JP2016526920A - Recombinant vector with optimized A-bulge - Google Patents

Abstract

The present disclosure describes a replication competent retroviral vector (RCR) for gene therapy and gene delivery. The RCR contains an IRES sequence with 6-7 A's in the A bulge of the branch region. The present disclosure includes a retroviral GAG protein; a retroviral POL protein; a retroviral envelope; a long terminal repeat (LTR) sequence at the 3 ′ end of the retroviral polynucleotide sequence, and a promoter sequence at the 5 ′ end of the retroviral polynucleotide. A recombinant replication competent retrovirus comprising a retroviral polynucleotide is provided. [Selection] Figure 5

Description

This application claims priority to US Provisional Application No. 61 / 862,433, filed Aug. 5, 2013, the disclosure of which is incorporated herein by reference.

  The present disclosure relates to compositions comprising such optimized IRES, including optimized internal ribosome entry sites (IRES), vectors. More specifically, the present disclosure relates to replication competent retroviral vectors for treating cell proliferative disorders. The present disclosure further relates to the use of such replication competent retroviral vectors for delivery and expression of heterologous nucleic acids.

Efficient delivery methods of genes and heterologous nucleic acids to cells and subjects have become researchers' goals for scientific development and potential treatment of diseases and disorders.
Include sequence listing

  This application is filed with a sequence listing in electronic format. The sequence listing was created on August 5, 2014 and is titled “00014-019WO1Sequence_ST25” and has a size of 202 Kb. Information in electronic format of the sequence listing is incorporated herein by reference in its entirety.

  The present disclosure relates to a recombinant replication competent retrovirus comprising a retroviral GAG protein; a retroviral POL protein; a retroviral envelope; a retroviral polynucleotide having a long terminal repeat at the 3 ′ end of the retroviral polynucleotide sequence. (LTR) sequence, said retroviral polynucleotide comprising at the 5 ′ end of the retroviral polynucleotide a promoter sequence suitable for expression in mammalian cells, a gag nucleic acid domain, a pol nucleic acid domain and an env nucleic acid domain; A cassette containing an internal ribosome entry site (IRES) comprising 6A's in the A bulge, wherein the IRES is operably linked to a heterologous polynucleotide, and the cassette is 5 'on the 3'LTR, and a retrovirus Located 3 'to the env nucleic acid domain encoding the envelope Cassette; and cis-acting sequences necessary for reverse transcription, packaging and integration in target cells, and RCR is higher after 6 passages compared to the vector containing SEQ ID NO: 21 (pACE) Provided is the above-mentioned recombinant replication competent retrovirus that maintains replication ability. In one embodiment, the virus infects the target cell multiple times resulting in an average copy number / diploid genome of 5 or greater. In any other embodiment of the above, the retroviral polynucleotide sequence is murine leukemia virus (MLV), Moloney murine leukemia virus (MoMLV), feline leukemia virus (FeLV), baboon endogenous retrovirus (BEV), swine Selected from the group consisting of endogenous virus (PERV), feline retrovirus RD114, squirrel monkey retrovirus, heterotrophic murine leukemia-related virus (XMRV), avian reticuloendotheliosis virus (REV), and gibbon leukemia virus (GALV) Derived from the virus. In other embodiments of any of the above, the retroviral envelope is a broad host MLV envelope. In other embodiments of any of the above, the retrovirus is a gamma retrovirus. In other embodiments of any of the above, the target cell is a cell with a cell proliferative disorder. In other embodiments of any of the above, the target cell is a neoplastic cell. In any other embodiment of the above, the cell proliferative disorder is lung cancer, colorectal cancer, breast cancer, prostate cancer, urinary tract cancer, uterine cancer, brain cancer, head and neck cancer, pancreatic cancer, melanoma, gastric cancer and ovary Selected from the group consisting of cancer, rheumatoid arthritis and other autoimmune diseases. In other embodiments of any of the above, the promoter sequence is associated with a growth regulatory gene. In other embodiments of any of the above, the promoter sequence comprises a tissue specific promoter sequence. In any other embodiment of the above, the tissue specific promoter sequence comprises at least one androgen response element (ARE). In other embodiments of any of the above, the promoter comprises a CMV promoter having nucleotide 1 to about nucleotide 582 of the sequence set forth in SEQ ID NO: 19, 20, 22, or 42, and can induce and initiate transcription. , May include one or more nucleobase modifications. In any other embodiment of the above, the promoter comprises a CMV-R-U5 domain polynucleotide. In any other embodiment of the above, the CMV-R-U5 domain comprises an early promoter from human cytomegalovirus linked to the MLV R-U5 region. In any other embodiment of the above, the CMV-R-U5 domain polynucleotide is from about nucleotide 1 to about nucleotide 1202 of the sequence set forth in SEQ ID NO: 19, 20, 22, or 42, or SEQ ID NO: 19, 20, The polynucleotide comprises a sequence that is at least 95% identical to the sequence set forth in 22 or 42, and the polynucleotide facilitates transcription of a nucleic acid molecule operably linked thereto. In other embodiments of any of the above, the gag polynucleotide is derived from a gamma retrovirus. In any other embodiment of the above, the gag nucleic acid domain is from about nucleotide number 1203 to about nucleotide 2819 of SEQ ID NO: 19, 20, 22, or 42, or at least 95%, 98%, 99%, or 99.8 relative thereto. Contains sequences with% identity. In other embodiments of any of the above, the pol domain of the polynucleotide is derived from a gamma retrovirus. In any other embodiment of the above, the pol domain comprises from about nucleotide number 2820 to about nucleotide 6358 of SEQ ID NO: 19, 20, 22, or 42, or at least 95%, 98%, 99%, or 99.9% thereto Including sequences having the same identity. In any other embodiment of the above, the env domain has from about nucleotide number 6359 to about nucleotide 8323 of SEQ ID NO: 19, 20, 22, or 42, or at least 95%, 98%, 99%, or 99.8% thereto Including sequences having the same identity. In any other embodiment of the above, the IRES consists of the sequence set forth in SEQ ID NO: 41. In any other of the above embodiments, the retroviral polynucleotide sequence comprises (i) the sequence set forth in SEQ ID NO: 42, or (ii) the sequence set forth in SEQ ID NO: 42 where T is U. In any other embodiment of the above, the heterologous nucleic acid encodes a polypeptide comprising a polynucleotide having the sequence set forth in SEQ ID NO: 3, 5, 11, 13, 15 or 17. In any other embodiment of the above, the heterologous nucleic acid comprises the sequence set forth in SEQ ID NO: 4. In other embodiments of any of the above, the heterologous nucleic acid is human codon optimized and encodes the polypeptide set forth in SEQ ID NO: 4. In any other of the above embodiments, the heterologous nucleic acid comprises the sequence set forth from about nucleotide number 8877 to about 9353 of SEQ ID NO: 19 or 22. In other embodiments of any of the above, the 3 ′ LTR is derived from a gamma retrovirus. In other embodiments of any of the above, the 3 ′ LTR comprises a U3-R-U5 domain. In any other of the above embodiments, the 3 ′ LTR is a sequence set forth from about nucleotide 9405 to about 9988 of SEQ ID NO: 19 or 22, or a sequence that is at least 95%, 98%, or 99.5% identical thereto including. In other embodiments of any of the above, the heterologous nucleic acid sequence encodes a biological response modifier, or an immunostimulatory cytokine. In any other embodiment of the above, the immunostimulatory cytokine is selected from the group consisting of interleukins 1-15, interferon, tumor necrosis factor (TNF), and granulocyte macrophage colony stimulating factor (GM-CSF). . In any other embodiment of the above, the immunostimulatory cytokine is interferon gamma. In other embodiments of any of the above, the heterologous nucleic acid encodes a polypeptide that converts a non-toxic prodrug to a toxic agent. In any other embodiment of the above, the polypeptide that converts a non-toxic prodrug to a toxic drug is thymidine kinase, purine nucleoside phosphorylase (PNP), or cytosine deaminase. In other embodiments of any of the above, the heterologous nucleic acid sequence encodes a receptor domain, antibody, or antibody fragment. In other embodiments of any of the above, the heterologous nucleic acid sequence comprises an inhibitory polynucleotide. In any other embodiment of the above, the inhibitory polynucleotide comprises a miRNA, RNAi, or siRNA sequence.

