WO2015021077A1 - Recombinant vector with optimized a-bulge - Google Patents

Recombinant vector with optimized a-bulge Download PDF

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Publication number
WO2015021077A1
WO2015021077A1 PCT/US2014/049831 US2014049831W WO2015021077A1 WO 2015021077 A1 WO2015021077 A1 WO 2015021077A1 US 2014049831 W US2014049831 W US 2014049831W WO 2015021077 A1 WO2015021077 A1 WO 2015021077A1
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sequence
replication competent
recombinant replication
competent retrovirus
retrovirus
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PCT/US2014/049831
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English (en)
French (fr)
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Amy H. LIN
Harry E. Gruber
Carlos Ibanez
Douglas J. Jolly
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Tocagen Inc.
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Priority to CA2920049A priority Critical patent/CA2920049A1/en
Priority to CN201480053358.9A priority patent/CN105579581A/zh
Priority to JP2016533383A priority patent/JP2016526920A/ja
Priority to EP14835030.9A priority patent/EP3030659A4/en
Priority to AU2014306093A priority patent/AU2014306093A1/en
Publication of WO2015021077A1 publication Critical patent/WO2015021077A1/en
Priority to US15/016,201 priority patent/US20160222412A1/en

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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C12N2310/141—MicroRNAs, miRNAs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2330/00—Production
    • C12N2330/50—Biochemical production, i.e. in a transformed host cell
    • C12N2330/51—Specially adapted vectors

Definitions

  • This disclosure relates to optimized internal ribosome entry sites (IRES) , compositions containing such optimized IRESs including vectors. More particularly, the disclosure relates to replication competent retroviral vectors for treating cell proliferative disorders. The disclosure further relates to the use of such replication competent retroviral vectors for delivery and expression of heterologous nucleic acids.
  • IRES internal ribosome entry sites
  • the disclosure provides recombinant replication competent retrovirus comprising: a retroviral GAG protein; a retroviral POL protein; a retroviral envelope; a retroviral polynucleotide comprising Long-Terminal Repeat (LTR) sequences at the 3' end of the retroviral polynucleotide sequence, a promoter sequence at the
  • the promoter being suitable for expression in a mammalian cell, a gag nucleic acid domain, a pol nucleic acid domain and an env nucleic acid domain; a cassette comprising an internal ribosome entry site (IRES) consisting of 6A' s in the A-bulge in the bifurcation region of the
  • IRES wherein the IRES is operably linked to a heterologous polynucleotide, wherein the cassette is positioned 5' to the 3' LTR and 3' to the env nucleic acid domain encoding the retroviral envelope; and cis-acting sequences necessary for reverse
  • the virus infects a target cell multiple times resulting in an average number of copies/diploid genome of 5 or greater.
  • the retroviral polynucleotide sequence is derived from a virus selected from the group consisting of murine leukemia virus (MLV) , Moloney murine leukemia virus (MoMLV) , Feline leukemia virus (FeLV) , Baboon endogenous retrovirus (BEV) , porcine endogenous virus (PERV) , the cat derived retrovirus RD114, squirrel monkey retrovirus,
  • MMV murine leukemia virus
  • MoMLV Moloney murine leukemia virus
  • FeLV Feline leukemia virus
  • BEV Baboon endogenous retrovirus
  • PERV porcine endogenous virus
  • the cat derived retrovirus RD114 squirrel monkey retrovirus
  • Xenotropic murine leukemia virus-related virus XMRV
  • REV avian reticuloendotheliosis virus
  • GALV Gibbon ape leukemia virus
  • the retroviral envelope is an amphotropic MLV envelope.
  • the retrovirus is a
  • the target cell is a cell having a cell proliferative disorder.
  • target cell is a neoplastic cell.
  • the cell proliferative disorder is selected from the group consisting of lung cancer, colon-rectum cancer, breast cancer, prostate cancer, urinary tract cancer, uterine cancer, brain cancer, head and neck cancer, pancreatic cancer, melanoma, stomach cancer and ovarian cancer, rheumatoid arthritis or other autoimmune disease.
  • the promoter sequence is associated with a growth regulatory gene.
  • the promoter sequence comprises a tissue-specific promoter sequence.
  • the tissue-specific promoter sequence comprises at least one androgen response element (ARE) .
  • the promoter comprises a CMV promoter having a sequence as set forth in SEQ ID NO: 19, 20, 22 or 42 from nucleotide 1 to about nucleotide 582 and may include modification to one or more nucleic acid bases and which is capable of directing and initiating transcription
  • the promoter comprises a CMV-R-U5 domain polynucleotide.
  • the CMV-R-U5 domain comprises the immediately early promoter from human cytomegalovirus linked to an MLV R-U5 region.
  • the CMV-R-U5 domain polynucleotide comprises a sequence as set forth in SEQ ID NO: 19, 20, 22 or 42 from about nucleotide 1 to about nucleotide 1202 or sequences that are at least 95% identical to a sequence as set forth in SEQ ID NO: 19, 20, 22 or 42, wherein the polynucleotide promotes transcription of a nucleic acid molecule operably linked thereto.
  • the gag polynucleotide is derived from a
  • the gag nucleic acid domain comprises a sequence from about nucleotide number 1203 to about nucleotide 2819 of SEQ ID NO: 19, 20, 22 or 42 or a sequence having at least 95%, 98%, 99% or 99.8% identity thereto.
  • the pol domain of the polynucleotide is derived from a gammaretrovirus. In another embodiment of any of the foregoing, the pol domain
  • the env domain comprises a sequence from about nucleotide number 6359 to about nucleotide 8323 of SEQ ID NO: 19, 20, 22 or 42 or a sequence having at least 95%, 98%, 99% or 99.8% identity thereto.
  • the IRES consists of the sequence set forth in SEQ ID NO: 41.
  • the retroviral polynucleotide sequence comprises (i) the sequence set forth in SEQ ID NO: 42 or (ii) the sequence as set forth in SEQ ID NO: 42, wherein T is U. In another embodiment of any of the
  • the heterologous nucleic acid comprises a polynucleotide having a sequence as set forth in SEQ ID NO: 3, 5, 11, 13, 15 or 17.
  • the heterologous nucleic acid encodes a polypeptide comprising a sequence as set forth in SEQ ID NO : 4.
  • the heterologous nucleic acid is human codon optimized and encodes a polypeptide as set forth in SEQ ID NO: 4.
  • the heterologous nucleic acid comprises a sequence as set forth in SEQ ID NO: 19 or 22 from about nucleotide number 8877 to about 9353.
  • the 3' LTR is derived from a gammaretrovirus .
  • the 3' LTR comprises a U3-R-U5 domain.
  • the 3' LTR comprises a sequence as set forth in SEQ ID NO: 19 or 22 from about nucleotide 9405 to about 9998 or a sequence that is at least 95%, 98% or 99.5% identical thereto.
  • the heterologous nucleic acid sequence encodes a biological response modifier or an immunopotentiating cytokine.
  • the immunopotentiating cytokine is selected from the group consisting of interleukins 1 through 15, interferon, tumor necrosis factor (TNF) , and granulocyte-macrophage-colony stimulating factor (GM- CSF) .
  • TNF tumor necrosis factor
  • GM- CSF granulocyte-macrophage-colony stimulating factor
  • the heterologous nucleic acid encodes a polypeptide that converts a nontoxic prodrug in to a toxic drug.
  • the polypeptide that converts a nontoxic prodrug in to a toxic drug is thymidine kinase, purine nucleoside phosphorylase (PNP) , or cytosine deaminase.
  • the heterologous nucleic acid sequence encodes a receptor domain, an antibody, or antibody fragment.
  • the heterologous nucleic acid sequence comprises an inhibitory polynucleotide.
  • the inhibitory polynucleotide comprises an miRNA, RNAi or siRNA sequence.
  • the disclosure also provides a recombinant retroviral polynucleotide genome for producing a replication competent retrovirus as described above.
  • the disclosure also provides a method of treating a cell proliferative disorder comprising contacting the subject with a recombinant replication competent retrovirus of the disclosure under conditions such that the cytosine deaminase polynucleotide is expressed and contacting the subject with 5-fluorocytosine .
  • the cell proliferative disorder is glioblastoma multiforme.
  • the cell proliferative disorder is selected from the group consisting of lung cancer, colon-rectum cancer, breast cancer, prostate cancer, urinary tract cancer, uterine cancer, brain cancer, head and neck cancer, pancreatic cancer, melanoma, stomach cancer and ovarian cancer .
  • the disclosure also provides a vector that expresses a heterologous gene in a mammalian cell from an ECMV IRES with 6As in the A bulge in the J-K bifurcation region.
  • the vector is a viral vector.
  • the vector is a retroviral replicating vector.
  • the vector is a retroviral replicating vector derived from a gamma-retrovirus .
  • the gamma-retrovirus is derived from one of Murine Leukemia Virus, Baboon Endogenous Virus, Gibbon Ape Leukemia virus, Feline leukemia virus.
  • the heterologous gene is a gene with a therapeutic activity in mammals
  • the therapeutic activity is an anticancer activity.
  • the heterologous gene is a prodrug activating gene.
  • the vector can express a heterologous gene in a mammalian cell from an ECMV IRES in the absence of the protein PTB-1.
  • the disclosure also provides a method of treating cancer, by administering a vector as described above.
  • the disclosure also provides a recombinant replication competent retrovirus comprising: a retroviral GAG protein; a retroviral POL protein; a retroviral envelope; a retroviral polynucleotide comprising Long-Terminal Repeat (LTR) sequences at the 3' end of the retroviral polynucleotide sequence, a promoter sequence at the 5' end of the retroviral polynucleotide, said promoter being suitable for expression in a mammalian cell, a gag nucleic acid domain, a pol nucleic acid domain and an env nucleic acid domain; a cassette comprising (i) a minimal internal ribosome entry site (IRES), wherein the minimal IRES is operably linked to a heterologous polynucleotide, (ii) a polIII promoter linked to an miRNA or (iii) a mini-promoter operably linked to a heterologous polynucleotide that is proceeds
  • the minimal IRES consists of a sequence from about base 123 to 544 of SEQ ID NO: 41. In another embodiment of any of the foregoing, the minimum IRES consists of a sequence from about base 183 to 544 of SEQ ID NO: 41. In another embodiment of any of the foregoing, the IRES has 6As in the A bulge. In another embodiment of any of the foregoing, the virus infects a target cell multiple times resulting in an average number of copies/diploid genome of 5 or greater.
  • the retroviral polynucleotide sequence is derived from a virus selected from the group consisting of murine leukemia virus (MLV) , Moloney murine leukemia virus (MoMLV) , Feline leukemia virus (FeLV) , Baboon endogenous retrovirus (BEV) , porcine endogenous virus (PERV) , the cat derived retrovirus RD114, squirrel monkey retrovirus, Xenotropic murine leukemia virus-related virus (XMRV) , avian reticuloendotheliosis virus (REV) , or Gibbon ape leukemia virus (GALV) .
  • MMV murine leukemia virus
  • MoMLV Moloney murine leukemia virus
  • FeLV Feline leukemia virus
  • BEV Baboon endogenous retrovirus
  • PERV porcine endogenous virus
  • the cat derived retrovirus RD114 squirrel monkey retrovirus
  • XMRV Xenotropic murine leuk
  • the retroviral envelope is an amphotropic MLV envelope.
  • the retrovirus is a gammaretrovirus .
  • the target cell is a cell having a cell
  • the target cell is a neoplastic cell.
  • the cell proliferative disorder is selected from the group consisting of lung cancer, colon-rectum cancer, breast cancer, prostate cancer, urinary tract cancer, uterine cancer, brain cancer, head and neck cancer, pancreatic cancer, melanoma, stomach cancer and ovarian cancer, rheumatoid arthritis or other autoimmune disease.
  • the promoter sequence is associated with a growth regulatory gene.
  • the promoter sequence comprises a tissue-specific promoter sequence.
  • the tissue-specific promoter sequence comprises at least one androgen response element (ARE) .
  • the promoter comprises a CMV promoter having a sequence as set forth in SEQ ID NO: 19, 20, 22 or 42 from nucleotide 1 to about nucleotide 582 and may include modification to one or more nucleic acid bases and which is capable of directing and initiating transcription.
  • the promoter comprises a CMV-R-U5 domain polynucleotide.
  • the CMV-R-U5 domain comprises the immediately early promoter from human cytomegalovirus linked to an MLV R-U5 region.
  • the CMV-R-U5 domain polynucleotide comprises a sequence as set forth in SEQ ID NO: 19, 20, 22 or 42 from about nucleotide 1 to about nucleotide 1202 or sequences that are at least 95% identical to a sequence as set forth in SEQ ID NO: 19, 20, 22 or 42, wherein the polynucleotide promotes transcription of a nucleic acid molecule operably linked thereto.
  • the gag polynucleotide is derived from a gammaretrovirus .
