG, IgM, IgA, IgD and IgE, chimeric immunoglobulins, humanized antibodies, single chain antibodies, T cell receptors, chimeric T cell receptors, single chain T cell receptors, class I and class II MHC molecules, as well as engineered immunoglobulins and MHC molecules.
• Useful gene products also include complement regulatory proteins such as complement regulatory proteins, membrane cofactor protein (MCP), decay accelerating factor (DAF), CR1, CF2 and CD59.
• CMP membrane cofactor protein
• DAF decay accelerating factor
• CR1, CF2 CR1, CF2
• Still other useful selected polynucleotides may produce a product such as any one of the receptors for the hormones, growth factors, cytokines, lymphokines, regulatory proteins and immune system proteins.
• the selected polynucleotide may be a receptor for cholesterol regulation, including the low density lipoprotein (LDL) receptor, high density lipoprotein (HDL) receptor, the very low density lipoprotein (VLDL) receptor, and the scavenger receptor.
• LDL low density lipoprotein
• HDL high density lipoprotein
• VLDL very low density lipoprotein
• the selected polynucleotide sequence useful in this invention also encodes products such as members of the steroid hormone receptor superfamily including glucocorticoid receptors and estrogen receptors, Vitamin D receptors and other nuclear receptors.
• useful polynucleotide sequences include sequences encoding transcription factors such as jun,fos, max, mad, serum response factor (SRF), AP-1, AP2, myb, MyoD and myogenin, ETS-box containing proteins, TFE3, E2F, ATF1, ATF2, ATF3, ATF4, ZF5, NFAT, CREB, HNF-4, C/EBP, SP1, CCAAT-box binding proteins, interferon regulation factor (IRF- 1), Wilms tumor protein, ETS-binding protein, ST AT, GATA-box binding proteins, e.g., GATA-3, and the forkhead family of winged helix proteins.
• transcription factors such as jun,fos, max, mad, serum response factor (SRF), AP-1, AP2, myb, MyoD and myogenin
• ETS-box containing proteins TFE3, E2F, ATF1, ATF2, ATF3, ATF4, ZF5, NFAT, CREB, HNF-4
• Other useful products which may be encoded by the selected polynucleotide sequence of the expression cassette include, carbamoyl synthetase I, ornithine transcarbamylase, arginosuccinate synthetase, arginosuccinate lyase, arginase, fumarylacetacetate hydrolase, phenylalanine hydroxylase, alpha- 1 antitrypsin, glucose- 6-phosphatase, porphobilinogen deaminase, factor VTII, factor IX, cystathione beta- synthase, branched chain ketoacid decarboxylase, albumin, isovaleryl-coA dehydrogenase, propionyl CoA carboxylase, methyl malonyl CoA mutase, glutaryl CoA dehydrogenase, insulin, beta-glucosidase, pyruvate carboxylate, hepatic phosphorylase, phosphorylase kinase,
• Still other useful polynucleotide-encoded products include, non- naturally occurring polypeptides, such as chimeric or hybrid polypeptides having a non- naturally occurring amino acid sequence containing insertions, deletions or amino acid substitutions, e.g., single-chain engineered immunoglobulins, antisense molecules and catalytic nucleic acids, such as ribozymes. Still other suitable polynucleotide sequences may be readily selected by one of skill in the art. The selection of the polynucleotide is not considered to be a limitation of this invention. As shown in the following examples, an illustrative selected polynucleotide sequence is the sequence encoding murine interleukin-12.
• the first strand are at least one, and desirably at least two, and preferably three or more transcriptional terminator sequences.
• Transcriptional terminators can be located anywhere on the DNA coding (i.e., the non-transcribed) strand, including within the expression cassette sequence, as long as transcription can proceed uninterrupted within the expression cassette on the DNA template ("second") strand and the biological function of the polynucleotide sequence in the expression cassette is not adversely effected upon expression.
• any modification may be made to the first strand to incorporate the transcription terminators, so long as transcription can take place from the one or more expression cassette complementary region(s) of the second "template” strand and the expressed protein or peptide retains its biological function.
• Preferred transcription terminators do not create secondary structures that interfere with transcription of the template strand.
• the function of these terminator sequences is to prevent unwanted transcription from occurring from the first strand, and also from the complementary second transcription template strand, when these strands of the DNA molecule are in the host cell.
• these first strand transcription terminators cannot impede or interrupt the necessary transcription from the second template strand or from an expression cassette intended to be transcribed from the first strand of a bicistronic molecule.
• the function of these terminator sequences prevents the formation of undesired transcripts which, if homologous to a polynucleotide sequence present in the host cell, could shut off or down regulate its normal transcription in the host cell.
• the function of these terminators prevents physical occlusion of the promoter, thereby increasing the efficacy of the expression of the selected polynucleotide.
• the first strand is bicistronic (i.e., contains two or more expression cassettes or each strand contains an expression cassette)
• the same constraints are placed upon the identity and location of the terminator sequence(s).
• each terminator sequence may be the same or a different terminator sequence.
• a terminator sequence may be a bacterial terminator, such as the 5S ribosomal RNA terminator, rrnB.
• a terminator sequence may be a bacterial terminator, such as the tryptophan operon terminator, trpA.
• a terminator sequence may be a bacteriophage terminator, e.g., a lambda 5S RNA terminator or the bacteriophage PI head protein terminator.
• Still another desirable terminator sequence which may be employed positioned 3 ' to a polyA site is a eukaryotic pause site for RNA pol II, such as the site described in P. Enriquez-Harris et al, EMBO J.. 10(7): 1833-1842 (1991).
• An ⁇ -globin terminator is an example of a terminator for RNA pol II and may also be employed as a terminator sequence in a first strand of this molecule.
• a histone mRNA processing signal such as that described in N. Chodchoy et al, Mol. Cell. Bio il(l):497-509 (Jan. 1991), is a useful terminator sequence for the first strand.
• the terminator for the mammalian gastrin gene, the gastrin terminator is also useful.
• Another useful terminator sequence is a polynucleic acid sequence that provides a ribozyme cleavage site followed by a pause site terminator sequence, as described above.