  The present disclosure also provides a recombinant retroviral polynucleotide genome for producing the replication competent retrovirus described above.

  The present disclosure relates to a cell proliferative disorder comprising contacting a subject with a recombinant replication competent retrovirus of the present disclosure under conditions in which a cytosine deaminase polynucleotide is expressed, and contacting a subject with 5-fluorocytosine. A method of treating is also provided. In other embodiments, the cell proliferative disorder is glioblastoma multiforme. In any other embodiment of the above, the cell proliferative disorder is lung cancer, colorectal cancer, breast cancer, prostate cancer, urinary tract cancer, uterine cancer, brain cancer, head and neck cancer, pancreatic cancer, melanoma, gastric cancer and ovary Selected from the group consisting of cancer.

  The present disclosure also provides vectors that express heterologous genes in mammalian cells from ECMV IRES having 6 A in the A bulge of the J-K branch region. In other embodiments, the vector is a viral vector. In other embodiments of any of the above, the vector is a retroviral replication vector. In other embodiments of any of the above, the vector is a retroviral replication vector derived from a gamma retrovirus. In any other embodiment of the above, the gamma retrovirus is derived from any one of murine leukemia virus, baboon endogenous virus, gibbon leukemia virus, feline leukemia virus. In any other embodiment of the above, the heterologous gene is a gene having therapeutic activity in a mammal. In other embodiments of any of the above, the therapeutic activity is anticancer activity. In other embodiments of any of the above, the heterologous gene is a prodrug activating gene. In other embodiments of any of the above, the vector may express a heterologous gene in mammalian cells from an ECMV IRES in the absence of PTB-1 protein.

  The present disclosure also provides a method for treating cancer by administering a vector as described above.

  The present disclosure relates to a recombinant replication competent retrovirus comprising a retroviral GAG protein; a retroviral POL protein; a retroviral envelope; a retroviral polynucleotide having a long terminal repeat at the 3 ′ end of the retroviral polynucleotide sequence. (I) a heterologous (LTR) sequence, a promoter sequence suitable for expression in mammalian cells at the 5 ′ end of a retroviral polynucleotide, a gag nucleic acid domain, a pol nucleic acid domain and an env nucleic acid domain; A minimal internal ribosome entry site (IRES) operably linked to a polynucleotide, (ii) a polIII promoter linked to a miRNA, or (iii) a heterologous poly that precedes or follows (ii) Contains a mini-promoter operably linked to the nucleotide A cassette located 5 ′ of the 3 ′ LTR and 3 ′ of the env nucleic acid domain encoding the retroviral envelope; and the cis action required for reverse transcription, packaging and integration in target cells Also provided is the above-described recombinant replication competent retrovirus comprising a sex sequence. In one embodiment, the minimum IRES consists of a sequence of about bases 123 to 544 of SEQ ID NO: 41. In any other embodiment of the above, the minimum IRES consists of a sequence of about bases 183 to 544 of SEQ ID NO: 41. In any other embodiment of the above, the IRES has 6 A's in the A bulge. In other embodiments of any of the above, the virus infects the target cell multiple times resulting in an average copy number / diploid genome of 5 or greater. In any other embodiment of the above, the retroviral polynucleotide sequence is murine leukemia virus (MLV), Moloney murine leukemia virus (MoMLV), feline leukemia virus (FeLV), baboon endogenous retrovirus (BEV), swine Selected from the group consisting of endogenous virus (PERV), feline retrovirus RD114, squirrel monkey retrovirus, heterotrophic murine leukemia-related virus (XMRV), avian reticuloendotheliosis virus (REV), and gibbon leukemia virus (GALV) Derived from the virus. In other embodiments of any of the above, the retroviral envelope is a broad host MLV envelope. In other embodiments of any of the above, the retrovirus is a gamma retrovirus. In other embodiments of any of the above, the target cell is a cell with a cell proliferative disorder. In other embodiments of any of the above, the target cell is a neoplastic cell. In any other embodiment of the above, the cell proliferative disorder is lung cancer, colorectal cancer, breast cancer, prostate cancer, urinary tract cancer, uterine cancer, brain cancer, head and neck cancer, pancreatic cancer, melanoma, gastric cancer and ovary Selected from the group consisting of cancer, rheumatoid arthritis and other autoimmune diseases. In other embodiments of any of the above, the promoter sequence is associated with a growth regulatory gene. In other embodiments of any of the above, the promoter sequence comprises a tissue specific promoter sequence. In any other embodiment of the above, the tissue specific promoter sequence comprises at least one androgen response element (ARE). In other embodiments of any of the above, the promoter comprises a CMV promoter having nucleotide 1 to about nucleotide 582 of the sequence set forth in SEQ ID NO: 19, 20, 22, or 42, and can induce and initiate transcription. , May include one or more nucleobase modifications. In any other embodiment of the above, the promoter comprises a CMV-R-U5 domain polynucleotide. In any other embodiment of the above, the CMV-R-U5 domain comprises an early promoter from human cytomegalovirus linked to the MLV R-U5 region. In any other embodiment of the above, the CMV-R-U5 domain polynucleotide is from about nucleotide 1 to about nucleotide 1202 of the sequence set forth in SEQ ID NO: 19, 20, 22, or 42, or SEQ ID NO: 19, 20, The polynucleotide comprises a sequence that is at least 95% identical to the sequence set forth in 22 or 42, and the polynucleotide facilitates transcription of a nucleic acid molecule operably linked thereto. In other embodiments of any of the above, the gag polynucleotide is derived from a gamma retrovirus. In any other embodiment of the above, the gag nucleic acid domain is from about nucleotide number 1203 to about nucleotide 2819 of SEQ ID NO: 19, 20, 22, or 42, or at least 95%, 98%, 99%, or 99.8 relative thereto. Contains sequences with% identity. In other embodiments of any of the above, the pol domain of the polynucleotide is derived from a gamma retrovirus. In any other embodiment of the above, the pol domain comprises from about nucleotide number 2820 to about nucleotide 6358 of SEQ ID NO: 19, 20, 22, or 42, or at least 95%, 98%, 99%, or 99.9% thereto Including sequences having the same identity. In any other embodiment of the above, the env domain has from about nucleotide number 6359 to about nucleotide 8323 of SEQ ID NO: 19, 20, 22, or 42, or at least 95%, 98%, 99%, or 99.8% thereto Including sequences having the same identity. In any other embodiment of the above, the heterologous nucleic acid comprises a polynucleotide having the sequence set forth in SEQ ID NO: 3, 5, 11, 13, 15 or 17. In other embodiments of any of the above, the heterologous nucleic acid encodes a polypeptide comprising the sequence set forth in SEQ ID NO: 4. In other embodiments of any of the above, the heterologous nucleic acid is human codon optimized and encodes the polypeptide set forth in SEQ ID NO: 4. In any other of the above embodiments, the heterologous nucleic acid comprises the sequence set forth from about nucleotide number 8877 to about 9353 of SEQ ID NO: 19 or 22. In other embodiments of any of the above, the 3 ′ LTR is derived from a gamma retrovirus. In other embodiments of any of the above, the 3 ′ LTR comprises a U3-R-U5 domain. In any other of the above embodiments, the 3 ′ LTR is a sequence set forth from about nucleotide 9405 to about 9988 of SEQ ID NO: 19 or 22, or a sequence that is at least 95%, 98%, or 99.5% identical thereto including. In other embodiments of any of the above, the heterologous nucleic acid sequence encodes a biological response modifier, or an immunostimulatory cytokine. In any other embodiment of the above, the immunostimulatory cytokine is selected from the group consisting of interleukins 1-15, interferon, tumor necrosis factor (TNF), and granulocyte macrophage colony stimulating factor (GM-CSF). . In any other embodiment of the above, the immunostimulatory cytokine is interferon gamma. In other embodiments of any of the above, the heterologous nucleic acid encodes a polypeptide that converts a non-toxic prodrug to a toxic agent. In any other embodiment of the above, the polypeptide that converts a non-toxic prodrug to a toxic drug is thymidine kinase, purine nucleoside phosphorylase (PNP), or cytosine deaminase. In other embodiments of any of the above, the heterologous nucleic acid sequence encodes a receptor domain, antibody, or antibody fragment. In other embodiments of any of the above, the heterologous nucleic acid sequence comprises an inhibitory polynucleotide. In any other embodiment of the above, the inhibitory polynucleotide comprises a miRNA, RNAi, or siRNA sequence.