  • the gag nucleic acid domain comprises a sequence from about nucleotide number 1203 to about nucleotide 2819 of SEQ ID NO: 19, 20, 22 or 42 or a sequence having at least 95%, 98%, 99% or 99.8% identity thereto.
  • the pol domain of the gag nucleic acid domain comprises a sequence from about nucleotide number 1203 to about nucleotide 2819 of SEQ ID NO: 19, 20, 22 or 42 or a sequence having at least 95%, 98%, 99% or 99.8% identity thereto.
  • the pol domain of the pol domain of the gag nucleic acid domain comprises a sequence from about nucleotide number 1203 to about nucleotide 2819 of SEQ ID NO: 19, 20, 22 or 42 or a sequence having at least 95%, 98%, 99% or 99.8% identity thereto.
  • polynucleotide is derived from a gammaretrovirus.
  • the pol domain comprises a sequence from about nucleotide number 2820 to about nucleotide 6358 of SEQ ID NO: 19, 20, 22 or 42 or a sequence having at least 95%, 98%, 99% or 99.9% identity thereto.
  • the env domain comprises a sequence from about nucleotide number 6359 to about nucleotide 8323 of SEQ ID NO: 19, 20, 22 or 42 or a sequence having at least 95%, 98%, 99% or 99.8% identity thereto.
  • the heterologous nucleic acid comprises a polynucleotide having a sequence as set forth in SEQ ID NO:3, 5, 11, 13, 15 or 17.
  • the heterologous nucleic acid encodes a polypeptide comprising a sequence as set forth in SEQ ID NO : 4.
  • the heterologous nucleic acid comprises a polypeptide having a sequence as set forth in SEQ ID NO : 4.
  • the heterologous nucleic acid is human codon optimized and encodes a polypeptide as set forth in SEQ ID NO: 4.
  • the heterologous nucleic acid comprises a sequence as set forth in SEQ ID NO: 19 or 22 from about nucleotide number 8877 to about 9353.
  • the 3' LTR is derived from a gammaretrovirus .
  • the 3' LTR comprises a U3-R-U5 domain.
  • the 3' LTR comprises a sequence as set forth in SEQ ID NO: 19 or 22 from about nucleotide 9405 to about 9998 or a sequence that is at least 95%, 98% or 99.5% identical thereto.
  • the heterologous nucleic acid sequence encodes a biological response modifier or an immunopotentiating cytokine.
  • the immunopotentiating cytokine is selected from the group consisting of interleukins 1 through 15, interferon, tumor necrosis factor (TNF) , and granulocyte-macrophage-colony stimulating factor (GM- CSF) .
  • TNF tumor necrosis factor
  • GM- CSF granulocyte-macrophage-colony stimulating factor
  • the heterologous nucleic acid encodes a polypeptide that converts a nontoxic prodrug in to a toxic drug.
  • the polypeptide that converts a nontoxic prodrug in to a toxic drug is thymidine kinase, purine nucleoside phosphorylase (PNP) , or cytosine deaminase.
  • the heterologous nucleic acid sequence encodes a receptor domain, an antibody, or antibody fragment.
  • the heterologous nucleic acid sequence comprises an inhibitory polynucleotide.
  • the inhibitory polynucleotide comprises an miRNA, RNAi or siRNA sequence.
  • Figure 1A-C shows replicating retroviral vectors containing IRES with various numbers of A' s in the A bulge and their titers.
  • ATG8 (AUG in RNA) and ATG9 are underlined; enlarged and underlined sequence represents the A bulge in the J-K bifurcation region; lower case letters indicate the 5' sequences in the polypyrimidine tract in the 3' IRES;
  • C Viral titer of RRV containing various numbers of As in the A bulge produced by infected HT1080 cells.
  • FIG. 2A-D shows cellular viral derived RNA and protein expression by RRV with various numbers of A' s in the A bulge.
  • A Schematic diagram of cellular viral RNA isoforms. Env2 primers and probe, and yCD2 primers and probe recognize both unspliced and spliced viral RNA in the env and the yCD2 region, respectively, were used to measure the level of cellular viral RNA by qRT-PCR. Filled triangles: env2 primer and probe set; open triangles: yCD2 primer and probe set.
  • B Immunoblot of yCD2 and GAPDH protein.
  • RNA and protein expression levels relative to the yCD2-6A vector were determined by flow cytometry using proper gating to exclude GFP-negative cells. GFP protein expression levels were quantified by using mean fluorescent intensity (D) Proviral vector copy number of infected U87-MG cells (MOI of 0.01) by qPCR.
  • Genomic DNA is isolated day 14 post infection at which the vector with 7A is expected to be maximally infected.
  • the data show that there is no significant difference in vector copy of number of maximally infected U87-MG cells. This is consistent with viral production data in which no significant effect on viral titer is observed among the variants.
  • Figure 3 shows a vector sequence (SEQ ID NO: 22) with an
  • Figure 4A-B shows vector stability data.
  • A Vectors stability in infected U87-MG cells (MOI of 0.01) by end-point PCR. Genomic DNA is isolated day 14 post infection and the IRES-yCD2 region is amplified using the primer set spanning the 3' of the env and 3'UTR region (Perez et al . , 2012) .
  • B Assessment of vector stability by serial infection. Approximately 10 5 naive U87-MG cells were initially infected with the viral vectors at a MOI of 0.1 and grown for 1 week to complete a single cycle of infection.
  • IRES-yCD2 region 100 of the 2 ml of viral supernatant from fully infected cells is used to infect naive cells and repeated up to 12 cycles.
  • Vector stability of the IRES-yCD2 region is assessed by PCR amplification of the integrated provirus from the infected cells.
  • the expected PCR product size is approximately 1.2 kb .
  • the appearance of any bands smaller than 1.2 kb indicate deletion in the IRES-yCD2 region.
  • Figure 5 shows a diagram of a construct of the disclosure designed with minimal IRESs (SEQ ID NO: 41 from base 123-139; and 183 to 198) .
  • PCR PCR
  • LCR ligase chain reaction
  • NASBA RNA polymerase mediated techniques
  • the disclosure provides methods and compositions useful for gene or protein delivery to a cell or subject. Such methods and compositions can be used to treat various diseases and disorders in a subject including cancer and other cell
  • IRESs Such optimized IRESs can be used in various vectors to facilitate protein expression.
  • the disclosure provides replication competent retroviral vectors for gene delivery. The disclosure demonstrates that commonly used IRESs containing 7A' s in the A-bulge in the J-K bifurcation region are not optimal and thus the disclosure provides an IRES with an optimal A bulge sequence having improved
  • polypeptide expression compared to IRESs with fewer (3-5) or more
  • An internal ribosome entry sites refers to a segment of nucleic acid that promotes the entry or retention of a ribosome during translation of a coding sequence usually 3' to the IRES.
  • the IRES may comprise a splice
  • IRESs lack a splice acceptor/donor site.
  • the entry of ribosomes into messenger RNA takes place via the cap located at the 5' end of all eukaryotic mRNAs .
  • the absence of a cap in some viral mRNAs suggests the existence of alternative structures permitting the entry of ribosomes at an internal site of these RNAs .
  • IRES on account of their function, have been identified in the 5' noncoding region of uncapped viral mRNAs, including, for example, that of picornaviruses such as poliomyelitis virus (Pelletier et al .
  • the disclosure provides the use of an optimized IRES in the context of a vector and more particularly a replication-competent retroviral (RCR) vector.
  • RCR replication-competent retroviral
  • the IRES from encephalomyocarditis virus (EMCV) has been studied extensively and is widely used in retroviral and other mammalian expression vectors.
  • the proper folding and secondary structure of the IRES dictate its functionality, and sequence changes may or may not affect this. Palmenberg and coworkers showed that, independent of the 5' -IRES region, the J-K elements in the 3' end of the IRES play a critical role in translation initiation, ( Figure 1A) .
  • the sequence of the IRES in various vectors can be found to contain various numbers of polyAs in the A-bulge. For example, Logg et al .
  • the number of A' s in the A-bulge affects the expression of an operably associated heterologous sequence.
  • the disclosure identifies an optimal number of A' s in the A-bulge as peaking at 6 A' s and expression decreasing slightly the further from the optimal number of A' s on either sides. For example, 4 A' s is less effective than 6 A' s and 8 A' s is less effective than 6 A' s .
  • an “optimized IRES” refers to an IRES derived from an encephalomyocarditis virus having 6As in the A- bulge of the J-K bifurcation region.
  • the optimized IRES can be part of a cassette that comprises a gene or sequence to be
  • heterologous polynucleotide or "gene”
  • the optimized IRES is operably linked and upstream of the heterologous polynucleotide sequence and is operably to cause translation of the linked heterologous polynucleotide.
  • the optimized IRES cassette demonstrates increased protein expression from a linked heterologous polynucleotide compared to a non- optimized IRES (e.g., and IRES having 3-5 or 7-8 A' s in the A- bulge) .
  • An optimized IRES or IRES-cassette can be cloned into any number of vectors for expression of a linked heterologous
  • vectors that can contain and be used with an optimized IRES or IRES-cassette of the disclosure include plasmids, expression vectors, viral vectors (replication defective and replication competent) and the like.
  • the disclosure provides an optimized
  • IRES comprising a sequence selected from the group consisting of:
  • heterologous nucleic acid sequence is operably linked to an optimized IRES consisting of, in one embodiment, 6 "As" in the A-bulge region.
  • heterologous nucleic acid sequence or transgene refers to (i) a sequence that does not normally exist in a wild-type retrovirus, (ii) a sequence that originates from a foreign species, (iii) a sequence that is not normally found downstream of an IRES, or (iv) if from the same species, it may be substantially modified from its original form. Alternatively, an unchanged nucleic acid sequence that is not normally expressed in a cell is a heterologous nucleic acid sequence .
  • the disclosure provides a vector comprising an optimized IRES in a cassette comprising an A-bulge in the J-K bifurcation region consisting of 6As operably linked to a polynucleotide sequence to be expressed.
  • an A-bulge consisting of 6A' s unexpectedly provides superior protein expression compared to similar IRES cassettes containing 3-5 or 7-8 A' s .
  • protein expression from a recombinant vector is important not only for in vitro protein production but also for therapeutic protein production in vivo.
  • Logg et al . J. Virol. 75:6989-6998, 2001
  • the optimized IRES cassette can be cloned into any number of art recognized vectors. Such vectors are described below, but include plasmids and viral vectors.
  • the disclosure contemplates an optimized IRES of the disclosure cloned into an expression vector wherein the optimized IRES is located just upstream (e.g., 0 to about 50 bp upstream) of a heterologous polynucleotide to be expressed.
  • the optimized IRES is located just upstream (e.g., 0 to about 50 bp upstream) of a heterologous polynucleotide to be expressed.
  • replication competent gamma retroviral vectors that are capable of infecting and spreading in mammalian tissue without the need for recombinant receptors or helper cells.
  • RCR vectors include gamma retroviruses such as mo-MLV, MLV, GALV, FELV and the like.
  • a typical gamma retrovirus comprises LTRs, gag, pol and env gene, and factors necessary for reverse transcription and integration into a host genome (e.g., psi factors) .
  • Modifications of the typical gamma retroviral vector have been performed for nearly 20 years including generating replication incompetent vectors, vectors carrying heterologous genes in various locations and vectors containing IRES cassettes. For example, Kasahara et al . describes the generation of a replication competent retroviral vector derived from MLV in U.S. Patent No.
  • the disclosure provides, in one embodiment, a replication competent gammaretroviral vector (RCR) comprising an optimized IRES cassette just downstream of the env gene and upstream of the 3' LTR, wherein the optimized IRES of the optimized IRES cassette consists of an A-bulge in the bifurcation region of 6As .
  • the RCR has increased protein expression compared to a vector containing an A-bulge having 3-5 or 7-8A's.
  • the disclosure provides vectors having an A-bulge in the J- K bifurcation region consisting of 6A' s compared to that found in prior replication competent retroviral vectors (e.g., see U.S.
  • 7A' s to 6A's provides increased protein production compared to that of 7A' s .
  • a vector comprising 6A' s would have improved protein expression of a heterologous gene linked to an IRES cassette having a "6A" A-bulge.
  • vector means the vehicle by which a DNA or RNA sequence (e.g., a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g., transcription and translation) of the introduced sequence.
  • Vectors typically comprise the DNA or RNA of a transmissible agent, into which foreign DNA or RNA encoding a protein is inserted by restriction enzyme
  • a common type of vector is a "plasmid", which generally is a self-contained molecule of double-stranded DNA that can readily accept additional (foreign) DNA and which can readily introduced into a suitable host cell.
  • plasmid a vector that can readily accept additional (foreign) DNA and which can readily introduced into a suitable host cell.
  • vectors including plasmid and fungal vectors, have been described for replication and/or expression in a variety of eukaryotic and prokaryotic hosts.
  • Non-limiting examples include pKK plasmids
  • Recombinant cloning vectors will often include one or more replication systems for cloning or expression, one or more markers for selection in the host, e.g., antibiotic resistance, and one or more expression cassettes .