• Terminor sequences for use in the first strand include a ribonucleic acid cleavage site followed 3' by a pause site terminator sequence.
• rho-dependent terminators e.g., C. E. Bogden et al, Mol. Cell, 3:487-493 (Apr.
• rho-independent terminators e.g., I. Gusarov and E. Nudler, Mol. Cell. 3:495-504 (Apr. 1999) are also sequences which may be employed for this purpose in the first strand.
• a padlock probe described in M. Nilsson et al, Science. 265:2085-2088 (1994) is a useful terminator for a double-stranded or partially double-stranded DNA molecule.
• a padlock is a circular single stranded polynucleotide sequence tortionally linked to the polynucleotide molecule of this invention by hybridization between at least 10 consecutive nucleotides of the padlock and at least 10 consecutive nucleotides on the first strand. See Fig. 1C and ID.
• at least one of the above- described terminator sequences is preferably located 5' to the promoter.
• this first strand terminator sequence is located between 1 to 50 nucleotides 5' of the promoter. In another desirable embodiment, this first strand terminator sequence is located between 20 to 40 nucleotides 5' of the promoter. In another desirable embodiment, this first strand terminator sequence is located between 10 to 30 nucleotides 5' of the promoter. In one embodiment, therefore, a padlock terminator (or any other suitable terminator or the complement of a ribonucleolytic or catalytic site) may be present within the 5' untranslated region of the first strand.
• a first strand terminator sequence is located 3 ' to the polyA site, outside of the expression cassette.
• This first strand terminator sequence is desirably located at least 100 to 150 nucleotides 3' from the polyA site, or immediately following the end of the polyA sequence, of the expression cassette.
• a padlock terminator (or any other suitable terminator or the complement of a ribonucleolytic or catalytic site) may be present within the 3' untranslated region of the first strand.
• both the 5' and 3' terminator sequences are employed, flanking an expression cassette on the first strand.
• RNA instability sequence in addition to the 5' and 3' terminator sequences that flank the expression cassette, additional optional terminator sequences are located on the first strand outside of the expression cassette sequence.
• at least one RNA instability sequence is located on the first strand, preferably outside of the expression cassette sequence.
• RNA instability sequence is located ' to the 3' flanking terminator sequence or 5' to the 5' flanking terminator sequence.
• Figs. 1A-1D illustrate schematic versions of several of the preferred embodiments.
• a padlock terminator (or any other suitable terminator or the complement of a ribonucleolytic or catalytic site) is located within the expression cassette, and more particularly within the coding sequence of the selected polynucleotide sequence.
• the padlock terminator is located in the coding sequence of the polynucleotide sequence. When in this position, the terminator sequence does not impede transcription from the portion of the second strand complementary to the expression cassette, nor effect the biological function of the protein to be expressed.
• the presence of the terminator sequence in the first strand cannot prevent or impede the transcription from the second strand, so that the selected polynucleotide sequence is always correctly transcribed and expressed, retaining its biological function, regardless of the position of the first strand terminator sequence.
• the terminator sequence within the coding region may be any of the above-identified terminators, including a padlock probe described above, provided that it not impede transcription from the second strand or effect the biological function of the expressed protein.
• the first strand avoids any modification which results in the introduction of an ATG (start codon) or a Kozak region. Such modifications, alone or in combination, will ideally prevent unwanted transcription of antisense RNA.
• ds RNA may be added, at one or more sites within the coding strand, the complement of an RNA instability sequence. Any undesired antisense RNA inadvertently formed from this strand will contain an instability sequence promoting RNA degradation and will not be available to hybridize with sense RNA.
• the first strand of the DNA molecule lacks any inverted complementary repeat sequences of greater than seven consecutive nucleotides in length located anywhere on the strand.
• a first strand contains a sequence of, for example, ATGCTTA
• This lack of inverted complementary repeat sequences avoids unwanted internal base pairing of sequences on the same strand at 37° C, a normal body temperature for a mammalian host cell. This is a particularly important when the DNA molecule is transcribed into RNA in the host cell or where the first strand is the single-stranded DNA molecule.
• the first strand of the DNA molecule lacks any inverted complementary repeat sequences of greater than four consecutive nucleotides in length located anywhere on the strand. This lack of inverted complementary repeat sequences avoids unwanted internal base pairing of sequences on the same strand at the lower body temperatures of some invertebrate species' host cell.
• the "wobble" nucleotides in the first strand can be manipulated to alter the nucleotide sequence and prevent or eliminate any inverted complementary repeats in the sequence.
• Such wobble nucleotides are generally in the third base position of most codons, but can be located in the first or second position. This manner of eliminating such repeats permits the DNA to avoid being transcribed into aberrant RNA structures that will form stable double-stranded regions, or will exhibit inverted complementary repeats or homology to essential host polynucleotide sequences.
• an embodiment of the first strand of the DNA molecule of this invention involves altering the wobble nucleotides in substantially all of the codons in that portion of the first strand that comprises the selected polynucleotide sequence, where there is a possibility that the selected polynucleotide sequence in the molecule is too homologous with a polynucleotide sequence already present in the host cell.
• the selected polynucleotide sequence thus encodes the same amino acid sequence, but provides a nucleotide sequence which is substantially nonhomologous to a desired polynucleotide sequence in a host cell or host organism.
• This area of non-complementarity in the "wobbled" codon prevents the transcript made from the DNA molecule in a host cell from undesirably or unintentionally shutting off or down-regulating a homologous polynucleotide sequence which is native to, or essential to, the transfected host cell. See Figs. 2A and 2B.
• substantially all wobble bases is meant wobbling enough codons to destroy the homology between the selected polynucleotide and any polynucleotide in the host cell, so as to prevent the shutting off of the polynucleotide sequence in the host cell.