  The details of one or more embodiments of the disclosure are set forth in the accompanying drawings and the detailed description below. Other features, objects, and advantages will be apparent from the detailed description, the drawings, and the claims.

FIGS. 1A-C show replicating retroviral vectors containing IRES with various numbers of A's in A bulge and their titers. (A) Predicted secondary structure of ECMV internal ribosome entry site. The sequence begins at position 680. Circled capital letters J, K, L, and M indicate a predetermined region in the IRES. Arrows indicate branch loops in the JK region. AUG8, AUG9, AUG10 and AUG11 are underlined. (B) Schematic diagram of A bulge in JK branch in ECMV IRES incorporated into RRV expressing yCD2 or GFP. Native ATG8 (AUG for RNA) and ATG9 are underlined; the enlarged and underlined sequence indicates the A bulge in the JK branch region; (C) Viral titer of RRV containing various numbers of A in A bulges produced by infected HT1080 cells. Figures 2A-D show protein expression by cell virus-derived RNA and RRV with various numbers of A's in the A bulge. (A) Schematic representation of cell virus RNA isoform. Env2 primer and probe, and yCD2 primer and probe recognize both unspliced and spliced RNA in the env and yCD2 regions, respectively, and are used to measure cellular viral RNA levels by qRT-PCR It was. Black triangle: env2 primer and probe set; White triangle: yCD2 primer and probe set. (B) Immunoblot of yCD2 and GAPDH proteins. Twenty micrograms of cell lysate was applied to each lane, and equal amounts of the test and blotting efficiency were controlled by detecting GAPDH, a ubiquitous marker. PC, positive control; NC, negative control. The graph shows RNA and protein expression levels for the yCD2-6A vector. (C) RNA and GFP expression levels for GFP-6A vector. The percentage of GFP positive cells was determined by flow cytometry using appropriate gating excluding GFP negative cells. GFP protein expression levels were quantified by using average fluorescence intensity. Continuation of Figures 2A-C. (D) Proviral vector copy number of infected U87-MG cells (0.01 MOI) by qPCR. Genomic DNA is isolated 14 days after infection when vectors with 7 A are predicted to be maximally infected. The data show that there is no significant difference in the vector copy number of the maximally infected U87-MG cells. This is consistent with virus production data where there is no significant effect on virus titer among mutants. FIG. 3 shows the vector sequence (SEQ ID NO: 22) with an A bulge containing 7A's underlined and bolded. This is a continuation of Figure 3-1. It is a continuation of Figure 3-2. This is a continuation of Figure 3-3. Figures 4A-B show virus stability data. (A) Virus stability in infected U87-MG cells (0.01 MOI) by endpoint PCR. Genomic DNA is isolated 14 days after infection and the IRES-yCD2 region is amplified using a primer set that spans the 3 ′ and 3 ′ UTR regions of env (Perez et al., 2012). (B) Evaluation of vector stability due to stage infection. Approximately 10 ⁶ naive U87-MG cells were first infected with the viral vector at a MOI of 0.1 and grown for 1 week to complete a single infection cycle. 100 μL of 2 ml of viral supernatant from fully infected cells was used to infect naïve cells and repeated up to 12 cycles. The stability of the IRES-yCD2 region vector is assessed by PCR amplification of the integrated provirus from infected cells. The size of the predicted PCR product is approximately 1.2 kb. The appearance of any band shorter than 1.2 kb indicates a deletion in the IRES-yCD2 region. FIG. 5 shows a schematic diagram of a designed construct of the present disclosure having minimal IRES (bases 123 to 139 of SEQ ID NO: 41; and 183 to 198).