  • express and expression mean allowing or causing the information in a gene or DNA sequence to become manifest, for example producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene, RNA or DNA sequence.
  • a DNA or RNA sequence is expressed in or by a cell to form an "expression product” such as a protein.
  • the expression product itself e.g. the resulting protein, may also be said to be “expressed” by the cell.
  • a polynucleotide or polypeptide is expressed recombinantly, for example, when it is expressed or produced in a foreign host cell under the control of a foreign or native promoter, or wherein a native gene in a native host cell is expressed under the control of a foreign promoter.
  • the disclosure provides modified retroviral vectors.
  • the modified retroviral vectors can be derived from members of the retroviridae family.
  • the Retroviridae family consists of three groups: the spumaviruses- (or foamy viruses) such as the human foamy virus (HFV) ; the lentiviruses, as well as visna virus of sheep; and the oncoviruses (although not all viruses within this group are oncogenic) .
  • the term "lentivirus” is used in its conventional sense to describe a genus of viruses containing reverse transcriptase.
  • the lentiviruses include the "immunodeficiency viruses” which include human immunodeficiency virus (HIV) type 1 and type 2 (HIV-1 and HIV-2) and simian immunodeficiency virus (SIV) .
  • the oncoviruses have historically been further subdivided into groups A, B, C and D on the basis of particle morphology, as seen under the electron microscope during viral maturation.
  • A-type particles represent the immature particles of the B- and D-type viruses seen in the cytoplasm of infected cells. These particles are not infectious.
  • B- type particles bud as mature virion from the plasma membrane by the enveloping of intracytoplasmic A-type particles.
  • B-type particles At the membrane they possess a toroidal core of 75 nm, from which long glycoprotein spikes project. After budding, B-type particles contain an eccentrically located, electron-dense core.
  • the prototype B-type virus is mouse mammary tumor virus (MMTV) . No intracytoplasmic particles can be observed in cells infected by C-type viruses.
  • the C-type viruses are the most commonly studied and include many of the avian and murine leukemia viruses (MLV) .
  • Bovine leukemia virus (BLV) and the human T-cell leukemia virus types I and II (HTLV-I/II) are similarly classified as C-type particles because of the morphology of their budding from the cell surface. However, they also have a regular hexagonal morphology and more complex genome structures than the prototypic C-type viruses such as the murine leukemia viruses (MLV) .
  • D-type particles resemble B-type particles in that they show as ring-like structures in the infected cell cytoplasm, which bud from the cell surface, but the virion incorporate short surface glycoprotein spikes. The electron-dense cores are also eccentrically located within the particles.
  • Mason Pfizer monkey virus (MPMV) is the prototype D-type virus .
  • Retroviruses have been classified in various ways but the nomenclature has been standardized in the last decade (see ICTVdB - The Universal Virus Database, v 4 on the World Wide Web
  • retroviral vector can comprise an Orthoretrovirus or more typically a gamma retrovirus vector.
  • Retroviruses are defined by the way in which they replicate their genetic material. During replication the RNA is converted into DNA. Following infection of the cell a double- stranded molecule of DNA is generated from the two molecules of RNA which are carried in the viral particle by the molecular process known as reverse transcription. The DNA form becomes covalently integrated in the host cell genome as a provirus, from which viral RNAs are expressed with the aid of cellular and/or viral factors. The expressed viral RNAs are packaged into particles and released as infectious virion. [ 0038 ] The retrovirus particle is composed of two identical RNA molecules. Each wild-type genome has a positive sense, single- stranded RNA molecule, which is capped at the 5' end and
  • the diploid virus particle contains the two RNA strands complexed with gag proteins, viral enzymes (pol gene products) and host tRNA molecules within a 'core' structure of gag proteins.
  • gag proteins viral enzymes (pol gene products)
  • host tRNA molecules within a 'core' structure of gag proteins.
  • Surrounding and protecting this capsid is a lipid bilayer, derived from host cell membranes and containing viral envelope (env) proteins.
  • the env proteins bind to a cellular receptor for the virus and the particle typically enters the host cell via receptor-mediated endocytosis and/or membrane fusion.
  • the viral RNA is copied into DNA by reverse transcription. This is catalyzed by the reverse transcriptase enzyme encoded by the pol region and uses the host cell tRNA packaged into the virion as a primer for DNA synthesis. In this way the RNA genome is converted into the more complex DNA genome .
  • the double-stranded linear DNA produced by reverse transcription may, or may not, have to be circularized in the nucleus.
  • the provirus now has two identical repeats at either end, known as the long terminal repeats (LTR) .
  • LTR long terminal repeats
  • the termini of the two LTR sequences produces the site recognized by a pol product--the integrase protein--which catalyzes integration, such that the provirus is always joined to host DNA two base pairs (bp) from the ends of the LTRs .
  • bp base pairs
  • a duplication of cellular sequences is seen at the ends of both LTRs, reminiscent of the integration pattern of transposable genetic elements.
  • Retroviruses can integrate their DNAs at many sites in host DNA, but different retroviruses have different integration site preferences. HIV-1 and simian
  • immunodeficiency virus DNAs preferentially integrate into expressed genes
  • murine leukemia virus (MLV) DNA preferentially integrates near transcriptional start sites (TSSs)
  • TSSs near transcriptional start sites
  • ASLV avian sarcoma leukosis virus
  • HTLV human T cell leukemia virus
  • Retroviruses J Virol 81:6731-6741; Lewinski MK, et al . (2006) Retroviral DNA integration: viral and cellular determinants of target-site selection. PLoS Pathog 2:e601).
  • RNA splicing and translation of the integrated viral DNA is mediated by host cell proteins. Variously spliced transcripts are generated. In the case of the human retroviruses HIV-1/2 and HTLV-I/II viral proteins are also used to regulate gene expression. The interplay between cellular and viral factors is a factor in the control of virus latency and the temporal sequence in which viral genes are expressed.
  • Retroviruses can be transmitted horizontally and vertically. Efficient infectious transmission of retroviruses requires the expression on the target cell of receptors which specifically recognize the viral envelope proteins, although viruses may use receptor-independent, nonspecific routes of entry at low efficiency. Normally a viral infection leads to a single or few copies of viral genome per cell because of receptor masking or down-regulation that in turn leads to resistance to superinfection (Ch3 pl04 in "Retroviruses", JM Coffin, SH Hughes, & HE Varmus 1997 Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY; Fan et al . J.Virol 28:802, 1978) . In addition, the target cell type must be able to support all stages of the replication cycle after virus has bound and penetrated.
  • provirus Vertical transmission occurs when the viral genome becomes integrated in the germ line of the host.
  • the provirus will then be passed from generation to generation as though it were a cellular gene.
  • endogenous proviruses become established which frequently lie latent, but which can become activated when the host is exposed to appropriate agents.
  • the disclosure provides recombinant replication competent retroviruses capable of infecting a target cell or target cell population multiple times resulting in an average number of copies/diploid genome of 5 or greater. The disclosure also provides methods of testing for this property.
  • the integrated DNA intermediate is referred to as a provirus.
  • Prior gene therapy or gene delivery systems use methods and retroviruses that require transcription of the provirus and assembly into infectious virus while in the presence of an appropriate helper virus or in a cell line
  • helper virus is not required for the production of the recombinant retrovirus of the disclosure, since the sequences for encapsidation are provided in the genome thus providing a replication competent retroviral vector for gene delivery or therapy.
  • the retroviral genome and the proviral DNA of the disclosure have at least three genes: the gag, the pol, and the env, these genes may be flanked by one or two long terminal (LTR) repeat, or in the provirus are flanked by two long terminal repeat (LTR) and sequences containing cis-acting sequences such as psi.
  • the gag gene encodes the internal structural (matrix, capsid, and nucleocapsid) proteins;
  • the pol gene encodes the RNA-directed DNA polymerase (reverse transcriptase), protease and integrase; and the env gene encodes viral envelope glycoproteins.
  • the 5' and/or 3' LTRs serve to promote transcription and polyadenylation of the virion RNAs .
  • the LTR contains all other cis-acting sequences necessary for viral replication.
  • Lentiviruses have additional genes including vif, vpr, tat, rev, vpu, nef, and vpx (in HIV-1, HIV-2 and/or SIV) .
  • Adjacent to the 5' LTR are sequences necessary for reverse transcription of the genome (the tRNA primer binding site) and for efficient encapsidation of viral RNA into particles (the Psi site) . If the sequences necessary for encapsidation (or packaging of retroviral RNA into infectious virion) are missing from the viral genome, the result is a cis defect which prevents encapsidation of genomic viral RNA.
  • This type of modified vector is what has typically been used in prior gene delivery systems (i.e., systems lacking elements which are required for encapsidation of the virion) as 'helper' elements providing viral proteins in trans that package a non-replicating, but packageable, RNA genome.
  • the disclosure provides vectors that contain an optimized IRES.
  • the optimized IRES is typically linked to a heterologous polynucleotide encoding, for example, a cytosine deaminase or mutant thereof, a thymidine kinase or mutant thereof, an miRNA or siRNA, a cytokine, an antibody binding domain etc., that can be delivered to a cell or subject.
  • the vector is a viral vector.
  • the viral vector can be an adenoviral vector, a measles vector, a herpes vector, a retroviral vector (including a lentiviral vector) , a rhabdoviral vector such as a Vesicular Stomatitis viral vector, a reovirus vector, a Seneca Valley Virus vector, a poxvirus vector (including animal pox or vaccinia derived vectors) , a parvovirus vector (including an AAV vector) , an alphavirus vector or other viral vector known to one skilled in the art (see also, e.g., Concepts in Genetic Medicine, ed.
  • the retroviral genome of the disclosure contains an optimized IRES comprising a cloning site downstream of the optimized IRES for insertion of a
  • the optimized IRES is located 3' to the env gene in a retroviral vector, but 5' to the desired heterologous polynucleotide and 5' to the 3' LTR.
  • the optimized IRES comprises an A-bulge with 6A' s .
  • a heterologous polynucleotide encoding a desired polypeptide may be operably linked to the optimized IRES.
  • the viral vector can be a replication competent retroviral vector obtained or derived from a
  • the replication competent retroviral vector comprises an optimized internal ribosomal entry site (IRES) comprising an A-bulge consisting of 6 A' s located 5' to a heterologous polynucleotide encoding, e.g., a cytosine deaminase (SEQ ID NO: 3), thymidine kinase (SEQ ID NO: 37), miRNA, siRNA, cytokine, receptor, antibody or the like.
  • a heterologous polynucleotide encodes a non- translated RNA such as siRNA, miRNA or RNAi then an IRES is not necessary, but may be included for another translated
  • an optimized IRES cassette containing the heterologous polynucleotide is 3' to a ENV
  • the viral vector is a retroviral vector capable of infecting target cells multiple times (e.g., 5 or more per diploid cell) .
  • the disclosure provides replication competent retroviral vectors having increased stability relative to prior retroviral vectors and containing an optimized IRES having 6A' s in the A- bulge. Such increased stability during infection and replication is important for the treatment of cell proliferative disorders.
  • the increased protein expression from the optimized A- bulge provides additional delivery of therapeutic proteins to a target cell/tissue.
  • the combination of transduction efficiency, transgene stability, transgene expression and target selectivity is provided by the replication competent retrovirus.
  • the compositions and methods provide insert stability and maintain transcription activity of the transgene and the translational viability of the encoded polypeptide .
  • any number of heterologous polynucleotide or nucleic acid sequences may be inserted into the vector or retroviral vector.
  • marker genes or reporter genes may be used,
  • polypeptide sequences including, antibiotic resistance and fluorescent molecules (e.g., GFP) .
  • Additional polynucleotide sequences encoding any desired polypeptide sequence may also be inserted into the vector of the disclosure. Where in vivo delivery of a heterologous nucleic acid sequence is sought both therapeutic and non-therapeutic sequences may be used.
  • the heterologous sequence can encode a therapeutic molecule including antisense molecules (miRNA, siRNA) or ribozymes directed to a particular gene associated with a cell proliferative disorder or other gene-associated disease or disorder
  • the heterologous sequence can be a suicide gene (e.g., HSV-tk or PNP or cytosine deaminase; either modified or unmodified, humanized or non-humanized) , a growth factor or a therapeutic protein (e.g., Factor IX, IL2, and the like) .
  • suicide gene e.g., HSV-tk or PNP or cytosine deaminase; either modified or unmodified, humanized or non-humanized
  • a growth factor or a therapeutic protein e.g., Factor IX, IL2, and the like
  • Other therapeutic proteins applicable to the disclosure are easily identified in the art .
  • the heterologous polynucleotide within the vector comprises a cytosine deaminase that has been optimized for expression in a human cell.
  • the cytosine deaminase comprises a sequence that has been human codon optimized and comprises mutations that increase the cytosine deaminase's stability (e.g., reduced degradation or increased thermo-stability) compared to a wild-type cytosine deaminase (see, e.g., SEQ ID NO:4) .