• the wobble bases may be altered by well-known mutational techniques. Where the DNA is synthesized, the wobble codon bases are deliberately made to avoid a match with native sequences in the intended host cell or organism. The wobble bases may be selected to incorporate preference codons to optimize codons for expression in a selected host cell, provided that the aberrant RNA structures discussed above are avoided. See, e.g., United States Patent No. 5,786,464, which teaches the selection of preferred codons. As used herein and as known in the art, the terms "homology" or
• homologous refers to the degree of sequence relatedness between two polypeptide or two polynucleotide sequences as determined by the identity of the match between two lengths of such sequences. Both identity and homology can be readily calculated by methods extant in the prior art [See, e.g., COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, A.M., ed., Oxford University Press, New York, (1988);
• BIOCOMPUTLNG INFORMATICS AND GENOME PROJECTS, Smith, D.W., ed., Academic Press, New York, (1993); COMPUTER ANALYSIS OF SEQUENCE DATA, PART I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, (1994); SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, von Heinje, G., Academic Press, (1987); and SEQUENCE ANALYSIS PRLMER, Gribskov, M. and
• the double-stranded or partially double-stranded DNA molecule of this invention comprises preferably a second "transcription template" strand. While the second strand is ordinarily 100% complementary to the first strand in plasmids produced in bacteria, this is not essential, as, e.g., where at least one of the strands is produced synthetically and combined with a substantially homologous strand. Provided that there is sufficient homology between the two strands that the T m is high enough to cause the strands to stay together under appropriate conditions, certain areas of non-homology with the first strand may be present on the second strand.
• the second strand comprises at least one second strand terminator sequence which terminates any transcription initiated on the second strand.
• the second strand terminator sequence is located on the second strand in a sequence of the second strand which is not complementary to the expression cassette sequence.
• the second strand of the DNA molecule contains more than one second strand terminator sequence. More preferably, the second strand contains from two terminator sequences to one terminator sequence for every 100 nucleotides of second strand outside of the sequence complementary to the expression cassette. Another preferred embodiment contains one terminator sequence for every 500 nucleotides of second strand in the region which is not complementary to the expression cassette of the first strand. If more than one second strand terminator sequence is used, the multiple terminator sequences may be interspersed in the region of the second strand which is not complementary to the expression cassette, as described above.
• the second antisense DNA strand may have some degree of non-homology which needs to be transcribed into the mRNA.
• a second strand that contains one or more transcription terminators or RNA instability sequences outside of the region complementary to the coding region on the first strand. Changes in the second antisense strand can ordinarily be made only insofar as such changes will nevertheless yield a functional polypeptide and will not adversely effect transcription.
• cis terminator or instability sequences may be encoded within the sense strand of a plasmid
• nucleotides on the second, antisense or transcribed strand must include only conservative or nonsense mutations or other mutations which do not change or do not adversely affect the resulting amino acid sequence. In such embodiments, the function of any expressed protein must not be adversely affected.
• Each terminator sequence on the second strand may be the same or each may each be a different terminator sequence. These terminator sequences include the sequences identified above as first strand terminator sequences.
• a terminator sequence may be a bacterial terminator, such as the rrnB or trpA.
• a terminator sequence may be a bacteriophage terminator, e.g., a lambda 5S RNA terminator or the bacteriophage PI head protein terminator.
• Still another desirable terminator sequence which may be employed is a polynucleic acid sequence that provides a ribozyme cleavage site or a ribonucleic acid cleavage site, followed 3' by a eukaryotic pause site, e.g., a pause site for RNA pol II or an ⁇ -globin terminator.
• a histone mRNA processing signal is a useful terminator sequence for the second strand.
• the terminator for the mammalian gastrin gene is also useful.
• Both rho-dependent terminators and rho-independent terminators are also sequences which may be employed for this purpose in the second strand.
• a padlock terminator sequence as described above is a useful terminator for the second strand if it is part of a double- stranded or partially double-stranded DNA molecule.
• the terminator sequence is located in the region of the second strand which is not complementary to the expression cassette, and is positioned immediately 3' with reference to the first strand poly A site (see Fig. IB).
• a second strand terminator sequence is located less than 200 nucleotides from the poly A site in the region of the second strand which is not complementary to the expression cassette sequence (see Fig. 1C).
• An alternative embodiment of the second strand contains a terminator sequence is located 5' with reference to the first strand promoter sequence (see Figs. 1 A-1C).
• a preferred embodiment contains multiple terminator sequences interspersed throughout the region of the second strand which is not complementary to the expression cassette of the first strand (see Figs. IB and 1C).
• the second strand of the DNA molecule involve the second strand additionally containing at least one RNA instability sequence located on the region of the second strand sequence which is not complementary to the expression cassette sequence. Preferably, more than a single instability sequence is present in that region of the second strand. Further, as with the first strand, the second strand preferably does not contain any complementary inverted repeat sequence of greater than seven nucleotides for use in mammalian cells or mammals or four nucleotides for use in invertebrate cells or organisms.
• the "wobble" nucleotides in the second strand can be manipulated to alter the nucleotide sequence and prevent or eliminate any inverted complementary repeats in the sequence. This manner of eliminating such repeats permits the DNA to avoid being transcribed into RNA that will form double-stranded regions.
• an embodiment of the second strand of the DNA molecule of this invention involves altering the wobble nucleotides in substantially all of the codons in that portion of the second strand that is complementary to the selected polynucleotide sequence, where there is a possibility that the selected polynucleotide sequence in the molecule is too homologous with a polynucleotide sequence already present in the host cell.
• the selected polynucleotide sequence thus encodes the same amino acid sequence, but provides a nucleotide sequence which is substantially nonhomologous to a desired polynucleotide sequence in a host cell or host organism.
• This area of non- complementarity in the "wobbled" codon prevents the transcript made from the DNA molecule in a host cell from undesirably or unintentionally shutting off or down- regulating a homologous polynucleotide sequence which is native to, or essential to, the transfected host cell. See Figs. 2A and 2B.
• substantially all wobble bases is meant wobbling enough codons to destroy the homology between the selected polynucleotide and any polynucleotide in the host cell, so as to prevent the shutting off of the polynucleotide sequence in the host cell.
• the wobble bases may be altered by well-known mutational techniques. Where the DNA is synthesized, the wobble bases are deliberately made to avoid a match with native sequences in the intended host cell or organism.