  As used in this specification and the appended claims, the singular forms include the plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells, reference to “agent” includes reference to one or more agents known to those of skill in the art, and so forth.

  Also, the use of “or” or “or” means “and / or” unless stated otherwise. Similarly, “including”, “including”, and variations thereof, and the like are interchangeable and are not intended to be limiting.

  Where the term “comprising” is used in the description of various embodiments, in some specific cases, certain embodiments may instead be referred to as “consisting essentially of” or “consisting of” It should be further understood that one skilled in the art understands that can be described using.

  Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, representative methods, devices, and materials are described herein.

  General textbooks describing molecular biological techniques useful in the present invention, including the use of vectors, promoters and many other related topics, include Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology Volume 152, (Academic Press, Inc., San Diego, Calif.) ("Berger"); Sambrook et al., Molecular Cloning--A Laboratory Manual, 2d ed., Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY , 1989 ("Sambrook") and Current Protocols in Molecular Biology, FM Ausubel et al., Eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 1999) (" Ausubel "). For example, in vitro amplification, including polymerase chain reaction (PCR), ligase chain reaction (LCR), Qβ replicase amplification and other RNA polymerase mediated techniques (e.g., NASBA) for the production of homologous nucleic acids of the present disclosure Examples of protocols sufficient to guide one of ordinary skill in the art include Berger, Sambrook, and Ausubel, and Mullis et al. (1987) US Pat.No. 4,683,202; Innis et al., Eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press Inc. San Diego, Calif.) ("Innis"); Arnheim & Levinson (Oct. 1, 1990) C & EN 36-47; The Journal Of NIH Research (1991) 3: 81-94; Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173; Guatelli et al. (1990) Proc. Nat'l. Acad. Sci. USA 87: 1874; Lomell et al. (1989) J. Clin. Chem 35 : 1826; Landegren et al. (1988) Science 241: 1077-1080; Van Brunt (1990) Biotechnology 8: 291-294; Wu and Wallace (1989) Gene 4: 560; Barringer et al. (1990) Gene 89: 117; And Sooknanan and Malek (1995) Biotechnology 13: 563- Seen at 564. An improved method for cloning nucleic acids amplified in vitro is described in Wallace et al., U.S. Pat. No. 5,426,039. Improved methods for amplifying large nucleic acids are summarized in Cheng et al. (1994) Nature 369: 684-685 and references cited therein, which generate PCR amplification products spanning 40 kb. Has been. It will be appreciated by those skilled in the art that virtually any RNA can be converted to double stranded DNA suitable for restriction enzyme digestion, PCR extension and sequencing using reverse transcriptase and polymerase. See, for example, Ausubel, Sambrook and Berger (all above).

  Publications discussed throughout the text are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior disclosure.

  The present disclosure provides methods and compositions useful for the delivery of genes or proteins to cells or subjects. Such methods and compositions can be used to treat a variety of disorders and diseases, including cancer and other cell proliferative diseases and disorders. In one aspect, the present disclosure provides an optimized IRES. Such optimized IRES can be used in a variety of vectors to facilitate protein expression. In other embodiments, the present disclosure provides replication competent retroviral vectors for gene delivery. The present disclosure shows that IRES containing 7A's in commonly used JK-branched A-bulges is not optimal, and therefore the present disclosure has less (3-5) or more (7-8) A An IRES having an optimal A bulge sequence with improved polypeptide expression relative to an IRES having:

  An internal ribosome entry site ("IRES") refers to a region of a nucleic acid that facilitates ribosome entry or retention during translation of the coding sequence, usually 3 'to the IRES. In some embodiments, the IRES may include a splicing acceptor / donor site, but a preferred IRES lacks a splicing acceptor / donor site. Normally, entry of ribosomal messenger RNA occurs through a cap located at the 5 'end of all eukaryotic mRNAs. However, there are exceptions to this general rule. The absence of caps in some viral mRNAs suggests the existence of alternative structures that allow ribosome entry at the internal site of these RNAs. To date, many of these structures, referred to as IRES because of their function, have been found in the 5 ′ non-coding region of uncapped viral mRNAs, such as poliovirus (Pelletier et al., 1988, Mol. Cell. Biol., 8, 1103-1112) and EMCV viruses (encephalomyocarditis virus (Jang et al., J. Virol., 1988, 62, 2636-2643)) have been identified. The present disclosure provides for the use of optimized IRES in the context of vectors, and more specifically replication competent retrovirus (RCR) vectors.

  An internal ribosome entry site (IRES) allows translation of viral RNA in a CAP-independent manner. IRES from encephalomyocarditis virus (EMCV) has been extensively studied and is widely used in retroviruses and other mammalian expression vectors. Appropriate folding and secondary structure of the IRES defines its functionality, and sequence changes may or may not affect this. Palmenberg and coworkers have shown that the J-K element at the 3 'end of the IRES plays an important role in the initiation of translation, independent of the 5'-IRES region (Figure 1A). The sequence of IRES in various vectors can be found to contain various numbers of poly A in A-bulges. For example, Logg et al. (J. Virol. 75: 6989-6998, 2001) has an IRES with 7 A instead of 6 adenosine residues (A, As) in the A bulge of the molecular region. (See, eg, Duke et al., J. Virol. 66: 1924-1932, 1992). As described more fully elsewhere herein, the number of A's in A-bulge affects the expression of heterologous sequences that are operably associated. For example, the present disclosure identifies an optimal number of A's for A-bulges that peak at 6A's and have slightly reduced expression on either side from the optimal number of A's. For example, 4A's is less effective than 6A's, and 8A's is less effective than 6A's.

  As used herein, “optimized IRES” refers to an IRES derived from encephalomyocarditis virus having 6 A in the A-bulge of the JK branch region. An optimized IRES can be part of a cassette that contains the gene or sequence to be expressed ("heterologous polynucleotide" or "gene"). In such cases, the optimized IRES is operably linked upstream of the heterologous polynucleotide and can function to cause translation of the linked heterologous polynucleotide. Optimized IRES cassettes show increased protein expression from ligated heterologous polynucleotides compared to non-optimized IRES (eg, IRES with 3-5 or 7-8 A's in the A-bulge). The optimized IRES or IRES cassette can be cloned into any number of vectors for expression of the linked heterologous polynucleotide. For example, vectors of the present disclosure that can include an optimized IRES or IRES cassette and that can be used with the optimized IRES or IRES cassette include plasmids, expression vectors, viral vectors (replication deletion and replication competent), and the like.