  • the heterologous polynucleotide encodes a fusion construct comprising a cytosine deaminase (either human codon optimized or non-optimized, either mutated or non-mutated) operably linked to a polynucleotide encoding a polypeptide having UPRT or OPRT activity (see, e.g., SEQ ID NO: 11, 13, 15 and 17) .
  • cytosine deaminase either human codon optimized or non-optimized, either mutated or non-mutated
  • polypeptide having UPRT or OPRT activity see, e.g., SEQ ID NO: 11, 13, 15 and 17
  • Examples of such polypeptides having cytosine deaminase and polynucleotides encoding such polypeptides can be found in International Publication No. WO 2010/045002, which is incorporated herein by reference.
  • a vector or replication competent retroviral vector can comprise a heterologous polynucleotide encoding a polypeptide comprising a cytosine deaminase (as described herein) and may further comprise a polynucleotide comprising a miRNA or siRNA molecule either as part of the primary transcript from the viral promoter or linked to a promoter, which can be cell-type or tissue specific.
  • heterologous amino acids [ 0055] In yet further embodiments, the heterologous amino acids
  • polynucleotide may comprise a cytokine such as an interleukin, interferon gamma or the like.
  • Cytokines that may expressed from a retroviral vector of the disclosure include, but are not limited to, IL-lalpha, IL-lbeta, IL-2 (SEQ ID NO:40), 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, IL- 16, IL-17, IL-18, IL-19, IL-20, and IL-21, anti-CD40, CD40L, IFN- gamma (human - SEQ ID NO: 38; mouse - SEQ ID NO: 39) and TNF-alpha, soluble forms of TNF-alpha, lymphotoxin-alpha (LT-alpha, also known as TNF-beta) , LT-beta (found in complex
  • Angiogenic proteins may be useful in some embodiments, particularly for protein production from cell lines.
  • angiogenic factors include, but are not limited to, Glioma Derived Growth Factor (GDGF) , Platelet Derived Growth
  • PDGF-A Platelet Derived Growth Factor-B
  • PIGF Placental Growth Factor
  • PIGF-2 Placental Growth Factor-2
  • VEGF Vascular Endothelial Growth Factor
  • VEGF-A Vascular Endothelial Growth Factor-A
  • VEGF-A Vascular Endothelial Growth Factor-2
  • VEGF-2 Vascular Endothelial Growth Factor B
  • VEGF-3 Vascular Endothelial Growth Factor B
  • VEGF-l 86 Vascular Endothelial Growth Factor B-l 86
  • VEGF-D Vascular Endothelial Growth Factor-D
  • VEGF-D Vascular Endothelial Growth Factor-E
  • VEGF-E Vascular Endothelial Growth Factor-E
  • Fibroblast Growth Factors may be delivered by a vector of the disclosure and include, but are not limited to, FGF-1, FGF-2, FGF- 3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF- 12, FGF-13, FGF-14, and FGF-15.
  • Hematopoietic growth factors may be delivered using vectors of the disclosure, such growth factors include, but are not limited to, granulocyte macrophage colony stimulating factor (GM-CSF) (sargramostim) , granulocyte colony stimulating factor (G-CSF) (filgrastim), macrophage colony stimulating factor (M-CSF, CSF-1) erythropoietin (epoetin alfa) , stem cell factor (SCF, c-kit ligand, steel factor) , megakaryocyte colony stimulating factor, PIXY321 (a GMCSF/IL-3) fusion protein and the like.
  • GM-CSF granulocyte macrophage colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • M-CSF macrophage colony stimulating factor
  • SCF stem cell factor
  • c-kit ligand steel factor
  • megakaryocyte colony stimulating factor PIXY321 (a GMCSF/IL-3)
  • MicroRNAs are small, non-coding RNAs . They are located within introns of coding or non-coding gene, exons of non- coding genes or in inter-genic regions. miRNA genes are
  • RNA polymerase II that generate precursor
  • pri-miRNA primary precursor miRNA
  • pre-miRNA miRNA precursor
  • Dicer another ribonuclease
  • a mature miRNA is approximately 21 nucleotides in length. It exerts in function by binding to the 3' untranslated region of mRNA of targeted genes and suppressing protein expression either by repression of protein translation or degradation of mRNA. miRNA are involved in biological processes including development, cell proliferation, differentiation and cancer progression.
  • miRNA profiling indicate that some miRNA expressions are tissue specific or enriched in certain tissues.
  • miR- 142-3p, miR-181 and miR-223 expressions have demonstrated to be enriched in hematopoietic tissues in human and mouse (Baskerville et al., 2005 RNA 11, 241-247; Chen et al . , 2004 Science 303, 83- 86) .
  • the target sequence of miR-142-3p is shown in SEQ ID NO: 35.
  • the target of miR-142-3p4X is shown in SEQ ID NO: 36.
  • miR-21 is overexpressed in glioblastoma, breast, lung, prostate, colon, stomach, esophageal, and cervical cancer, uterine leiomyosarcoma, DLBCL, head and neck cancer.
  • let-7 members of let-7 have reported to be down-regulated in glioblastoma, lung, breast, gastric, ovary, prostate and colon cancers. Re-establishment of homeostasis of miRNA expression in cancer is an imperative
  • miRNA replacement might represent an equally efficacious strategy.
  • the most therapeutically useful miRNAs are the ones expressed at low levels in tumors but at high level, and therefore tolerated, in normal tissues.
  • miRNAs that are down-regulated in cancers could be useful as anticancer agents. Examples include mir-128-1/2 (SEQ ID NO:31 and 32 respectively), let-7, miR-26, miR-124, and miR-137
  • miR-128 expression has reported to be enriched in the central nervous system and has been observed to be down-regulated in glioblastomas (Sempere et al . , 2004 Genome Biology 5:R13.5-11; Godlewski et al . , 2008 Cancer Res 68: (22) 9125-9130).
  • miR-128 is encoded by two distinct genes, miR-128-1 and miR-128-2. Both are processed into identical mature sequence.
  • Bmi-l F3a have been reported to be the direct targets of miR-128 (Godlewski et al., 2008 Cancer Res 68: (22) 9125-9130; Zhang et al . , 2009 J. Mol Med 87:43-51) .
  • Bmi-l expression has been observed to be up-regulated in a variety of human cancers, including gliomas, mantle cell lymphomas, non-small cell lung cancer B-cell non- Hodgkin's lymphoma, breast, colorectal and prostate cancer.
  • Bmi-l has been demonstrated to be required for the self-renewal of stem cells from diverse tissues, including neuronal stem cells as well as "stem-like" cell population in gliomas.
  • Replication-defective retroviral and lentiviral vectors have been used to stably express pri-miRNA by a polymerase II promoter such as CMV or LTR and demonstrated production of mature miRNA.
  • a polymerase II promoter such as CMV or LTR
  • type III RNA polymerase III promoters such as the U6 and the HI promoter in non-replicative retroviral and lentiviral vectors has been used widely to express functional small interference RNA (siRNA) producing a short hairpin structured RNA (Brora erg-White et al.
  • the loop sequence is cleaved by Dicer producing the mature siRNAs that are 21-22 nucleotides in length.
  • shRNA can be stably expressed in cells to down-regulate target gene
  • SEQ ID NO: 33 and 34 comprise a pre-miR-128 linked to an HI promoter.
  • an optimized IRES comprising 6A' s in the A-bulge can be used in combination with a core promoter, wherein an optimized IRES is operably linked to a first
  • heterologous coding sequence and the core promoter or minipromoter is linked to a second heterologous coding sequence or an siRNA, miRNA, or shRNA sequence (see, e.g., WO 2014/066700, incorporated herein by reference) .
  • a “core promoter” refers to a minimal promoter comprising about 50-100 bp and lacks enhancer elements.
  • core promoters include, but are not limited to, SCP1, AdML and CMV core promoters. More particularly, where a core-promoter cassette is present a second cassette (e.g., a second mini-promoter cassette, a polIII promoter cassette or IRES cassette) will be present.
  • a vector comprising a cassette with a core promoter specifically excludes the use of SCP1, AdML and CMV core promoters, but rather utilize designer core promoters as described further herein and below.
  • Core promoters include certain viral promoters.
  • Viral promoters are promoters that have a core sequence but also usually some further accessory elements.
  • the early promoter for SV40 contains three types of elements: a TATA box, an initiation site and a GC repeat (Barrera-Saldana et al . , EMBO J, 4:3839-3849, 1985; Yaniv, Virology, 384:369-374, 2009).
  • the TATA box is located approximately 20 base-pairs upstream from the transcriptional start site.
  • the GC repeat regions is a 21 base-pair repeat containing six GC boxes and is the site that determines the direction of transcription.
  • This core promoter sequence is around 100 bp. Adding an additional 72 base-pair repeats, thus making it a “mini-promoter, " is useful as a transcriptional enhancer that increase the functionality of the promoter by a factor of about 10.
  • the SP1 protein interacts with the 21 bp repeats it binds either the first or the last three GC boxes. Binding of the first three initiates early expression, and binding of the last three initiates late expression.
  • the function of the 72 bp repeats is to enhance the amount of stable RNA and increase the rate of
  • a core promoter typically encompasses -40 to +40 relative to the +1 transcription start site (Juven-Gershon and Kadonaga, Dev. Biol. 339:225-229, 2010), which defines the location at which the RNA polymerase II machinery initiates transcription.
  • RNA polymerase II interacts with a number of
  • transcription factors that bind to DNA motifs in the promoter.
  • transcriptions factors include, but are not limited to, TFIIA
  • RNA polymerase I IA transcription factor for RNA polymerase I A
  • TFIIB transcription factor for RNA polymerase I A
  • TFIID transcription factor for RNA polymerase I IA
  • TFIIE transcription factor for RNA polymerase I IA
  • TFIIH transcription factor for RNA polymerase I H
  • the pRC/CMV core promoter consists of a TATA box and is 81 bp in length
  • the CMV core promoter consists of a TATA box and a initiator site
  • the SCP synthetic core promoters (SCP1 and SCP2) consist of a TATA box, an Inr (initiator), an MTE site (Motif Ten Element) , and a DPE site (Downstream promoter element) and is about 81 bp in length.
  • the SCP synthetic promoter has improved expression compared to the simple pRC/CMV core promoter.
  • mini-promoter refers to a regulatory domain that promotes transcription of an operably linked gene or coding nucleic acid sequence.
  • the mini- promoter includes the minimal amount of elements necessary for effective transcription and/or translation of an operably linked coding sequence.
  • a mini-promoter can comprise a "core promoter" in combination with additional
  • the mini-promoter or modified core promoter will be about 100-600 bp in length while a core promoter is typically less than about lOObp (e.g., about 70-80 bp) .
  • the cassette will typically comprise an enhancer element or another element either upstream or downstream of the core promoter sequence that facilitates expression of an operably linked coding sequence above the expression levels of the core promoter alone.
  • modified core promoters derived from cellular elements as determined for "core promoter" elements ( ⁇ 100, ⁇ 200, ⁇ 400 or ⁇ 600 bp) that allow ubiquitous expression at significant levels in target cells and are useful for stable incorporation into vectors, in general, and replicating retroviral vectors, in particular, to allow efficient expression of transgenes.
  • mini- promoters comprising core promoters plus minimal enhancer sequences and/or Kozak sequences to allow better gene expression compared to a core-promoter lacking such sequences that are still under 200, 400 or 600bp.
  • mini-promoters include modified core promoters and naturally occurring tissue specific promoters such as the elastin promoter (specific for pancreatic acinar cells, (204 bp; Hammer et al . , Mol Cell Biol., 7:2956-2967, 1987) and the promoter from the cell cycle dependent ASK gene from mouse and man (63-380 bp; Yamada et al . , J. Biol. Chem., 277: 27668-27681, 2002).
  • tissue specific promoters such as the elastin promoter (specific for pancreatic acinar cells, (204 bp; Hammer et al . , Mol Cell Biol., 7:2956-2967, 1987) and the promoter from the cell cycle dependent ASK gene from mouse and man (63-380 bp; Yamada et al . , J. Biol. Chem., 277: 27668-27681, 2002).
  • Ubiquitously expressed small promoters also include viral promoters such as the SV40 early and late promoters (about 340 bp) , the RSV LTR promoter (about 270 bp) and the HBV X gene promoter (about 180 bp) (e.g., R Anish et al . , PLoS One, 4: 5103, 2009) that has no canonical "TATTAA box” and has a 13 bp core sequence of 5'- CCCCGTTGCCCGG-3' (SEQ ID NO:43).
  • the therapeutic cassette comprising at least one mini-promoter cassette will have expression levels that exceed, are about equal to, or about about 1 fold to 2.5 fold less than the expression levels of an IRES cassette present in an RRV.
  • transcription for example, there is either a single major transcription start site or several start sites within a narrow region of several nucleotides. Focused transcription is the predominant mode of transcription in simpler organisms. In dispersed transcription, there are several weak transcription start sites over a broad region of about 50 to 100 nucleotides. Dispersed transcription is the most common mode of transcription in
  • dispersed transcription is observed in about two-thirds of human genes.