• the DNA molecules of this invention permits the nucleotide sequence which is transcribed within the cell to become a single stranded RNA sense or anti-sense strand which lacks the capacity to form any significant double strandedness.
• the DNA molecule can provide a single stranded RNA sequence comprising both a sense polynucleotide sequence and an anti-sense polynucleotide sequence, optionally separated by a non-base paired polynucleotide sequence.
• RNA molecules of the invention may be prepared and used in the methods described in detail below. II. RNA Molecules of the Invention
• composition according to this invention is a substantially single- stranded RNA molecule, comprising a sequence of ribonucleic acids having a 5' end and a 3' end, which is designed to prevent the formation of stable double-stranded RNA or partially double-stranded RNA molecules in the host cell.
• this molecule can contain a 5'ATG codon.
• a 5' ATG is not required, e.g., for catalytic RNA molecules, such as ribozymes or antisense RNA.
• regulatory regions such as Kozak sequences precede the 5' ATG codon.
• This single-stranded RNA molecule may be a linear molecule. Alternatively, this single-stranded RNA molecule may be circular.
• This single stranded RNA molecule comprises a selected polynucleotide sequence having a selected biological function when translated in a host cell.
• the selected polynucleotide sequence may be a coding sequence, that is, it is translated to express a protein or a functional fragment thereof.
• the selected sequence may be non-coding, but may have a regulatory function or other biological function.
• the selected polynucleotide sequence with the biological function may be selected from the sequences described above for the first strand of the DNA molecule.
• This single-stranded RNA polynucleotide sequence is between about 100 to 10,000 polynucleotides in length. At present the sequence is most desirably at least 200 polynucleotides in length, but it can range in one embodiment from 200 to 8000 polynucleotides in length. In another embodiment, the RNA molecule can be less than 7500 polynucleotides in length. In still another embodiment the RNA molecule can have a sequence length less than about 5000 polynucleotides. In yet another embodiment the RNA molecule can have a sequence length less than about 2000 polynucleotides. In still another embodiment the RNA molecule can have a sequence length less than about 1000 polynucleotides. In yet another embodiment the RNA molecule can have a sequence length less than about 750 polynucleotides.
• the single-stranded RNA molecule may possess a small hairpin sequence at the 3' end, i.e., a sequence of no more than 5 nucleotides of double-strandedness. In other embodiments, any hairpin sequence which may have originally been present as part of the RNA molecule is deleted from the molecule.
• the RNA sequence lacks any inverted complementary repeat sequences of greater than seven consecutive nucleotides in length located anywhere on the strand.
• a first strand contains a sequence of, for example, AUGCUUA
• This lack of inverted complementary repeat sequences avoids unwanted internal base pairing of sequences on the same strand at 37° C, a normal body temperature for a mammalian host cell.
• the single-stranded RNA molecule lacks any inverted complementary repeat sequences of greater than four consecutive nucleotides in length located anywhere on the strand. This lack of inverted complementary repeat sequences avoids unwanted internal base pairing of sequences on the same strand at the lower body temperatures of some invertebrate species' host cell.
• the "wobble" nucleotides in the strand can be manipulated to alter the nucleotide sequence and prevent or eliminate any inverted complementary repeats in the sequence. This manner of eliminating such repeats prevents the formation of aberrant double-stranded RNA regions.
• an embodiment of the RNA molecule of this invention involves altering the wobble nucleotides in substantially all of the codons in that portion of the strand that is translated into the selected polynucleotide sequence, where there is a possibility that the selected polynucleotide sequence in the molecule is too homologous with a polynucleotide sequence already present in the host cell.
• the selected polynucleotide sequence thus encodes the same amino acid sequence, but provides a nucleotide sequence which is substantially nonhomologous to a desired polynucleotide sequence in a host cell or host organism. This area of non-complementarity in the "wobbled" codon prevents the undesirable or unintentional shutting off or down-regulating of a homologous polynucleotide sequence which is native to, or essential to, the transfected host cell.
• substantially all wobble bases is meant wobbling enough codons to destroy the homology between the selected polynucleotide and any polynucleotide in the host cell, so as to prevent the shutting off of the polynucleotide sequence in the host cell.
• the wobble bases may be altered by well-known mutational techniques.
• the wobble bases are deliberately made to avoid a match with native sequences in the intended host cell or organism.
• the single stranded RNA molecule contains a chromosomal copy of a gene in the intended host cell
• the single stranded RNA is prepared to be indistinguishable from the chromosomal RNA.
• the molecule contains no heterologous sequences flanking the selected polynucleotide sequence, which latter sequence is identical in ribonucleic acid sequence with the native sequence. This molecule is designed to avoid aberrently produced, partially double-stranded RNA with heterologous flanking sequences, which may cause the cell to shut off the chromosomal gene.
• the single-stranded RNA molecule comprises a cap at the 5' end of the molecule. In another embodiment, the single-stranded RNA molecule has no cap at the 5' end of the sequence.
• the single-stranded RNA molecule comprises a polyA sequence at the 3' end of the molecule. In another embodiment, the single-stranded RNA molecule has no polyA sequence at the 3 ' end of the sequence.
• the single-stranded RNA molecule has attached at the 3 ' hydroxy group, a modification which functions as a chain terminator to block the extension of a hairpin (a double- stranded region).
• a modification which functions as a chain terminator to block the extension of a hairpin a double- stranded region.
• chemical moieties include, without limitation, dideoxynucleotides (ddNTPs), 3' amino nucleotide triphosphates, 3' methyl nucleotide triphosphates, and 3' phosphorylthioate nucleotide triphosphates.
• ddNTPs dideoxynucleotides
• 3' amino nucleotide triphosphates 3' methyl nucleotide triphosphates
• 3' phosphorylthioate nucleotide triphosphates 3' phosphorylthioate nucleotide triphosphates.
• Other chain terminators may be readily selected by those of skill in the art
• RNA sequences which provide a topological knot or lariat at the 3' end which also functions to prevent chain extension. These structures can be prepared according to Smith and Nikonowicz, Biochem. 37: 13486-13498 (1998).