  In one embodiment, the disclosure provides: (i) a sequence having 95% identity to SEQ ID NO: 41 and having 6A's in the JK branch region; (ii) a sequence set forth in SEQ ID NO: 41 comprising 6A's in the branch region Truncate with improved polypeptide expression against a similar IRES having 7 A in the branch region, starting at any point after base pair 1 to about base 183 of SEQ ID NO: 41 and continuing to 544 An IRES (eg, about 123-544 or 183-544 of SEQ ID NO: 41); or (iii) the sequence set forth in SEQ ID NO: 41, and (iv) An optimized IRES comprising a sequence selected from the group consisting of:

  The heterologous nucleic acid sequence is operably linked to an optimized IRES consisting of six “A” s in the A-bulge region in one embodiment. As used herein, the term “heterologous” nucleic acid sequence or transgene includes (i) a sequence that is not normally present in a wild-type retrovirus, (ii) a sequence that is derived from a heterologous species, and (iii) is not normally found downstream of an IRES. Sequence, or (iv) when derived from the same species refers to something that can be substantially modified from its original form. Alternatively, an unaltered nucleic acid sequence that is not normally expressed in a cell is a heterologous nucleic acid sequence.

  In one embodiment, the present disclosure provides a vector comprising an optimized IRES in a cassette comprising an A bulge of 6 A in the JK branch region operably linked to the polynucleotide sequence to be expressed. As described in more detail below, A-bulges consisting of 6A's surprisingly provide superior protein expression compared to similar IRES cassettes containing 3-5 or 7-8 A's . As will be appreciated, particularly in gene delivery, protein expression from recombinant vectors is important not only for in vitro protein production, but also for production of therapeutic proteins in vitro. For example, Logg et al. (J. Virol. 75: 6989-6998, 2001) includes an IRES that has 7 adenosine residues (A, As) instead of 6 A's in the A bulge of the JK branch region. Is described.

  The optimized IRES cassette can be cloned into any number of art-recognized vectors. Such vectors are described below and include plasmids and viral vectors. For example, the present disclosure contemplates an optimized IRES of the present disclosure that has been cloned into an expression vector and located immediately upstream (eg, 0 to about 50 bp upstream) of the heterologous polynucleotide to be expressed. Of particular interest is the use of replication competent gamma retroviral vectors that can infect and propagate mammalian tissue without the need for recombinant receptors or helper cells. Examples of such RCR vectors include mo-MLV, MLV, GALV, and FELV. A typical gamma retrovirus contains the LTR, gag, pol, and env genes and factors necessary for reverse transcription and integration in the host genome (eg, psi factors). Modifications of typical gamma retroviral vectors have been carried out for about 20 years, including the production of replication incompetent vectors, vectors with heterologous genes at various locations, and vectors containing IRES cassettes. For example, US Pat. No. 6,410,313 to Kasahara et al. Describes the production of an MLV-derived replicating retroviral vector having an IRES cassette downstream of the env gene and upstream of the 3 ′ LTR. Gruber et al. (US Pat. No. 8,722,867) describes further optimized vectors that contain an IRES cassette immediately downstream of the env gene and upstream of the 3 ′ LTR. In Gruber et al., The IRES cassette displays 7 A A-bulges in the JK branch region.

  The disclosure provides, in one embodiment, a replication competent gamma retroviral vector comprising an optimized IRES cassette immediately downstream of the env gene and upstream of the 3 ′ LTR, wherein the optimized IRES of the optimized IRES cassette is 6 It consists of A-bulges in the branch region of A pieces. In a further embodiment, the RCR has increased protein expression relative to an A-bulge containing vector having 3-5 or 7-8 A's.

  The present disclosure describes conventional replication competent retroviral vectors (eg, US Patent Publication Nos. 2011 / 0287020-A1; and 2011 / 0217267-A1, which show 7A's in the A-bulge, incorporated herein by reference; Provided are vectors with an A-bulge consisting of 6A's in the JK branch region compared to those found in (see). Surprisingly, a single A change (ie, 7A's to 6A's) results in increased protein production compared to that of 7A's. Thus, vectors containing 6A's have improved protein expression of a heterologous gene linked to an IRES cassette with "6 A" A-bulges.

  The terms “vector”, “vector construct” and “expression vector” refer to the introduction of a DNA or RNA sequence (eg, a foreign gene) into a host cell, thereby transforming the host and expressing (eg, transcription and translation) of the introduced sequence. It means a medium (vehicle) that can be promoted. Vectors typically contain transmissible mediator DNA or RNA into which heterologous DNA or RNA encoding the protein is inserted by restriction enzyme technology. A typical example of a vector is a “plasmid”, which is generally a self (built-in) double-stranded DNA molecule that can easily accept additional (heterologous) DNA and is easily introduced into a suitable host cell. can do. A number of vectors have been described, including plasmids and fungal vectors for replication and / or expression in a variety of prokaryotic and eukaryotic hosts. As non-limiting examples, pKK plasmids (Clonetech), pUC plasmids, pET plasmids (Novagen, Inc., Madison, Wis. ), PRSET or pREP plasmid (Invitrogen, San Diego, Calif.), Or pMAL plasmid (New England Biolabs, Beverly, Mass.) And many suitable host cells. A recombinant cloning vector will often contain one or more replication systems for cloning or expression, one or more markers for selection in the host, eg, antibiotics, and one or more expression cassettes.

  The terms “express” and “expression” mean to allow or cause the manifestation of information of a gene or DNA sequence, for example the activity of cellular functions involved in the transcription and translation of the corresponding gene, RNA or DNA sequence It means the production of protein by chemicalization. A DNA or RNA sequence is expressed in or by a cell to form an “expression product” such as a protein. For example, the resulting expression product, such as the resulting protein, may itself be “expressed” by the cell. A polynucleotide or polypeptide is expressed recombinantly, for example, in a heterologous host cell under the control of a heterologous or native promoter, or when expressed or produced in a natural host cell under the control of a heterologous promoter. Is done.

  The present disclosure provides modified retroviral vectors. The modified retroviral vector may be derived from a member of the Retroviridae family. Retroviridae consists of three groups: spumaviruses (or foamy virus) such as human foamy virus (HFV); lentivirus and sheep visnavirus; and oncovirus (included in this group) Not all viruses are carcinogenic). The term “lentivirus” is conventionally used to refer to the genus of viruses having reverse transcriptase. Lentiviruses include “immunodeficiency viruses” including human immunodeficiency virus (HIV) type 1 and type 2 (HIV-1 and HIV-2) and simian immunodeficiency virus (SIV). Oncoviruses have been historically subdivided into groups A, B, C and D, based on the morphology of the particles found in the electron microscope during virus maturation. Type A particles represent immature particles of type B and D viruses found in the cytoplasm of infected cells. These particles are not infectious. B-type particles bud from the plasma membrane as mature virions by enveloping A-type particles in the cytoplasm. In this membrane, the virus has a donut-shaped 75 nm nucleus from which a long glycoprotein spike protrudes. After budding, type B particles have high electron density nuclei located eccentrically. The prototype for type B virus is mouse mammary tumor virus (MMTV). In cells infected with type C virus, no particles are found in the cytoplasm. Instead, mature particles bud directly from the cell surface through a crescent “C” shaped concentrate, which then closes itself and is enveloped by the plasma membrane. Spikes of envelope glycoproteins are seen along with uniformly high electron density nuclei. Budding can occur from the surface plasma membrane or directly into intracellular vacuoles. Type C viruses are the most studied and include many of the avian and murine leukemia viruses (MLV). Bovine leukemia virus (BLV) and human T cell leukemia virus types I and II (HTLV-I / II) are similarly classified as type C particles based on the morphology of budding from the cell surface. However, they also have a regular hexagonal form and a more complex genomic structure than prototype C viruses such as murine leukemia virus (MLV). D-type particles are similar to B-type particles in that they exhibit a ring-like structure in the cytoplasm of infected cells, budding from the cell surface, while virions take up a short surface glycoprotein spike. Moreover, the nucleus of high electron density is eccentrically located in the particle. Mason Pfizer monkey virus (MPMV) is a prototype of a D-type virus.