  • focused transcription tends to be associated with regulated promoters, whereas dispersed transcription is typically observed in vertebrates.
  • Table 1 Binding sites that can contribute to a focused core promoter (almost always with a "TATA box and a single transcription start site (TSS) ) , or a dispersed promoter without a TATA box, usually with a DPE element (see R. Dickstein,
  • Table 2 sets forth oligonucleotides that can be used to construct and clone enhancer elements into core promoter regions.
  • the modified/optimized core promoters of the disclosure can include a core sequence with the addition of elements from Table 1 and may further include enhancers cloned as set forth in Table 2. In doing so, the size of the mini-promoter may be increased. However, the final mini-promoter should not exceed 600 bp and will typically be about 100 bp, 200 bp, 300 bp, 400 bp, 500 bp and any integer there between.
  • AP-1 activating protein-1
  • NF- ⁇ nuclear factor ⁇ .
  • the disclosure provides a recombinant replication competent retrovirus capable of infecting a non- dividing host cell, a host dividing cell, or a host cell having a cell proliferative disorder.
  • the recombinant replication competent retrovirus of the disclosure comprises a polynucleotide sequence encoding a viral GAG, a viral POL, a viral ENV, a heterologous polynucleotide preceded by an optimized internal ribosome entry site (IRES) having 6 A' s in the A-bulge of the IRES encapsulated within a virion.
  • IRES optimized internal ribosome entry site
  • the recombinant vector of the disclosure is capable of transferring a nucleic acid sequence into a target cell.
  • the phrase "non-dividing" cell refers to a cell that does not go through mitosis. Non-dividing cells may be blocked at any point in the cell cycle, (e.g., Go /Gi , Gi /S , G2/M) , as long as the cell is not actively dividing.
  • a dividing cell can be treated to block cell division by standard techniques used by those of skill in the art, including, irradiation, aphidocolin treatment, serum starvation, and contact inhibition.
  • a recombinant lentivirus vector is capable of infecting non-dividing cells.
  • pre-existing non-dividing cells in the body include neuronal, muscle, liver, skin, heart, lung, and bone marrow cells, and their derivatives.
  • onco-retroviral vectors can be used.
  • dividing cell is meant a cell that undergoes active mitosis, or meiosis.
  • dividing cells include stem cells, skin cells (e.g., fibroblasts and keratinocytes) , gametes, and other dividing cells known in the art.
  • skin cells e.g., fibroblasts and keratinocytes
  • gametes e.g., gametes
  • dividing cell are cells having cell proliferative disorders, such as neoplastic cells.
  • cell proliferative disorder refers to a condition characterized by an abnormal number of cells. The condition can include both
  • cell proliferative disorders include disorders associated with an overgrowth of connective tissues, such as various fibrotic conditions, including scleroderma, arthritis and liver cirrhosis. Cell proliferative disorders include neoplastic disorders such as head and neck carcinomas.
  • Head and neck carcinomas would include, for example, carcinoma of the mouth, esophagus, throat, larynx, thyroid gland, tongue, lips, salivary glands, nose, paranasal sinuses, nasopharynx, superior nasal vault and sinus tumors, esthesioneuroblastoma, squamous cell cancer, malignant melanoma, sinonasal undifferentiated carcinoma (SNUC) , brain (including glioblastomas) or blood neoplasia. Also included are carcinoma's of the regional lymph nodes including cervical lymph nodes,
  • prelaryngeal lymph nodes prelaryngeal lymph nodes, pulmonary j uxtaesophageal lymph nodes and submandibular lymph nodes (Harrison's Principles of Internal
  • cancer types include, but are not limited to, lung cancer, colon-rectum cancer, breast cancer, prostate cancer, urinary tract cancer, uterine cancer lymphoma, oral cancer, pancreatic cancer, leukemia, melanoma, stomach cancer, skin cancer and ovarian cancer.
  • the cell proliferative disease also includes rheumatoid arthritis (O'Dell NEJM 350:2591 2004) and other auto-immune disorders (Mackay et al NEJM 345:340 2001) that are often characterized by inappropriate proliferation of cells of the immune system.
  • host cells transfected with a replication competent retroviral vector of the disclosure are provided.
  • Host cells include eukaryotic cells such as yeast cells, insect cells, or animal cells.
  • Host cells also include prokaryotic cells such as bacterial cells.
  • the host cells have been modified or selected to be continuously grown in serum free suspension (see, e.g., U.S. Patent Publ . No. 2012/0087894-A1, which is incorporated herein by reference) .
  • engineered host cells that are transduced (transformed or transfected) with a vector provided herein (e.g., a replication competent retroviral vector) .
  • the engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants, or amplifying a coding polynucleotide. Culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to those skilled in the art and in the references cited herein, including, e.g., Sambrook, Ausubel and Berger, as well as e.g., Freshney (1994) Culture of Animal Cells: A Manual of Basic Technique, 3rd ed. (Wiley-Liss, New York) and the references cited therein.
  • mammalian cells such as CHO, COS, BHK, HEK 293 br Bowes melanoma etc.
  • human cells or cell lines will be used; however, it may be desirable to clone vectors and polynucleotides of the disclosure into non-human host cells for purposes of sequencing, amplification and cloning.
  • a targeting polynucleotide sequence is included as part of a recombinant retroviral vector of the disclosure.
  • the targeting polynucleotide sequence is a targeting ligand (e.g., peptide hormones such as heregulin, a single-chain antibody, a receptor or a ligand for a receptor) , a tissue-specific or cell-type specific regulatory element (e.g., a tissue-specific or cell-type specific promoter or enhancer) , or a combination of a targeting ligand and a tissue-specific/cell-type specific regulatory element.
  • a targeting ligand e.g., peptide hormones such as heregulin, a single-chain antibody, a receptor or a ligand for a receptor
  • tissue-specific or cell-type specific regulatory element e.g., a tissue-specific or cell-type specific promoter or enhancer
  • a combination of a targeting ligand and a tissue-specific/cell-type specific regulatory element
  • the targeting ligand is operably linked to the env protein of the retrovirus, creating a chimeric retroviral env protein.
  • the viral GAG, viral POL and viral ENV proteins can be derived from any suitable retrovirus (e.g., MLV or lentivirus-derived) .
  • the viral ENV protein is non-retrovirus-derived (e.g., CMV or VSV) .
  • the retroviral vector is targeted to the cell by binding to cells having a molecule on the external surface of the cell.
  • This method of targeting the retrovirus utilizes expression of a targeting ligand on the coat of the retrovirus to assist in targeting the virus to cells or tissues that have a receptor or binding molecule which interacts with the targeting ligand on the surface of the retrovirus.
  • the virus injects its nucleic acid into the cell and the retrovirus genetic material can integrate into the host cell genome.
  • the disclosure includes in one embodiment, a chimeric env protein comprising a retroviral ENV protein operably linked to a targeting polypeptide.
  • the targeting polypeptide can be a cell specific receptor molecule, a ligand for a cell specific receptor, an antibody or antibody fragment to a cell specific antigenic epitope or any other ligand easily identified in the art which is capable of binding or interacting with a target cell.
  • targeting polypeptides or molecules examples include bivalent antibodies using biotin-streptavidin as linkers (Etienne-Julan et al., J. Of General Virol., 73, 3251-3255 (1992); Roux et al . , Proc . Natl. Acad. Sci USA 86, 9079-9083 (1989)), recombinant virus containing in its envelope a sequence encoding a single-chain antibody variable region against a hapten (Russell et al .
  • LDL lipoprotein
  • SNV spleen necrosis virus
  • the recombinant retrovirus of the disclosure is genetically modified in such a way that the virus is targeted to a particular cell type (e.g., smooth muscle cells, hepatic cells, renal cells, fibroblasts, keratinocytes, mesenchymal stem cells, bone marrow cells, chondrocyte, epithelial cells, intestinal cells, mammary cells, neoplastic cells, glioma cells, neuronal cells and others known in the art) such that the virus is targeted to a particular cell type (e.g., smooth muscle cells, hepatic cells, renal cells, fibroblasts, keratinocytes, mesenchymal stem cells, bone marrow cells, chondrocyte, epithelial cells, intestinal cells, mammary cells, neoplastic cells, glioma cells, neuronal cells and others known in the art) such that the virus is targeted to a particular cell type (e.g., smooth muscle cells, hepatic cells, renal cells,
  • recombinant genome of the retroviral vector is delivered to a target non-dividing, a target dividing cell, or a target cell having a cell proliferative disorder.
  • targeting uses cell- or tissue-specific regulatory elements to promote expression and
  • the transferred retrovirus genetic material is then transcribed and translated into proteins within the host cell.
  • the targeting regulatory element is typically linked to the 5' and/or 3' LTR, creating a chimeric LTR.
  • the disclosure provides in one embodiment a replication competent retrovirus that does not require helper virus or additional nucleic acid sequence or proteins in order to propagate and produce virion.
  • the nucleic acid sequences of the retrovirus of the disclosure encode a group specific antigen and reverse transcriptase, (and integrase and protease-enzymes necessary for maturation and reverse transcription) , respectively, as discussed above.
  • the viral gag and pol can be derived from a lentivirus, such as HIV or an oncovirus or gammaretrovirus such as MoMLV.
  • the nucleic acid genome of the retrovirus of the disclosure includes a sequence encoding a viral envelope (ENV) protein.
  • the env gene can be derived from any retroviruses.
  • the env may be an amphotropic envelope protein which allows transduction of cells of human and other species, or may be an ecotropic envelope protein, which is able to transduce only mouse and rat cells. Further, it may be desirable to target the recombinant virus by linkage of the envelope protein with an antibody or a particular ligand for targeting to a receptor of a particular cell-type.
  • retroviral vectors can be made target specific by inserting, for example, a glycolipid, or a protein.
  • the env gene is derived from a non-retrovirus (e.g., CMV or VSV) .
  • retroviral-derived env genes include, but are not limited to: Moloney murine leukemia virus (MoMuLV) , Harvey murine sarcoma virus
  • HaMuSV murine mammary tumor virus
  • MoMTV murine mammary tumor virus
  • GaLV gibbon ape leukemia virus
  • HAV human immunodeficiency virus
  • RSV Rous Sarcoma Virus
  • Other env genes such as Vesicular stomatitis virus
  • VSV (Protein G)
  • CMV cytomegalovirus envelope
  • HA influenza virus hemagglutinin
  • the retroviral genome is derived from an onco-retrovirus , and more particularly a mammalian onco- retrovirus.
  • the retroviral genome is derived from a gamma retrovirus, and more particularly a mammalian gamma retrovirus.
  • derived is meant that the parent
  • polynucleotide sequence is an wild-type oncovirus which has been modified by insertion or removal of naturally occurring sequences
  • polynucleotide encoding a polypeptide or inhibitory nucleic acid of interest, swapping of a more effective promoter from a different retrovirus or virus in place of the wild-type promoter and the like) .
  • the disclosure provides a retrovirus that is replication-competent.
  • the disclosure provides a retrovirus having tissue-specific promoter elements at the 5' end of the retroviral genome.
  • tissue-specific regulatory elements/sequences are in the U3 region of the LTR of the retroviral genome, including for example cell- or tissue-specific promoters and enhancers to neoplastic cells (e.g., tumor cell-specific enhancers and
  • promoters include tetracycline, and inducible promoters (e.g., tetracycline) .
  • Modified versions of the CMV promoter that are less potent have also been used when reduced levels of expression of the transgene are desired.
  • retroviral promoters such as the LTRs from MLV or MMTV can be used.
  • Other viral promoters that can be used include SV40, RSV LTR, HIV-1 and HIV-2 LTR, adenovirus promoters such as from the E1A, E2A, or MLP region, AAV LTR, cauliflower mosaic virus, HSV-TK, and avian sarcoma virus.
  • tissue specific or selective promoters may be used to effect transcription in specific tissues or cells so as to reduce potential toxicity or undesirable effects to non-targeted tissues.
  • promoters such as the PSA, probasin, prostatic acid phosphatase or prostate-specific glandular
  • kallikrein may be used to target gene expression in the prostate.
  • the Whey accessory protein WAP
  • WAP Whey accessory protein
  • Other promoters/regulatory domains that can be used are set forth in Table 3.
  • tissue-specific regulatory elements are regulatory elements (e.g., promoters) that are capable of driving
  • tissue-specific promoters may have a detectable amount of "background” or “base” activity in those tissues where they are silent.
  • the degree to which a promoter is selectively activated in a target tissue can be expressed as a selectivity ratio (activity in a target tissue/activity in a control tissue) .
  • a tissue specific promoter useful in the practice of the disclosure typically has a selectivity ratio of greater than about 5.
  • the selectivity ratio is greater than about 15.
  • RRCR RRCR
  • promoters that are hormone or cytokine regulatable For example in therapeutic applications where the indication is a gonadal tissue where specific steroids are produced or routed to, use of androgen or estrogen regulated promoters may be advantageous.
  • promoters that are hormone regulatable include MMTV, MT-1, ecdysone and RuBisco.