• Still other embodiments of single-stranded RNA molecules of this invention include RNA sequences which contain two or more of the above modifications.
• an RNA molecule of this invention may have a 5' cap and a polyA tail; or no 5' cap, no inverted repeats, and a polyA tail.
• Still another embodiment has no inverted repeats, a chain terminator on the 3' end and no poly A tail.
• One of skill in the art may combine others of the modifications described above to prepare an RNA molecule of this invention.
• RNA molecules that are double stranded or partially double stranded to prevent the inadvertent shutting off or down-regulation of homologous polynucleotide sequences which are native to the host cell or otherwise essential to the host cell or organism.
• RNA molecules of the invention may be prepared and used in the methods and compositions described in detail below.
• DNA and RNA molecules described above may be designed and produced by use of known teachings of the art. Both of these molecules may be modified as described above to enhance expression of the selected sequence and prevent transcription of undesirable or aberrant polynucleotide species, thereby avoiding the unintended shutting off of polynucleotide sequences of the host cell or host organism.
• These polynucleotide molecules may be designed by resort to conventional techniques such as those described in Sambrook, cited above or in Promega Protocols and Applications Guide, (3rd ed. 1996), eds. Doyle, ISBN No. 1-882274-57-1. For example, these molecules may be produced by enzymatic synthetic methods or chemical synthetic methods in vitro.
• an RNA molecule is made in vitro by conventional enzymatic synthetic methods using, for example, the bacteriophage T7, T3 or SP6 RNA polymerases according to the conventional methods described by such texts as cited above
• an RNA or DNA molecule of this invention may be prepared in a host cell transfected with a plasmid containing the T7 promoter and another plasmid containing the T7 RNA polymerase
• these molecules may be made by chemical synthetic methods in vitro [see, e g , Q Xu et al, Nucl Acids Res . 24(18) 3643-4 (Sept 1996), N Naryshkin et al, Bioorg Khim .
• these molecules may be produced by recombinant means in a culture of host cells, and isolated therefrom
• the DNA or RNA molecules useful in this invention can be made in a recombinant microorganism, e g , bacteria and yeast or in a recombinant host cell, e g , mammalian cells, and isolated from the cultures thereof by conventional techniques See, e g , the techniques described in Sambrook, cited above, which is exemplary of laboratory manuals that detail these techniques, and the techniques described in US Patent Nos 5,824,538, 5,877,159 and 5,643,771, incorporated herein by reference
• the circular RNA molecule can be prepared according to the techniques described in S Wang et al, Nucl Acids Res . 22(12) 2326-33 (June 1994), Y
• DNA and/or RNA molecules prepared or synthesized in vitro may be directly delivered to the host cell or to the host organism as polynucleotide molecules.
• desired DNA or RNA molecules may be provided to host cells in live, attenuated or killed, inactivated recombinant bacteria which are designed to contain the sequences necessary for the required DNA or RNA molecules of this invention.
• recombinant bacterial cells, fungal cells and the like can be prepared by using conventional techniques such as described in US Patent Nos. 5,824,538; 5,877,159 and 65,643,771, incorporated herein by reference.
• Microorganisms useful in preparing these delivery agents include those listed in the above cited reference, including, without limitation, Escherichia coli, Bacillus subtilis, Salmonella typhimurium, and various species of Pseudomonas, Streptomyces, Staphylococcus and Shigella.
• the DNA or RNA molecules may be formed in the host cell or in the host by live, attenuated or killed, inactivated viruses, and particularly recombinant viruses carrying the required DNA or RNA polynucleotide sequence discussed above. Such viruses may be designed similarly to recombinant viruses presently used to deliver genes to cells for gene therapy and the like.
• viruses or viral sequences which may be manipulated to provide an RNA or DNA molecule to a host cell in vivo are, without limitation, alphavirus, adenovirus, adeno-associated virus, baculoviruses, delta virus, pox viruses, hepatitis viruses, herpes viruses, papova viruses (such as SV40), poliovirus, pseudorabies viruses, retroviruses, vaccinia viruses, positive and negative stranded RNA viruses, viroids, and virusoids, or portions thereof.
• These various viruses may be designed by applying conventional techniques such as described in M. Di Nocola et al, Cancer Gene Then. 5(6):350-6 (1998), among others, with the teachings of the present invention.
• Formation of the desired, above-described DNA or RNA molecules in the host cell may also occur in live, attenuated or killed, inactivated donor cells which have been transfected or infected in vitro with a synthetic RNA molecule or a DNA molecule or a recombinant virus as described above. These donor cells may then be administered to the host, as described in detail below.
• donor cells are desirably cells of the host species as the host into which they are intended to be delivered, e.g., mammalian cells, such as C127, 3T3, CHO, HeLa, human kidney 293, BHK cell lines, and COS-7 cells, are useful host cells for mammals.
• Such donor cells can be made using techniques similar to those described in, e.g., Emerich et al, J. Neurosc 16: 5168-81 (1996). Even more preferred, the donor cells may be harvested from the specific host to be treated and made into donor cells by ex vivo manipulation, akin to adoptive transfer techniques, such as those described in D. B. Kohn et al, Nature Med.. 4(7V775-80 (1998Y
• the molecules of this invention can also be made into a mixture of synthetic RNA molecules or synthetic DNA delivery molecules as described above, or as or in recombinant bacteria, cells, and viruses.
• the composition of the mixture may be readily selected by one of skill in the art.
• compositions of this invention are useful in an in vitro or tissue culture method for enhancing the efficiency of expression of a selected polynucleotide sequence in a host cell, or similarly useful if the same method (except for the recovery step) is employed to express the selected polynucleotide efficiently in vivo or ex vivo.
• One embodiment of the method comprises the step of transfecting the host cell with a double stranded or partially double stranded DNA molecule or an RNA molecule described above, thereby inhibiting the formation of aberrant polynucleotide species transcribed or translated from the polynucleotide molecule in the host cell.
• this method enables the expression and recovery of maximal amounts of the product encoded by the polynucleotide product from the host cell.