  Retroviruses can be classified in various ways, but the nomenclature has been standardized over the past 10 years (ICTVdB-The Universal Virus Database, v 4 on the World Wide Web (www) at ncbi.nlm.nih.gov/ICTVdb/ ICTVdB / and textbook “Retroviruses” See Eds Coffin, Hughs and Varmus, Cold Spring Harbor Press 1997; reference is made to the disclosure thereof and incorporated herein. In one embodiment, the replication competent retroviral vector may comprise an orthoretrovirus or more typically a gamma retroviral vector.

  Retroviruses are defined by a method that replicates their genetic material. During replication, RNA is converted to DNA. Following infection of the cell, double-stranded DNA molecules are made from a bimolecular RNA molecule in the viral particle by a molecular process known as reverse transcription. The DNA is covalently integrated into the host cell genome as a provirus, from which viral RNA is expressed using cellular and / or viral factors. The expressed viral RNA is packaged in particles and released as infectious virions.

  Retroviral particles are composed of two identical RNA molecules. Each wild-type genome has a positive sense that is a single-stranded RNA molecule that is capped at the 5 'end and polyadenylated at the 3' end. The diploid viral particle contains two RNA strands complexed with the host tRNA molecule within the gag protein, the viral enzyme (the product of the pol gene) and the “nuclear” structure of the gag protein. Surrounding and protecting this capsid is a lipid bilayer derived from the host cell membrane and containing the viral envelope (env) protein. The env protein binds to a cell-side receptor for the virus, and typically the particles enter the host cell by receptor-mediated endocytosis and / or membrane fusion.

  After the outer envelope is lost, the viral RNA is copied into the DNA by reverse transcription. This is catalyzed by the reverse transcriptase encoded in the pol region and uses host cell tRNA packaged in virions as primers for DNA synthesis. In this way, the RNA genome is converted into a more complex DNA genome.

  Double-stranded linear DNA produced by reverse transcription may have to be circularized in the nucleus. The provirus has two identical repeats known as long terminal repeats (LTR) at both ends. The ends of these two LTR sequences form a site that is recognized by the pol product that catalyzes the integration-the integrase protein-so that the provirus always enters the host DNA with two base pairs from the end of the LTR. Cell sequence replication is observed at the ends of both LTRs, reminiscent of the pattern of integration of transposable genetic elements. Retroviruses can integrate their DNA into many places in the host DNA, but different retroviruses have different integration site preferences. HIV-1 and simian immunodeficiency virus DNA are preferentially integrated into the expressed gene, mouse leukemia virus (MLV) DNA is preferentially integrated near the transcription start site (TSS), and avian sarcoma leukemia virus (ASLV) and human T-cell leukemia virus (HTLV) DNA are almost randomly integrated and gene preference is minimal (Derse D et al. (2007) Human T-cell leukemia virus type 1 integration target sites in the human 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).

  Transcription, RNA splicing and translation of integrated viral DNA is mediated by host cell proteins. Various spliced transcripts are produced. In the case of human retroviruses HIV-1 / 2 and HTLV-I / II, viral proteins are also used to regulate gene expression. The interaction between cellular and viral factors is a factor in controlling the viral latency and the temporal order in which viral genes are expressed.

  Retroviruses can be transmitted both horizontally and vertically. Efficient infectious transmission of retroviruses requires expression in the target cell of a receptor that specifically recognizes the viral envelope protein, but the virus is receptor-independent, nonspecific, and less efficient An approach route may be used. Usually viral infection results in one or a few viral genome copies per cell for receptor masking or suppression of expression resulting in resistance to superinfection (Ch3 p104 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). Furthermore, the target cell type must be able to support all stages of the replication cycle after the virus binds and enters. Vertical transmission occurs when the viral genome is integrated into the host germline. Provirus is transmitted from generation to generation as if it were a cellular gene. Thus, an endogenous provirus is established that is often latent but activated when the host is exposed to the appropriate agent.

  In many situations where the recombinant replicating competent retrovirus is used therapeutically, it is advantageous to have a high level of expression of the transgene encoded by the recombinant replicating competent retrovirus. For example, using a gene such as the cytosine deaminase gene that activates the prodrug, a higher level of CD protein can be expressed in the cell so that the prodrug 5-FC can be efficiently converted by 5-FU. It is advantageous to do. Similarly, high level expression of siRNA or shRNA results in more efficient suppression of target gene expression. For cytokines or single chain antibodies (scAbs), it is usually advantageous to express cytokines or scAbs at high levels. In addition, if there are several copies of mutations that inactivate or impair the activity of the vector or transgene, multiple copies of the vector in the target cell will result in efficient expression of the intact transgene. It is advantageous to have the multiple copies to get high probability. The present disclosure provides a recombinant replication competent retrovirus that infects a target cell or population of target cells multiple times, resulting in an average copy number of 5 or more on average for a diploid genome. The present disclosure also provides a method for testing this property. Also provided is a method of treating a cell proliferative disorder using a recombinant replication competent retrovirus that infects a target cell or population of target cells multiple times and produces an average copy number of 5 or greater for a diploid genome.

  As mentioned above, the integrated DNA intermediate is called a provirus. In previous gene therapy or gene delivery systems, provirus transcription and infection in the presence of a suitable helper virus or in a cell line containing the appropriate sequence that allows encapsidation without co-production of contaminating helper virus. Methods and retroviruses that require assembly into sex viruses are used. As described below, helper viruses are not required for production of the disclosed recombinant retroviruses. This is because the sequences necessary for encapsidation are provided in the genome, thus obtaining a replication competent retroviral vector for gene delivery and therapy.

  Other existing replication competent retroviral vectors are also unstable and tend to lose sequence upon horizontal and vertical propagation to infected or host cells and upon replication. This can be due in part to the presence of extra nucleotide sequences that contain repetitive sequences or reduce the efficiency of the polymerase.