  • Other hormone regulated promoters such as those responsive to thyroid, pituitary and adrenal hormones may be used.
  • Cytokine and inflammatory protein responsive promoters that could be used include K and T Kininogen
  • glycoprotein Prowse and Baumann, 1988
  • alpha-1 antitypsin lipoprotein lipase
  • angiotensinogen Ron et al . , 1990
  • fibrinogen c-jun (inducible by phorbol esters, TNF- alpha, UV radiation, retinoic acid, and hydrogen peroxide)
  • collagenase induced by phorbol esters and retinoic acid
  • metallothionein heavy metal and glucocorticoid inducible
  • Stromelysin inducible by phorbol ester, interleukin-1 and EGF
  • alpha-2 macroglobulin alpha-1 antichymotrypsin
  • Tumor specific promoters such as osteocalcin, hypoxia-responsive element (HRE) , MAGE-4 , CEA, alpha-fetoprotein, GRP78/BiP and tyrosinase may also be used to regulate gene expression in tumor cells.
  • HRE hypoxia-responsive element
  • MAGE-4 MAGE-4
  • CEA alpha-fetoprotein
  • GRP78/BiP tyrosinase
  • Adipsin (Spiegelman et al . , 1989) acetyl-CoA carboxylase (Pape and Kim,
  • tissue specific promoters While not restricted in activity to a single tissue type, may nevertheless show selectivity in that they may be active in one group of tissues, and less active or silent in another group. Such promoters are also termed "tissue specific", and are contemplated for use with the disclosure. For example, promoters that are active in a variety of central nervous system (CNS) neurons may be therapeutically useful in protecting against damage due to stroke, which may affect any of a number of different regions of the brain. Accordingly, the tissue-specific regulatory elements used in the disclosure, have applicability to regulation of the heterologous proteins as well as an applicability as a targeting polynucleotide sequence in the present retroviral vectors.
  • CNS central nervous system
  • the disclosure provides plasmids comprising a recombinant retroviral derived construct.
  • the plasmid can be directly introduced into a target cell or a cell culture such as NIH 3T3 or other tissue culture cells. The resulting cells release the retroviral vector into the culture medium .
  • the disclosure provides in one embodiment, a recombinant replication competent retrovirus (RCR) comprising an optimized IRES cassette.
  • the retroviral polynucleotide sequence is derived from a virus selected from the group consisting of murine leukemia virus (MLV) , Moloney murine leukemia virus (MoMLV) , Feline leukemia virus (FeLV) , Baboon endogenous retrovirus (BEV) , porcine endogenous virus (PERV) , the cat derived retrovirus RD114, squirrel monkey retrovirus, Xenotropic murine leukemia virus-related virus
  • the RCR comprises a retroviral GAG protein; retroviral POL protein; a retroviral envelope (which can be chimeric, ecotropic and amphotropic) ; a retroviral polynucleotide comprising Long-Terminal Repeat (LTR) sequences at the 3' end of the retroviral polynucleotide sequence, gag, pol and env genes and an optimized IRES cassette (and/or optional additional elements including core promoter, inhibitory nucleic acid such as miRNA and the like) and a promoter within the LTR at the 5' end of the retroviral polynucleotide.
  • LTR Long-Terminal Repeat
  • the 3' LTR comprises a sequence that is at least 98% identical to the sequence from about nucleotide 9405 to about 9998 of SEQ ID NO: 19, 22 or 42.
  • the promoter sequence at the 5' end of the retroviral polynucleotide is suitable for expression in a mammalian cell.
  • the promoter, gag, pol and env domains comprise a sequence that is at least 98% identical to the sequence from about 1 to about 8323 of SEQ ID NO: 19, 22 or 42 and wherein the retroviral polynucleotide lacks 70 base pairs of MLV sequence downstream form the 3'LTR compared to a vector of SEQ ID NO: 21
  • a cassette comprising an optimized internal ribosome entry site (IRES) comprising a sequence that is at least 98% identical to the sequence from about 8327 to 8875 of SEQ ID NO: 19, 22 or 42 and consisting of 6As in the A-bulge in the J-K bifurcation region.
  • IRS optimized internal ribosome entry site
  • the optimized IRES is operably linked to a heterologous polynucleotide, wherein the cassette is positioned 5' to the 3 ' LTR and 3 ' to the env nucleic acid domain encoding the retroviral envelope and lacking small repeats on either side of the cassette compared to the pACE vector of SEQ ID NO:21 (pACE-CD) .
  • the vector includes cis-acting sequences necessary for reverse transcription, packaging and integration in a target cell.
  • the RCR maintains higher replication competency after 6 passages compared to a vector comprising SEQ ID NO: 21 (pACE) and wherein when the heterologous polynucleotide is expressed it produces at least 20%, 30%, 40%, 50% or more expressed heterologous polypeptide compared to a pAC3-yCD2 (SEQ ID NO: 22) vector.
  • the RCR infects a target cell multiple times resulting in an average number of copies/diploid genome of 5 or greater.
  • the retroviral envelope is an amphotropic MLV envelope.
  • the promoter comprises a CMV promoter having a sequence as set forth in SEQ ID NO: 19, 20, 22 or 42 from nucleotide 1 to about nucleotide 582 and may include modification to one or more nucleic acid bases and which is capable of directing and initiating transcription.
  • the promoter comprises a CMV-R-U5 domain polynucleotide.
  • the CMV-R-U5 domain comprises the immediately early promoter from human cytomegalovirus linked to an MLV R-U5 region.
  • the CMV-R-U5 domain polynucleotide comprises a sequence as set forth in SEQ ID NO: 19, 20, 22 or 42 from about nucleotide 1 to about nucleotide 1202 or sequences that are at least 99% identical to a sequence as set forth in SEQ ID NO: 19, 20, 22 or 42, wherein the polynucleotide promotes transcription of a nucleic acid molecule operably linked thereto.
  • the gag nucleic acid domain comprises a sequence from about nucleotide number 1203 to about nucleotide 2819 of SEQ ID NO: 19, 22 or 42 or a sequence having at least 99% or 99.8% identity thereto.
  • the pol domain of the polynucleotide is derived from a gammaretrovirus .
  • the pol domain comprises a sequence from about nucleotide number 2820 to about nucleotide 6358 of SEQ ID NO: 19, 22 or 42 or a sequence having at least 99% or 99.9% identity thereto.
  • the env domain comprises a sequence from about nucleotide number 6359 to about nucleotide 8323 of SEQ ID NO: 19, 22 or 42 or a sequence having at least 99% or 99.8% identity thereto.
  • the IRES comprises a sequence as set forth in SEQ ID NO: 41.
  • the heterologous nucleic acid comprises a polynucleotide having a sequence as set forth in SEQ ID NO: 3, 5, 11, 13, 15 or 17.
  • heterologous nucleic acid encodes a polypeptide comprising a sequence as set forth in SEQ ID NO : 4.
  • the heterologous nucleic acid is human codon optimized and encodes a polypeptide as set forth in SEQ ID NO: 4.
  • the heterologous nucleic acid comprises a sequence as set forth in SEQ ID NO: 19, 22 or 42 from about nucleotide number 8877 to about 9353.
  • the 3' LTR comprises a U3-R-U5 domain.
  • the 3' LTR comprises a sequence as set forth in SEQ ID NO: 19, 22 or 42 from about nucleotide 9405 to about 9998 or a sequence that is at least 95%, 98% or 99.5% identical thereto.
  • the disclosure provides a retroviral polynucleotide comprising SEQ ID NO: 42.
  • the retroviral polynucleotide of SEQ ID NO: 42 is an RNA sequence wherein T is replaced with U.
  • a retroviral RNA polynucleotide according to SEQ ID NO: 42, wherein T is U is encapsulated in a viral capsid.
  • the retroviral polynucleotide can further comprise and miRNA, siRNA or shRNA sequence to be delivered to a target cell.
  • the miRNA, siRNA or shRNA can be operably linked to a polIII promoter.
  • the miRNA may be located upstream or downstream of the optimized IRES cassette.
  • the heterologous polynucleotide can be any number of coding sequences including cytokines, immunopotentiating agents, thymidine kinase, cytosine deaminase, purine nucleoside phophorylase , receptors, antibody and fragments etc.
  • the disclosure also provides a method of treating a cell proliferative disorder comprising contacting the subject with a retrovirus as described herein.
  • heterologous polynucleotide linked to the optimized IRES comprises cytosine deaminase activity and contacting the subject with 5- fluorocytosine .
  • the retrovirus infects a cell resulting in integration of a polynucleotide comprising SEQ ID NO: 42.
  • the cell proliferative disorder is glioblastoma multiforme.
  • proliferative disorder is selected from the group consisting of lung cancer, colon-rectum cancer, breast cancer, prostate cancer, urinary tract cancer, uterine cancer, brain cancer, head and neck cancer, pancreatic cancer, melanoma, stomach cancer and ovarian cancer.
  • the method can include a combination therapy, wherein a subject to be treated is contacted with a retrovirus and further contacted with an anticancer agent or chemotherapeutic agent.
  • the anticancer or chemotherapeutic agent can be selected from the group consisting of bevacizumab, pegaptanib, ranibizumab, sorafenib, sunitinib, AE-941, VEGF Trap, pazopanib, vandetanib, vatalanib, cediranib, fenretinide, squalamine, INGN-241, oral tetrathiomolybdate , tetrathiomolybdate, Panzem NCD, 2- methoxyestradiol, AEE-788, AG-013958, bevasiranib sodium, AMG-706, axitinib, BIBF-1120, CDP-791, CP-547632, PI-88, SU-14813, SU-6668, XL-647, XL-999, IMC-1121B, ABT-869, BAY-57-9352, BAY-73-4506, BMS- 582664,
  • a retrovirus is administered from about 10 3 to 10 7 TU/g brain weight. In another embodiment, the retrovirus is administered from about 10 4 to 10 6 TU/g brain weight.
  • the disclosure provides a polynucleotide construct comprising from 5' to 3' : a promoter or regulatory region useful for initiating transcription; a psi packaging signal; a gag encoding nucleic acid sequence, a pol encoding nucleic acid sequence; an env encoding nucleic acid sequence; an internal ribosome entry site nucleic acid sequence comprising 6 A' s in the A-bulge; a heterologous polynucleotide encoding a marker,
  • polynucleotide construct of the disclosure e.g., a recombinant replication competent retroviral polynucleotide
  • a recombinant replication competent retroviral polynucleotide are engineered depending in part upon the desired host cell, expression timing or amount, and the heterologous polynucleotide.
  • replication competent retroviral construct of the disclosure can be divided up into a number of domains that may be individually modified by those of skill in the art.
  • the promoter can comprise a CMV promoter having a sequence as set forth in SEQ ID NO: 19, 20, 22 or 42 from nucleotide 1 to about nucleotide 582 and may include modification to one or more (e.g., 2-5, 5-10, 10-20, 20-30, 30-50 or more nucleic acid bases) so long as the modified promoter is capable of directing and initiating transcription.
  • the promoter or regulatory region comprises a CMV-R-U5 domain
  • the CMV-R-U5 domain comprises the immediately early promoter from human cytomegalovirus to the MLV R-U5 region.
  • the CMV-R-U5 domain polynucleotide comprises a sequence as set forth in SEQ ID NO: 19, 20, 22 or 42 from about nucleotide 1 to about nucleotide 1202 or sequences that are at least 95% identical to a sequence as set forth in SEQ ID NO: 19, 20, 22 or 42 from about nucleotide 1 to about nucleotide 1202, wherein the polynucleotide promotes transcription of a nucleic acid molecule operably linked thereto.
  • polynucleotide may be derived from any number of retroviruses, but will typically be derived from an oncoretrovirus and more
  • the gag domain comprises a sequence from about nucleotide number 1203 to about nucleotide 2819 of a sequence as set forth in SEQ ID NO: 19, 20, 22 or 42 or a sequence having at least 95%, 98%, 99% or 99.8% (rounded to the nearest 10 th ) identity thereto.
  • the pol domain of the polynucleotide may be derived from any number of
  • the pol domain comprises a sequence from about nucleotide number 2820 to about nucleotide 6358 of a sequence as set forth in SEQ ID NO: 19, 20, 22 or 42 or a sequence having at least 95%, 98%, 99% or 99.9% (roundest to the nearest 10 th ) identity thereto.
  • the env domain of the polynucleotide may be derived from any number of retroviruses, but will typically be derived from an oncoretrovirus or gamma-retrovirus and more particularly from a mammalian oncoretrovirus or gamma-retrovirus.
  • the env coding domain comprises an amphotropic env domain. In one embodiment the env domain comprises a sequence from about
  • optimized IRES domain of the polynucleotide may be obtained from any number of internal ribosome entry sites.
  • optimized IRES is derived from an encephalomyocarditis virus.
  • the optimized IRES domain comprises as set forth in SEQ ID NO: 41 or a sequence having at least 95%, 98%, or 99% (roundest to the nearest 10 th ) identity thereto so long as the domain allows for entry of a ribosome and comprises 6 A' s in the A- bulge.