• the host cell may be conventionally lysed and the product collected; or if the product is secreted, it may be collected from media by conventional techniques.
• the methods involve administering an effective amount of a pharmaceutical composition comprising a polynucleotide molecule as described above, an optional agent that facilitates polynucleotide uptake in a cell, and a suitable pharmaceutically acceptable carrier to a mammalian subject.
• a pharmaceutical composition comprising a polynucleotide molecule as described above, an optional agent that facilitates polynucleotide uptake in a cell, and a suitable pharmaceutically acceptable carrier to a mammalian subject.
• the compositions of this invention for pharmaceutical use desirably contain the DNA or RNA molecules, or mixtures thereof, in a pharmaceutically acceptable carrier, with additional optional components for pharmaceutical delivery.
• the specific formulation of the pharmaceutical composition depends upon the form of the active agent.
• Suitable pharmaceutically acceptable carriers facilitate administration of the polynucleotide compositions of this invention, but are physiologically inert and/or nonharmful.
• Carriers may be selected by one of skill in the art. Such carriers include but are not limited to, sterile saline, phosphate buffered saline, dextrose, sterilized water, glycerol, ethanol, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, olive oil, sesame oil, and water and combinations thereof.
• the carrier or diluent may include a time delay material, such as glycerol monostearate or glycerol distearate alone or with a wax.
• slow release polymer formulations can be used. The formulation should suit also the mode of administration. Selection of an appropriate carrier in accordance with the mode of administration is routinely performed by those skilled in the art.
• Such molecules of the invention may be introduced into cells as polynucleotides, by well known techniques for introducing DNA into cells.
• the molecules in the case of phage and viral vectors, may also be and preferably are introduced into cells as packaged or encapsidated DNA or RNA virus by well known techniques for infection and transduction.
• Viral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.
• composition contains a polynucleotide molecule, e.g., a DNA molecule, plasmid, viral vector, or recombinant virus, or multiple copies of the polynucleotide or different polynucleotides, etc., as described above, the composition may desirably be formulated as "naked" polynucleotide with only a carrier.
• a polynucleotide molecule e.g., a DNA molecule, plasmid, viral vector, or recombinant virus, or multiple copies of the polynucleotide or different polynucleotides, etc.
• compositions desirably contain optional polynucleotide facilitating agents or "co-agents", such as a local anaesthetic, a peptide, a lipid including cationic lipids, a liposome or lipidic particle, a polycation such as polylysine, a branched, three- dimensional polycation such as a dendrimer, a carbohydrate, a cationic amphiphile, a detergent, a benzylammonium surfactant, or another compound that facilitates polynucleotide transfer to cells.
• polynucleotide facilitating agents or co-agents such as a local anaesthetic, a peptide, a lipid including cationic lipids, a liposome or lipidic particle, a polycation such as polylysine, a branched, three- dimensional polycation such as a dendrimer, a carbohydrate, a cationic am
• the facilitating agent used is a local anesthetic, preferably bupivacaine
• an amount of from about 0.1 weight percent to about 1.0 weight percent based on the total weight of the polynucleotide composition is preferred.
• PCT US98/08799 for delivery in vesicular complexes
• PCT US98/22841 which teaches the incorporation of benzylammonium surfactants as co-agents, administered in an amount of between about 0.001-0.03 weight %, the teachings of which are hereby incorporated by reference.
• composition is other than a polynucleotide, e.g., is a transfected donor cell or a bacterium as described above, the composition may also contain other additional agents, such as those discussed in US Patents No. 5,824,538; 5,643,771; 5,877,159, incorporated herein by reference.
• compositions are, adjuvants, preservatives, chemical stabilizers, or other antigenic proteins.
• stabilizers, adjuvants, and preservatives are optimized to determine the best formulation for efficacy in the target human or animal.
• Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol.
• Suitable stabilizing ingredients which may be used include, for example, casamino acids, sucrose, gelatin, phenol red, N-Z amine, monopotassium diphosphate, lactose, lactalbumin hydrolysate, and dried milk.
• a conventional adjuvant is used to attract leukocytes or enhance an immune response.
• adjuvants include, among others, Ribi, mineral oil and water, aluminum hydroxide, Amphigen, Avridine, L121/squalene, D-lactide-polylactide/glycoside, pluronic plyois, muramyl dipeptide, killed Bordetella, and saponins, such as Quil A.
• agents which may function as transfecting agents and/or replicating agents and/or inflammatory agents and which may be co-administered with the composition of this invention include growth factors, cytokines and lymphokines such as alpha-interferon, gamma-interferon, platelet derived growth factor (PDGF), colony stimulating factors, such as G-CSF, GM-CSF, tumor necrosis factor (TNF), epidermal growth factor (EGF), and the interleukins, such as IL-1, IL-2, IL-4, IL-6, JJ -8, IL-10 and IL-12.
• growth factors such as alpha-interferon, gamma-interferon, platelet derived growth factor (PDGF), colony stimulating factors, such as G-CSF, GM-CSF, tumor necrosis factor (TNF), epidermal growth factor (EGF), and the interleukins, such as IL-1, IL-2, IL-4, IL-6, JJ -8, IL-10 and
• fibroblast growth factor may also be administered in conjunction with the compositions of the invention.
• surface active agents such as immune-stimulating complexes (ISCOMS), Freund's incomplete adjuvant, LPS analog including monophosphoryl Lipid A (MPL), muramyl peptides, quinone analogs and vesicular complexes such as squalene and hyaluronic acid may also be administered in conjunction with the compositions of the invention.
• compositions may also contain other additives suitable for the selected mode of administration of the composition.
• these compositions can contain additives suitable for administration via any conventional route of administration, including without limitation, parenteral administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intra-pulmonary administration, rectal administration, vaginal administration, and the like. All such routes are suitable for administration of these compositions, and may be selected depending on the agent used, patient and condition treated, and similar factors by an attending physician.
• composition of the invention may also involve lyophilized polynucleotides, which can be used with other pharmaceutically acceptable excipients for developing powder, liquid or suspension dosage forms, including those for intranasal or pulmonary applications.