  The retroviral genome and proviral DNA in this disclosure has at least three genes: gag, pol, and env, which genes are flanked by one or two long terminal repeats (LTR) or provirus Is adjacent to a sequence containing two long terminal repeats (LTR) and a cis-acting sequence such as psi. The gag gene encodes a protein with an internal structure (substrate, capsid and nucleocapsid); the pol gene encodes an RNA-dependent DNA polymerase (reverse transcriptase), protease and integrase; the env gene is the viral envelope It encodes a glycoprotein. The 5 ′ and / or 3 ′ LTR serves to promote transcription and polyadenylation of virion RNA. 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 essential for reverse transcription of the genome (tRNA primer binding site) and sequences necessary for efficient encapsidation into viral RNA particles (Psi site). When sequences essential for encapsidation (or packaging of retroviral RNA into infectious virions) are deleted from the viral genome, this results in cis deficiency and prevents encapsidation of genomic viral RNA. This type of engineered vector is a “helper” that provides viral proteins that package RNA genomes that can be packaged but cannot replicate in traditional gene delivery systems (i.e., systems lacking elements necessary for virion encapsidation) in trans. It has been commonly used as an element.

  The present disclosure provides a vector comprising an optimized IRS. The optimized IRES is typically linked to a heterologous polynucleotide, eg, cytosine deaminase or variant thereof, thymidine kinase or variant thereof, miRNA or siRNA, cytokine, antibody binding domain, etc. that can be delivered to a cell or subject. In one embodiment, the vector is a viral vector. Virus vectors include adenovirus vectors, measles virus vectors, herpes virus vectors, retrovirus vectors (including lentivirus vectors), vesicular stomatitis virus vectors and other rhabdovirus vectors, reovirus vectors, Seneca Valley virus vectors, poxvirus vectors. (Including vectors derived from animal poxillus or vaccinia), parvovirus vectors (including AAV vectors), alphavirus vectors or other viral vectors known to those skilled in the art (eg, Concepts in Genetic Medicine, ed. Boro Dropulic and Barrie Carter, Wiley, 2008, Hoboken, NJ .; The Development of Human Gene Therapy, ed. Theodore Friedmann, Cold Springs Harbor Laboratory Press, Cold springs Harbor, New York, 1999; Gene and Cell Therapy, ed Nancy Smyth Templeton, Marcel Dekker I nc., New York, New York, 2000 and Gene Therapy: Therapeutic Mechanism and Strategies, ed.Nancy Smyth Templetone and Danilo D Lasic, Marcel Dekker, Inc., New York, New York, 2004; Gene and Cell Therapy: Therapeutic Mechanisms See also, and Strategies, Third Edition, ed. Nancy Smyth Templeton, CRC Press, 2008; the disclosures of which are incorporated herein by reference).

  In one embodiment, the retroviral genome of the present disclosure comprises an optimized IRES that includes a cloning site for insertion of a desired / heterologous polynucleotide downstream of the optimized IRES. In one embodiment, the optimized IRES is 3 'to the env gene of the retroviral vector, but is located 5' to the desired heterologous polynucleotide and 5 'to the 3' LTR. In all of the above embodiments, the optimized IRES includes A-bulges with 6A's. A heterologous polynucleotide encoding a desired polypeptide can be operably linked to an optimized IRES.

  In one embodiment, the viral vector is derived from or derived from a gamma retrovirus capable of infecting replicating mammalian cells. The replication competent retroviral vector is located on the 5 ′ side of a heterologous polynucleotide encoding, for example, cytosine deaminase (SEQ ID NO: 3), thymidine kinase (SEQ ID NO: 37), miRNA, siRNA, cytokine, receptor, or antibody. It contains an optimized internal ribosome entry site (IRES) containing an A-bulge consisting of 6A's. If the heterologous polynucleotide encodes an untranslated RNA such as siRNA, miRNA or RNAi, an IRES can be included, but not necessarily, for other translated polynucleotides. In one embodiment, the optimized IRES cassette comprising the heterologous polynucleotide is 3 ′ to the ENV polynucleotide of the retroviral vector, but is 5 ′ to the 3 ′ LTR. In one embodiment, the viral vector is a retroviral vector that can infect target cells multiple times (eg, 5 or more diploid cells).

  The present disclosure provides replicative competent retroviral vectors with increased stability compared to previous and retroviral vectors containing optimized IRES with 6A's in the A-bulge. Such increased stability during infection and replication is important for the treatment of cell proliferative disorders. Furthermore, increased protein expression from optimized A-bulges results in further delivery of therapeutic proteins to target cells / tissues. A combination of transduction efficiency, transgene stability, transgene expression and target selectivity is provided by replication competent retroviruses. The present compositions and methods provide insertion stability and maintain the transcriptional activity of the transgene and the transcriptional effectiveness of the encoded polypeptide.

  Any number of heterologous polynucleotide or nucleic acid sequences can be inserted into the vector or retroviral vector, depending on the intended use of the vector or retroviral vector of the present disclosure. For example, commonly used marker or reporter genes, including antibiotic resistance and fluorescent molecules (eg, GFP) can be used for in vitro studies. Additional polynucleotide sequences encoding any desired polynucleotide sequence can also be inserted into the vectors of the present disclosure. For purposes of in vivo delivery of heterologous nucleic acid sequences, both therapeutic and non-therapeutic sequences can be used. For example, the heterologous sequence may encode a therapeutic molecule including an antisense molecule (miRNA, siRNA), or a ribozyme for a particular gene associated with a cell proliferative disorder or other gene-related disease or disorder; (Eg, HSV-tk or PNP or cytosine deaminase; whether modified or unmodified, humanized or non-humanized), growth factor or therapeutic protein (eg Factor IX, IL2 etc.). Other therapeutic proteins that can be utilized in the present disclosure are readily identified in the art. Other therapeutic proteins that can be applied to the present disclosure are readily identified in the art.

  In one embodiment, the heterologous polynucleotide in the vector comprises cytosine deaminase optimized for expression in human cells. In further embodiments, the cytosine deaminase comprises a sequence optimized for human codons and also increases the stability of cytosine deaminase (eg, reduced degradation or increased thermal stability) compared to wild-type cytosine deaminase. ) Mutations (see, eg, SEQ ID NO: 4). In another embodiment, the heterologous polynucleotide is a cytosine deaminase (either human codon optimized or non-optimized, mutated or non-optimized) operably linked to a polynucleotide encoding a polypeptide having UPRT or OPRT activity. Encodes a fusion construct comprising any of the variants) (see, eg, SEQ ID NOS: 11, 13, 15 and 17). Examples of such polypeptides having cytosine deaminase and polynucleotides encoding the polypeptides can be found in International Publication No. WO2010 / 045002, the contents of which are incorporated herein by reference.