  • the heterologous domain can comprise a cytosine deaminase (CD) of the disclosure.
  • the CD polynucleotide comprises a human codon optimized sequence.
  • the CD polynucleotide encodes a mutant polypeptide having cytosine deaminase, wherein the mutations confer increased thermal stabilization that increase the melting temperature (T m ) by 10 °C allowing sustained kinetic activity over a broader
  • the disclosure comprises a human codon
  • the heterologous domain may be followed by a polypurine rich domain.
  • the 3' LTR can be derived from any number of retroviruses, typically an oncoretrovirus and preferably a mammalian oncoretrovirus. In one embodiment, the 3' LTR comprises a U3-R-U5 domain. In yet another embodiment the LTR comprises a sequence as set forth in SEQ ID NO: 19, 20, 22 or 42 from about nucleotide 9405 to about 9998 or a sequence that is at least 95%, 98% or 99.5% (rounded to the nearest 10 th ) identical thereto .
  • the disclosure also provides a recombinant retroviral vector comprising from 5' to 3' a CMV-R-U5, fusion of the immediate early promoter from human cytomegalovirus to the MLV R-U5 region; a PBS, primer binding site for reverse transcriptase; a 5' splice site; a ⁇ packaging signal; a gag, ORF for MLV group specific antigen; a pol, ORF for MLV polymerase polyprotein; a 3' splice site; a 4070A env, ORF for envelope protein of MLV strain 4070A; an optimized IRES, consisting of 6A' s in the A-bulge; a modified cytosine deaminase (thermostabilized and codon optimized) or human codon optimized thymidine kinase; a PPT, polypurine tract; and a U3-R-U5, MLV long terminal repeat.
  • a recombinant retroviral vector comprising from 5
  • the disclosure also provides a retroviral vector comprising a sequence as set forth in SEQ ID NO: 42 (or SEQ ID NO: 42 wherein T can be U) comprising an optimized A-bulge for expression.
  • the optimized A-bulge of the IRES consists of 6A' s .
  • the retroviral vectors can be used to treat a wide range of disease and disorders including a number of cell proliferative diseases and disorders (see, e.g., U.S. Pat. Nos . 4,405,712 and 4,650,764; Friedmann, 1989, Science, 244:1275-1281; Mulligan, 1993, Science, 260:926-932, R. Crystal, 1995, Science 270:404-410, each of which are incorporated herein by reference in their entirety, see also, The Development of Human Gene Therapy, Theodore
  • the disclosure also provides gene therapy for the treatment of cell proliferative disorders. Such therapy would achieve its therapeutic effect by introduction of an appropriate therapeutic polynucleotide (e.g., antisense, ribozymes, suicide genes, siRNA) , into cells of subject having the proliferative disorder. Delivery of polynucleotide constructs can be achieved using the recombinant retroviral vector of the disclosure, particularly if it is based on MLV, which is capable of infecting dividing cells.
  • an appropriate therapeutic polynucleotide e.g., antisense, ribozymes, suicide genes, siRNA
  • the therapeutic methods e.g., the gene therapy or gene delivery methods as described herein can be performed in vivo or ex vivo. It may be preferable to remove the majority of a tumor prior to gene therapy, for example surgically or by radiation. In some aspects, the retroviral therapy may be preceded or followed by surgery, chemotherapy or radiation therapy.
  • the disclosure provides a recombinant retrovirus capable of infecting a non-dividing cell, a dividing cell or a neoplastic cell, therein the recombinant retrovirus comprises a viral GAG; a viral POL; a viral ENV; a heterologous nucleic acid operably linked to an IRES consisting of 6A' s in the A-bulge; and cis-acting nucleic acid sequences necessary for packaging, reverse transcription and integration.
  • the recombinant retrovirus can be a lentivirus, such as HIV, or can be an oncovirus. As described above for the method of producing a recombinant retrovirus, the
  • recombinant retrovirus of the disclosure may further include at least one of VPR, VIF, NEF, VPX, TAT, REV, and VPU protein. While not wanting to be bound by a particular theory, it is believed that one or more of these genes/protein products are important for increasing the viral titer of the recombinant retrovirus produced
  • NEF Newcastle Equivalent Fidel Virus
  • the disclosure also provides a method of nucleic acid transfer to a target cell to provide expression of a particular nucleic acid (e.g., a heterologous sequence) . Therefore, in another embodiment, the disclosure provides a method for introduction and expression of a heterologous nucleic acid in a target cell comprising infecting the target cell with the recombinant virus of the disclosure and expressing the heterologous nucleic acid in the target cell.
  • the target cell can be any cell type including dividing, non-dividing, neoplastic, immortalized, modified and other cell types recognized by those of skill in the art, so long as they are capable of infection by a retrovirus.
  • nucleic acid sequence e.g., the heterologous nucleic acid sequence
  • nucleic acid sequence give rise, for example, to an antisense or ribozyme molecule.
  • modulate envisions the suppression of expression of a gene when it is over- expressed, or augmentation of expression when it is under- expressed.
  • nucleic acid sequences that interfere with the gene's expression at the translational level can be used. This approach utilizes, for example, antisense nucleic acid, ribozymes, or triplex agents to block transcription or translation of a specific mRNA, either by masking that mRNA with an antisense nucleic acid or triplex agent, or by cleaving it with a ribozyme.
  • nucleic acid encoding a biological response modifier e.g., a cytokine
  • a biological response modifier e.g., a cytokine
  • immunopotentiating agents including nucleic acids encoding a number of the cytokines
  • interleukins 1 through 15 are interleukins 1 through 15, as well as other response modifiers and factors described elsewhere herein. Also included in this category, although not necessarily working according to the same mechanisms, are interferons, and in particular gamma interferon, tumor necrosis factor (TNF) and granulocyte-macrophage-colony stimulating factor
  • TNF tumor necrosis factor
  • GM-CSF GM-CSF
  • Other polypeptides include, for example, angiogenic factors and anti-angiogenic factors. It may be desirable to deliver such nucleic acids to bone marrow cells or macrophages to treat enzymatic deficiencies or immune defects. Nucleic acids encoding growth factors, toxic peptides, ligands, receptors, or other physiologically important proteins can also be introduced into specific target cells.
  • the disclosure can be used for delivery of heterologous polynucleotides that promote drug specific targeting and effects.
  • HER2 a member of the EGF receptor family, is the target for binding of the drug trastuzumab (HerceptinTM, Genentech) .
  • trastuzumab is a mediator of antibody-dependent cellular
  • ADCC cytotoxicity
  • Enhancement of expression of HER2 by introduction of vector expressing HER2 or truncated HER2 (expressing only the extracellular and transmembrane domains) in HER2 low tumors may facilitate optimal triggering of ADCC and overcome the rapidly developing resistance to Herceptin that is observed in clinical use.
  • PCR Yamamoto et al . , Nature 319:230- 234, 1986; Chen et al., Cane. Res., 58:1965-1971, 1998) or chemically synthesized (BioBasic Inc., Markham, Ontario, Canada) and inserted between the IRES and yCD2 gene in the vector pAC3-yCD2 SEQ ID NO: 19 (e.g., between about nucleotide 8876 and 8877 of SEQ ID NO: 19) .
  • the yCD gene can be excised and replaced with a polynucleotide encoding a HER2 polypeptide or fragment thereof.
  • a further truncated HER2 with only the Herceptin binding domain IV of the ECD and TM domains (approximately 290 bp from position 1910 to 2200) can be amplified or chemically synthesized and used as above (Landgraf 2007; Garrett et al . , J. of Immunol., 178:7120-7131, 2007) .
  • a further modification of this truncated form with the native signal peptide (approximately 69 bp from position 175-237) fused to domain IV and the TM can be chemically synthesized and used as above.
  • the resulting viruses can be used to treat a cell proliferative disorder in a subject in combination with trastuzumab or trastuzumab and 5-FC.
  • HER2 and the modifications described above can be expressed in a separate vector containing a different ENV gene or other appropriate surface protein.
  • This vector can be replication competent (Logg et al . J.Mol Biol. 369:1214 2007) or non-replicative "first generation" retroviral vector that encodes the envelope and the gene of interest (Emi et al . J.Virol 65:1202 1991) . In the latter case the pre-existing viral infection will provide complementary gag and pol to allow infective spread of the "non-replicative" vector from any previously infected cell.
  • ENV and glycoproteins include xenotropic and polytropic ENV and glycoproteins capable of infecting human cells, for example ENV sequences from the NZB strain of MLV and glycoproteins from MCF, VSV, GALV and other viruses (Palu 2000, Baum et al . , Mol . Therapy, 13 ( 6) : 1050-1063 , 2006).
  • a polynucleotide can comprise a sequence wherein the GAG and POL and yCD2 genes of SEQ ID NO: 19 are deleted, the ENV corresponds to a xenotropic ENV domain of NZB MLV or VSV-g, and the IRES or a promoter such as RSV is operatively linked directly to HER2, HER2 ECDTM, HER2 ECDIVTM, or HER2 SECDIVTM.
  • proliferative disorder in a subject in combination with trastuzumab or trastuzumab and 5-FC in combination with trastuzumab or trastuzumab and 5-FC.
  • Another aspect of the development of resistance to trastuzumab relates to the interference with intracellular signaling required for the activity of trastuzumab.
  • Resistant cells show loss of PTEN and lower expression of p27kipl [Fujita, Brit J. Cancer, 94:247, 2006; Lu et al . , Journal of the National Cancer Institute, 93(24): 1852-1857, 2001; Kute et al . , Cytometry Part A 57A: 86-93, 2004) .
  • a polynucleotide encoding PTEN can be recombinantly generated or chemically synthesized
  • the PTEN encoding polynucleotide (SEQ ID NO: 25) can be synthesized as above and inserted between the IRES and yCD2 sequences or with a linker as previously described.
  • PTEN can be expressed in a separate vector containing a different ENV gene or other appropriate surface protein.
  • This vector can be replication competent (Logg et al . J.Mol Biol. 369:1214 2007) or non-replicative "first generation" retroviral vector that encodes the envelope and the gene of interest (Emi et al . , J.Virol 65:1202 1991) . In the latter case the pre-existing viral infection will provide complementary gag and pol to allow infective spread of the "non-replicative" vector from any previously infected cell.
  • Alternate ENV and glycoproteins include xenotropic and polytropic ENV and glycoproteins capable of infecting human cells, for example ENV sequences from the NZB strain of MLV and glycoproteins from MCF, VSV, GALV and other viruses (Palu, Rev Med Virol. 2000, Baum, Mol. Ther. 13(6):1050- 1063, 2006) .
  • a polynucleotide can comprise a sequence wherein the GAG and POL and yCD2 genes of SEQ ID NO: 19 are deleted, the ENV corresponds to a xenotropic ENV domain of NZB MLV or VSV-g, and the IRES or a promoter such as RSV is operatively linked directly to PTEN .
  • a polynucleotide encoding p27kipl (SEQ ID NO:27 and 28) can be chemically synthesized (BioBasic Inc.,
  • the p27kipl encoding polynucleotide can be synthesized as above and inserted between the IRES consisting of 6A' s in the A-bulge and yCD2 sequences or with a linker as previously described or in place of the yCD2 gene.
  • p27kipl can be expressed in a separate vector containing a different ENV gene or other appropriate surface protein.
  • This vector can be replication competent (Logg et al . J. Mol Biol. 369:1214 2007) or non-replicative "first generation" retroviral vector that encodes the envelope and the gene of interest (Emi et al . J.Virol 65:1202 1991) . In the latter case the pre-existing viral infection will provide complementary gag and pol to allow infective spread of the "non-replicative" vector from any previously infected cell.
  • ENV and glycoproteins include xenotropic and polytropic ENV and glycoproteins capable of infecting human cells, for example ENV sequences from the NZB strain of MLV and glycoproteins from MCF, VSV, GALV and other viruses (Palu 2000, Baum 2006, supra) .
  • ENV sequences from the NZB strain of MLV and glycoproteins from MCF, VSV, GALV and other viruses for example, a
  • polynucleotide can comprise a sequence wherein the GAG and POL and yCD2 genes of SEQ ID NO: 19 are deleted, the ENV corresponds to a xenotropic ENV domain of NZB MLV or VSV-g, and the IRES consisting of 6A' s in the A-bulge or a promoter such as RSV is operatively linked directly to p27kipl.
  • CD20 is the target for binding of the drug rituximab (RituxanTM, Genentech) .
  • Rituximab is a mediator of complement-dependent cytotoxicity (CDC) and ADCC.
  • CDC complement-dependent cytotoxicity
  • Cells with higher mean fluorescence intensity by flow cytometry show enhanced sensitivity to rituximab (van Meerten et al . , Clin Cancer Res 2006; 12 (13) : 4027-4035, 2006).
  • Enhancement of expression of CD20 by introduction of vector expressing CD20 in CD20 low B cells may facilitate optimal triggering of ADCC.