• lyophilized polynucleotides which can be used with other pharmaceutically acceptable excipients for developing powder, liquid or suspension dosage forms, including those for intranasal or pulmonary applications.
• routes of administration for these compositions may be combined, if desired, or adjusted.
• the pharmaceutical compositions of the invention are prepared for administration to mammalian subjects in the form of, for example, liquids, powders, aerosols, tablets, capsules, enteric coated tablets or capsules, or suppositories.
• the compositions of the present invention when used as pharmaceutical compositions, can comprise about 1 ng to about 20 mgs of polynucleotide molecules e.g., the synthetic RNA molecules or DNA molecules, plasmids, viral vectors, recombinant viruses, and mixtures thereof.
• the compositions contain about 10 ng to about 10 mgs of polynucleotide sequences.
• the pharmaceutical compositions contain about 0.1 to about 500 ⁇ g polynucleotide sequences. In some preferred embodiments, the compositions contain about 1 to about 350 ⁇ g polynucleotide sequences. In still other preferred embodiments, the pharmaceutical compositions contain about 25 to about 250 ⁇ g of the polynucleotide sequences. In some preferred embodiments, the vaccines and therapeutics contain about 100 ⁇ g of the polynucleotide sequences.
• compositions of the present invention in which the DNA or RNA molecules are delivered in donor cells or bacterium can be delivered in dosages of between about 1 cell to about 10 7 cells/dose.
• a suitable vector-based composition contains between lxlO 2 pfu to lxl 0 12 pfu per dose.
• the above dosage ranges are guidelines only.
• the pharmaceutical compositions are administered in an amount effective to treat or prevent the diseases, disorders or infections for which it is designed.
• the amount of the pharmaceutical composition in a dosage unit employed will be determined empirically, based on the response of cells in vitro and response of experimental animals to the compositions of this invention It will be appreciated that optimum dosage will be determined by standard methods for each treatment modality and indication. Thus the dose, timing, route of administration, and need for readministration of these compositions may be determined by one of skill in the art, taking into account the condition being treated, its severity, complicating conditions, and such factors as the age, and physical condition of the mammalian subject, the employment of other active compounds, and the like.
• Still another embodiment of this invention is the use of the DNA and RNA molecules of this invention to prevent the unintentional or undesirable shutting off or down regulating of polynucleotide sequences which are essential or desirable in a host cell transfected with a polynucleotide molecule containing a polynucleotide sequence homologous to a polynucleotide sequence native to the host cell.
• This method comprises the steps of administering an effective amount of a pharmaceutical composition comprising a polynucleotide molecule as described above, an optional agent that facilitates polynucleotide uptake in a cell, and a suitable pharmaceutically acceptable carrier.
• compositions, pharmaceutical compositions, dosages and modes of administration described above are particularly desirable for the treatment of a variety of disorders that plague vertebrates, especially mammals, but also avians and other fowl, as well as invertebrates, such as fish, including infections by heterologous pathogenic organisms, either extracellular or intracellular pathogens.
• compositions of this invention are useful in preventing infection of a host with a pathogen, or in treating cancers. Further, these compositions are useful in the treatment of inherited or genetic disorders by expressing efficiently a polynucleotide protein or function which the host lacks.
• compositions described above, and the general methods of using these compositions to enhance the expression of a selected polynucleotide sequence in a host cell and thereby reduce the production or transcription or aberrant sequences can also be applied to a variety of research, and in vitro applications, where such efficient expression is essential.
• These compositions and methods may also be applied to manipulate plant cells and insect cells, and the hosts may similarly be plants and insects, among the other organisms mentioned previously.
• application of this method can be used to make cell lines of mammalian, bacterial, yeast, fungal, insect, plant, and other origins which efficiently produce a selected polynucleotide product. These molecules may also be employed to increase the efficiency of stable cell lines used to produce certain polynucleotide- encoded products.
• Such manipulated cells may be employed in producing recombinant proteins for pharmaceutical use in animals, for vaccine use in animals, as well as for a variety of uses in the agricultural fields. Such cells may also be employed in conventional testing assays for drugs or other useful compounds, or for drug screening and development assays, etc. Still other uses are expected to be obvious to the person of skill in the art given the teachings herein.
• compositions and using the compositions of this invention illustrate methods for preparing the compositions and using the compositions of this invention to increase the efficiency of transcription and thus expression of a selected polynucleotide sequence, and to reduce or inhibit expression of aberrant (unintended) polynucleotide sequences. It is understood by one of skill in the art, that other selections for the various selected polynucleotides of the compositions, and RNA and/or DNA molecules may be readily made as taught by this specification. These examples are illustrative only and do not limit the scope of the invention.
• EXAMPLE 1 A DNA Plasmid Containing Terminator Sequences on the Coding Strand using Mouse Interleukin 12 (mIL12) p40 as the Selected Polynucleotide
• the plasmid sequence is modified by the insertion of terminator sequences to prevent the formation of aberrant RNA.
• the cloning sites in the mIL12 expression vector are shown in Fig. 6, as a Hpal site at nt#4700 (downstream of the polyA site), and an Sphl site at nt#2568 (upstream of the CMV promoter/enhancer/ RSV enhancer module).
• the plasmid encoding mIL12 (Fig. 6) is modified to contain sequences (elements) that cause termination of elongation.
• the terminator sequences identified below are placed upstream to the human CMV promoter at the promoter cloning site, and at about 150 bases downstream of the SV40 polyadenylation sequence. When the terminator is used upstream of the promoter, a polyadenylation sequence is also cloned 150 base pairs upstream of the terminator sequence, since some termination sequences efficiently perform only when associated with a polyA site.
• the terminator sequences inserted into the plasmids include the following: Bacterial polymerase terminators which are rho dependent and rho independent are trpA (Genbank accession #E02304) and rmBTlT2 (Amersham-Pharmacia Biotech catalog #27-4925-01, Brosius, J., Gene, 27: 151 (1984), Genbank accession #U13859), respectively. These sequences are isolated from existing vectors available from Pharmacia Biotech or these sequences are readily synthesized (Genbank accession #E02304). The histone terminator [Genbank accession #Z46261, nt 750 to 765, Mol. Cell. BioL. 497-509 (Jan.
• dsDNA are synthesized.
• the 33 base pair trpA ds DNA sequences are synthesized (Genbank accession #E02304), and the 1139 base pair PvuII fragment containing the r/wBTlT2 terminator fragment is isolated from pKK232-8 vector (Amersham-Pharmacia Biotech). These nucleotide fragments are cloned into either of the restriction sites or both the Sphl and the Hpal sites in the mIL12 vector (Fig. 6).
• Each of these plasmid constructs are purified.
• One hundred micrograms of each of the test plasmids is injected into mouse hind limb muscle tissue.
• Two control sets of mice are also used: one set of control mice is injected with similar amounts of starting plasmid, and another set is injected with the backbone plasmid that does not contain the p40 cDNA sequence.
• Plasmids containing the terminators result in higher levels of EL12p40 in serum than control plasmids, while plasmids that contain terminators on both sides of the expression cassette are expected to express IL12p40 maximally. Plasmids containing the terminator sequences on the coding strand are anticipated to result in the increased expression of IL-12 and an increased longevity of expression of the IL-12 vis a vis the prior art plasmids.
• the plasmid encoding mIL12 (Fig. 6) is similarly modified to contain sequences (elements) that cause termination of elongation on the non-coding strand. These elements are placed on the non-coding strand in positions upstream of the CMV promoter at the Sphl cloning site, and about -150 bases downstream relative to the SV40 polyadenylation sequence at the Hpal cloning site.
• Example 1 are cloned into either of the restriction sites or both the Sphl and the Hpal sites in the mIL12 vector on the non-coding strand. Each of these constructs will be purified. One hundred micrograms of each of the plasmids is injected into mouse hind limb muscle tissue. Expression of the mIL12/p40 chain is analyzed in sera of inoculated mice by ELIS A as described in Example 1. This example uses two control sets of mice: one set of control mice is injected with similar amounts of starting plasmid, and another set is injected with the backbone plasmid that does not contain the p40 cDNA sequence.
• Plasmids containing any one terminator on the non-coding strand are expected to result in higher levels of IL12p40 in serum than control plasmids, while plasmids that contain terminators on both sides of the expression cassette on the non-coding strand are expected to express IL12p40 maximally.
• the plasmid constructs that contain terminators on the non-coding strand result in increased expression of IL12 and over a longer period than plasmids of the prior art when compared to the controls.
• EXAMPLE 3 Terminator Sequences on the Coding and the Non-Coding Strands
• the plasmid of Examples 1 and 2 (Fig. 6) encoding mTL12 is further modified to contain sequences (elements) that cause termination of elongation on both the coding and non-coding strands of the double-stranded DNA molecule.
• Two such elements are placed upstream to the CMV promoter at the promoter cloning site (Sphl site), and two elements at the position -150 bases downstream of the SV40 polyadenylation sequence (Hpal site), such that one element is effective on the coding strand, and the other element on the non-coding strand.
• the cloning sites and the terminator sequences are the same as employed in Example 1.
• the plasmid of Examples 1 and 2 is further modified by adding a padlock terminator, as described in Nilsson et al, Science. 265:2085-2088 (1994) at one or more sites on the sense or coding strand within the cistron, including within the coding region.
• a padlock terminator is added at one, two or more sites within the IL-12 coding sequence.
• Additional optional padlocks may be added within the antisense in either the 5' or 3' untranslated regions.
• constructs that contain terminators on both the coding and the non-coding strands are anticipated to express maximal amounts of IL12 and over a longer time period than control plasmids
• RNA instability sequence UUAUUUAUU [A M Curatola et al, Mol Cell Biol . 15(11) 6331-6340 (Nov 1995), A M Zubiaga et al, Mol Cell Biol . 15(4) 2219-2230 (Apr 1995)] is synthesized as dsDNA and cloned into the coding or non-coding or both strands of the plasmid of Fig 6 and Example 1 at the Hpal site, downstream from the polyA site and/or at the Sphl site upstream of the promoter
• RNA molecules that contain these instability sequences degrade rapidly
• Each of these constructs are purified One hundred micrograms of each of the plasmids is injected into mouse hind limb muscle tissue Expression of the mIL12/p40 chain is analyzed in sera of inoculated mice by ELIS A as described above Two control sets of animals are used One set of control mice is injected with similar amounts of starting plasmid (without the RNA instability sequence), and another set is injected with the backbone plasmid that does not contain the p40 cDNA sequence Plasmids containing the instability sequence are expected to result in higher levels of IL12p40 in serum than control plasmids, while plasmids that contain higher numbers of RNA instability sequence are expected to express more IL12p40.
• EXAMPLE 5 The Absence of Aberrant RNA Synthesis from Modified Constructs
• the SCMV promoter and the CMV promoter are placed outside the expression cassette in opposing orientations in plasmids that contain terminator or instability sequences similar to those described above in Examples 1-4, and in control plasmids that do not contain terminators and RNA instability sequences.
• These plasmids are used to transfect human rhabdomyo sarcoma (RD) cells (a human cell line available from the American Type Culture Collection).
• RD rhabdomyo sarcoma
• RNA derived from transfected cells is analyzed. For example, Northern blots are probed with sequences derived from within the sequence in the Region A, i.e., that sequence between the two terminators •' and ⁇ 2 , in the schematic of Fig. 7. Northern blot analysis of RNA electrophoresed following RibonucleaseA digestion, is performed using a probe derived from within the Region A sequences. RibonucleaseA should digest ssRNA but not dsRNA.
• RNA from Region A None or highly decreased amounts of RNA from the Region A are detected in RNA preparations of cells transfected with plasmids that contain aberrant RNA suppressive elements (terminators and RNA instability sequences). RNA from Region A is detected in Northern blots of RNA preparations of cells transfected with the control plasmid. Furthermore, only in control plasmids that do not contain aberrant RNA suppressive elements are dsRNAs detected following ribonuclease digestion.

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