  • a polynucleotide encoding CD20 (SEQ ID NO:29 and 30) can be chemically synthesized (BioBasic Inc.,
  • the CD20 encoding polynucleotide can be synthesized as above and inserted between the IRES consisting of 6A' s in the A-bulge and yCD2 sequences or with a linker as previously described.
  • the CD20 sequence can be inserted into the pAC3-yCD2 vector after excision of the CD gene by Psil and Notl digestion.
  • a polynucleotide encoding CD20 (SEQ ID NO : 29 and 30) can be chemically synthesized (BioBasic Inc., Markham, Canada) and inserted into a vector containing a non amphotropic ENV gene or other appropriate surface protein (Tedder et al., PNAS, 85:208-212, 1988).
  • Alternate ENV and glycoproteins include xenotropic and polytropic ENV and glycoproteins capable of infecting human cells, for example ENV sequences from the NZB strain of MLV and glycoproteins from MCF, VSV, GALV and other viruses [Palu 2000, Baum 2006] .
  • a polynucleotide can comprise a sequence wherein the GAG and POL and yCD2 genes of SEQ ID NO: 19 are deleted, the ENV corresponds to a xenotropic ENV domain of NZB MLV or VSV-g, and the IRES consisting of 6A' s in the A-bulge or a promoter such as RSV is operatively linked directly to CD20.
  • phosphorylase expression is low in 5-FU resistant cancer cells compared to sensitive lines (Wang et al . , Cancer Res., 64:8167- 8176, 2004) .
  • Large population analyses show correlation of enzyme levels with disease outcome (Fukui et al . , Int'l. J. OF Mol . Med., 22:709-716, 2008) .
  • Coexpression of CD and other pyrimidine anabolism enzymes (PAE) can be exploited to increase the activity and therefore therapeutic index of fluoropyrimidine drugs.
  • the disclosure provides methods for treating cell proliferative disorders such as cancer and neoplasms comprising administering an RCR vector of the disclosure followed by treatment with a chemotherapeutic agent or anti-cancer agent.
  • the RCR vector is administered to a subject for a period of time prior to administration of the chemotherapeutic or anti-cancer agent that allows the RCR to infect and replicate.
  • the subject is then treated with a chemotherapeutic agent or anti-cancer agent for a period of time and dosage to reduce proliferation or kill the cancer cells.
  • a chemotherapeutic agent or anti-cancer agent for a period of time and dosage to reduce proliferation or kill the cancer cells.
  • chemotherapeutic or anti-cancer agent reduces, but does not kill the cancer/tumor (e.g., partial remission or temporary remission), the subject may then be treated with a non-toxic therapeutic agent
  • cytotoxic gene e.g., cytosine deaminase
  • the RCR vectors of the disclosure are spread during a replication process of the tumor cells, such cells can then be killed by treatment with an anti-cancer or
  • chemotherapeutic agent and further killing can occur using the RCR treatment process described herein.
  • the heterologous gene can comprise a coding sequence for a target antigen (e.g., a cancer antigen) .
  • a target antigen e.g., a cancer antigen
  • cells comprising a cell proliferative disorder are infected with an RCR comprising a heterologous polynucleotide encoding the target antigen to provide expression of the target antigen (e.g., overexpression of a cancer antigen) .
  • An anticancer agent e.g., an anticancer agent
  • the targeting cognate moiety can be operably linked to a cytotoxic agent or can itself be an anticancer agent.
  • a cancer cell infected by the RCR comprising the targeting antigen coding sequences increases the expression of target on the cancer cell resulting in increased efficiency/efficacy of cytotoxic targeting.
  • an RCR of the disclosure can comprise a coding sequence comprising a binding domain (e.g., an antibody, antibody fragment, antibody domain or receptor ligand) that specifically interacts with a cognate antigen or ligand.
  • the RCR comprising the coding sequence for the binding domain can then be used to infect cells in a subject comprising a cell
  • proliferative disorder such as a cancer cell or neoplastic cell.
  • the infected cell will then express the binding domain or antibody.
  • An antigen or cognate operably linked to a cytotoxic agent or which is cytotoxic itself can then be administered to a subject.
  • the cytotoxic cognate will then selectively kill infected cells expressing the binding domain.
  • the binding domain itself can be an anti-cancer agent.
  • an antibody refers to a protein that includes at least one immunoglobulin variable domain or immunoglobulin variable domain sequence.
  • an antibody can include a heavy (H) chain variable region (abbreviated herein as VH) , and a light (L) chain variable region (abbreviated herein as VL) .
  • an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions.
  • the term “antibody” encompasses antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab fragments, F(ab' ) 2, a Fd fragment, a Fv fragments, and dAb fragments) as well as complete antibodies.
  • the disclosure provides a method of treating a subject having a cell proliferative disorder.
  • the subject can be any mammal, and is preferably a human.
  • the subject is contacted with a recombinant replication competent retroviral vector of the disclosure.
  • the contacting can be in vivo or ex vivo.
  • Methods of administering the retroviral vector of the disclosure are known in the art and include, for example, systemic administration, topical administration, intraperitoneal administration, intra-muscular administration, intracranial, cerebrospinal, as well as
  • the disclosure includes various pharmaceutical compositions useful for treating a cell proliferative disorder.
  • the pharmaceutical compositions according to the disclosure are prepared by bringing a retroviral vector containing a heterologous polynucleotide sequence useful in treating or modulating a cell proliferative disorder according to the disclosure into a form suitable for administration to a subject using carriers, excipients and additives or auxiliaries. Frequently used carriers or
  • auxiliaries include magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, milk protein, gelatin, starch, vitamins, cellulose and its derivatives, animal and vegetable oils, polyethylene glycols and solvents, such as sterile water, alcohols, glycerol and polyhydric alcohols.
  • Intravenous vehicles include fluid and nutrient replenishers .
  • Preservatives include
  • compositions include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like, as described, for instance, in Remington's Pharmaceutical Sciences, 15th ed. Easton: Mack Publishing Co., 1405-1412, 1461-1487 (1975) and The National Formulary XIV., 14th ed. Washington: American Pharmaceutical Association (1975), the contents of which are hereby incorporated by reference.
  • pH and exact concentration of the various components of the pharmaceutical composition are adjusted according to routine skills in the art. See Goodman and Gilman's The Pharmacological Basis for Therapeutics
  • a retroviral vector useful in treating a cell proliferative disorder will include an amphotropic ENV protein, GAG, and POL proteins, a promoter sequence in the U3 region retroviral genome, and all cis- acting sequence necessary for replication, packaging and
  • RRVs containing various numbers of A' s in the A bulge of the J-K bifurcation region were generated to have 4, 5, 6, 7, 8, 10 or 12As in the A-bulge in the J-K bifurcation region.
  • Each construct was generated by DNA synthesis (BioBasics Inc.) of the entire IRES cassette with a Mlu I at the 5' end and a Psi I at the 3' end, respectively, for direct replacement of the equivalent cassette in the RRV backbone ( Figure IB) . All DNA fragments were confirmed by sequencing analysis prior and post cloning into the RRV backbone .
  • the RRV constructs containing the yCD2 transgene were designated using the name of the transgene followed by the number of A' s in the A bulge (e.g., yCD2-4A contains yCD2 transgene and 4As in the A bulge in the IRES) .
  • RRVs containing various numbers of A' s in the A bulge produce similar titers.
  • Virus stock was produced by transient transfection of 293T cells using calcium phosphate precipitation method. Viral supernatant was collected approximately 42 hours post transfection. Viral infection to determine titers was performed. Viral supernatant of each vector was subsequently used to infect HT1080 cells to generate RRV-producer cells. The viral titers obtained were measured before infecting naive U87-MG cells.
  • Figure 1C shows that HT1080 cells infected with RRVs containing various numbers of As produced similar levels of virus, suggesting that the number of the As in the bifurcation loop does not affect viral replication.
  • bifurcation region express similar levels of transcripts but different levels of protein expression.
  • the viral supernatant from HT1080 cells was then used to infect naive U87-MG cells at multiplicity of infection (MOI) of 0.1.
  • MOI multiplicity of infection
  • day 10 post infection when the cells were fully infected, cellular viral RNA levels were measured by quantitative real-time polymerase chain reaction (qRT- PCR) , and protein expression level of yCD2 was examined by immunoblotting (Perez et al . , 2012) .
  • the cellular viral RNA expression levels were measured using two different primer sets, located in the env (5'Env2: 5' -ACCCTCAACCTCCCCTACAAGT-3 ' , 3'Env2: 5' -GTTAAGCGCCTGATAGGCTC-3' , probe: 5 ' FAM-AGCCACCCCCAGGAACTGGAGA TAGA-3'BHQ) and in yCD2 region (5'yCD2: 5 ' -ATCATCATGTACGGCATCCCTAG- 3', 3'yCD2: 5 ' -TGAA CTGCTTCATCA GCTTCTTAC-3 ' , probe: 5'FAM- TCATCGTCAACAACCACCACCTCGT-3 ' BHQ) , respectively, ( Figure 2A) .
  • the relative level of RNA from each vector was calculated using 2- AA(ct) method with respect to the vector containing the 6As .
  • the cellular viral RNA level ratios range from 0.8 to 1.1 ( Figure 2B) ,
  • intracellular CD enzymatic activity was also measure by adding 5 FC to the cultures and measuring 5-FU after an hour. The differences in activity were ranked similarly to the Western blot data, but were not as marked. This can be attributed to limitations in a cell-based assay and to the low availability of intracellular 5-FC which was below the K m for the enzyme in the assay utilized.
  • Fig 1C The GFP expression levels were measured using flow cytometry by gating the GFP-positive cells.
  • the mean fluorescent intensity (MFI) of each vector was normalized to the cellular viral RNA level and calculated relative to the GFP-6A vector.
  • the results ( Figure 2C) from this set of vector were consistent with those observed with yCD2 vectors ( Figure 2B) and the vectors containing the 6As expresses the highest level of protein from the transgene in both sets of vectors. Furthermore, due to the sensitivity of the detection method, a remarkable difference in GFP expression level was revealed, showing approximately 96% and 99% decrease in GFP expressed by the vectors containing the lOAs and 12As, respectively. In both sets of the vectors, RRV with 7As showed an approximately 30% decrease in protein expression.
  • the vector variants with 4, 5, 8, 10 and 12As also require binding of PBT to the polypyrimidine tract for efficient protein translation and that these vector variants significantly distort the secondary and tertiary structure of the IRES and thus compromise the binding of PBT and/or other transacting factors to the polypyrimidine tract, and hence diminish the PBT-mediated rescue of translational activity.
  • the mutations in the number of adenosine residues in the A-bulge has not been described in EMCV.
  • the RRV constructs containing the yCD2 transgene were designated using the name of the transgene followed by the number of A' s in the A bulge (i.e. yCD2-4A contains yCD2 transgene and 4As in the A bulge in the IRES) .

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WO2021030251A1 (en) 2019-08-12 2021-02-18 Purinomia Biotech, Inc. Methods and compositions for promoting and potentiating t-cell mediated immune responses through adcc targeting of cd39 expressing cells
WO2021074695A1 (en) 2019-10-16 2021-04-22 Avacta Life Sciences Limited PD-L1 INHIBITOR - TGFβ INHIBITOR BISPECIFIC DRUG MOIETIES.
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WO2022165001A1 (en) 2021-01-29 2022-08-04 Merck Sharp & Dohme Llc Compositions of programmed death receptor 1 (pd-1) antibodies and methods of obtaining the compositions thereof
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EP4467650A2 (en) 2017-07-07 2024-11-27 Avacta Life Sciences Limited Scaffold proteins
WO2021030251A1 (en) 2019-08-12 2021-02-18 Purinomia Biotech, Inc. Methods and compositions for promoting and potentiating t-cell mediated immune responses through adcc targeting of cd39 expressing cells
WO2021074695A1 (en) 2019-10-16 2021-04-22 Avacta Life Sciences Limited PD-L1 INHIBITOR - TGFβ INHIBITOR BISPECIFIC DRUG MOIETIES.
WO2021249786A1 (en) 2020-06-09 2021-12-16 Avacta Life Sciences Limited Sars-cov2 diagnostic polypeptides and methods
WO2022165001A1 (en) 2021-01-29 2022-08-04 Merck Sharp & Dohme Llc Compositions of programmed death receptor 1 (pd-1) antibodies and methods of obtaining the compositions thereof
WO2022234003A1 (en) 2021-05-07 2022-11-10 Avacta Life Sciences Limited Cd33 binding polypeptides with stefin a protein
WO2023057567A1 (en) 2021-10-07 2023-04-13 Avacta Life Sciences Limited Pd-l1 binding affimers
WO2023057946A1 (en) 2021-10-07 2023-04-13 Avacta Life Sciences Limited Serum half-life extended pd-l1 binding polypeptides
WO2023212483A1 (en) 2022-04-29 2023-11-02 Purinomia Biotech, Inc. Methods and compositions for treating eosinophil driven diseases and disorders

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