  In other embodiments, the vector or replication competent retroviral vector may comprise a heterologous polynucleotide encoding a polypeptide comprising cytosine deaminase (as described herein), and further primary transcription of a viral promoter. It may further comprise a polynucleotide comprising a miRNA or siRNA molecule linked to a promoter that may be part of the product or may be cell type or tissue specific.

  In yet another embodiment, the heterologous polynucleotide may comprise an interleukin or a cytokine such as interferon gamma. Cytokines that can be expressed from the retroviral vectors of the present disclosure include, but are not limited to, IL-1α, IL-1β, 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-γ (human-SEQ ID NO: 38; mouse-SEQ ID NO: 39) and TNF-α, soluble TNF-α, lymphotoxin- α (also known as LT-α, TNF-β), LT-β (found as a complex heterotrimer of LT-α2-β), OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-γ (International Publication No. WO 96/14328), AIM-I (International Publication No. WO 97/33899), Endokine α (International Publication No. WO 98/07880), OPG, and Neutro Cain α (International Publication No.WO 98/18921), OX40, and Nerve Growth Factor (NGF), Soluble Fas, CD30, CD27, CD40 and 4-IBB, TR2 (International Publication No.WO 96/34095), DR3 ( International publication number WO 97/33904), DR4 (International Publication No.WO 98/32856), TR5 (International Publication No.WO 98/30693), TRANK, TR9 (International Publication No.WO 98/56892), TR10 (International Publication No.WO) 98/54202), 312C2 (WO 98/06842), and TR12, and soluble CD154, CD70, and CD153. In some embodiments, angiogenic proteins may be useful, particularly for protein production from cell lines. Such angiogenic factors include, but are not limited to, glioma-derived growth factor (GDGF), platelet-derived growth factor-A (PDGF-A), platelet-derived growth factor-B (PDGF-B), placenta Growth factor (PIGF), placental growth factor 2 (PIGF-2), vascular endothelial growth factor (VEGF), vascular endothelial growth factor A (VEGF-A), vascular endothelial growth factor 2 (VEGF-2), vascular endothelial growth factor There are B (VEGF-3), vascular endothelial growth factor B-186 (VEGF-B186), vascular endothelial growth factor D (VEGF-D), and vascular endothelial growth factor E (VEGF-E). Fibroblast growth factor may be delivered by the vectors of the present disclosure, including but 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 the vectors of the present disclosure, including but not limited to granulocyte macrophage colony stimulating factor (GM-CSF) (sargramostim), granule Sphere colony stimulating factor (G-CSF) (filgrastim), macrophage colony stimulating factor (M-CSF, CSF-1), erythropoietin (epoetin alfa), stem cell factor (SCF, c-kit ligand, hematopoietic stem cell factor) ), Megakaryocyte colony-stimulating factor, PIXY321 (a GMCSF / IL-3) fusion protein and the like.

MicroRNA (miRNA) is a small non-coding RNA. They are located in introns of coding or non-coding genes, exons of non-coding genes, or intergenic regions. The miRNA gene is transcribed by RNA polymerase II, which produces a precursor polynucleotide called early precursor miRNA (pri-miRNA). In the nucleus, pri-miRNAs are processed by the ribonuclease Drosha to produce miRNA precursors (pre-miRNAs) that form short hairpin structures. Subsequently, pre-miRNA moves to the cytoplasm via Exportin 5 and is further processed by another ribonuclease called Dicer to produce active mature miRNA.

  The mature miRNA is approximately 21 nucleotides in length. It exerts its function by binding to the 3 ′ untranslated region of the mRNA of the target gene and suppressing protein expression by either inhibiting protein translation or degrading mRNA. miRNAs are involved in biological processes including development, cell proliferation, differentiation, and cancer progression. Studies of miRNA profiling have shown that the expression of some miRNAs is tissue specific or abundant in some tissues. For example, expression of miR-142-3p, miR-181 and miR-223 has been shown to be abundant in human and mouse hematopoietic tissues (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 sequence of miR-142-3p4X is shown in SEQ ID NO: 36.

  Some miRNAs have been observed to be up-regulated (oncogenic miRNA) or down-regulated (repressor) in some tumors (Spizzo et al., 2009 Cell 137, 586e1). For example, miR-21 is overexpressed in glioblastoma, breast cancer, lung cancer, prostate cancer, colon cancer, gastric cancer, esophageal cancer, and cervical cancer, uterine leiomyosarcoma, DLBCL, head and neck cancer. In contrast, let-7 members have been reported to be down-regulated in glioblastoma, lung cancer, breast cancer, gastric cancer, ovarian cancer, prostate cancer, and colon cancer. Reestablishing homeostasis of miRNA expression in cancer is an inevitable mechanism for inhibiting or reversing cancer progression.

  In cancer, antisense-mediated inhibition (antigomers) of oncogenic miRNAs has attracted attention in the development of potential therapeutic approaches as a result of vital functions being modified by miRNAs. However, miRNA replacement may represent an equally effective strategy. In this approach, the most therapeutically useful miRNAs are those that are expressed at low levels in tumors, but are usually expressed at high levels in tissues and are therefore acceptable.

  MiRNAs that are down-regulated in cancer may be useful as anticancer agents. Examples include mir-128-1 / 2 (SEQ ID NOs: 31 and 32, respectively), let-7, miR-26, miR-124, and miR-137 (Esquela-Kerscher et al., 2008 Cell Cycle 7, 759- 764; Kumar et al., 2008 Proc Natl Acad Sci USA 105, 3903-3908; Kota et al., 2009 Cell 137, 1005-1017; Silber et al., 2008 BMC Medicine 6:14 1-17). miR-128 expression has been reported to be strong in the central nervous system and has been shown to be down-regulated in glioblastoma (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 different genes, miR-128-1 and miR-128-2. Both are processed to the same mature sequence. Bmi-1 and E2F3a have been reported to be 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 ). In addition, Bmi-1 expression is upregulated in various human cancers including glioma, mantle cell lymphoma, non-small cell lung cancer, B-cell non-Hodgkin lymphoma, breast cancer, colorectal cancer and prostate cancer It is recognized that Furthermore, Bmi-1 has been demonstrated to be required for the self-renewal of stem cells from different tissues, including neural stem cells, and “stem cell-like” cell populations in glioma.

  There are many in vitro demonstrations of the potential for miRNA-mediated inhibition of cellular functions, but as is true for other molecules, these can be used as oligonucleotides or as viral vectors for in vivo effects. It is difficult to deliver as efficiently as necessary (eg Li et al., Cell Cycle 5: 2103-2109 2006).

  Replication deficient retroviruses and lentiviral vectors were used to stably express pri-miRNA by polymerase II promoters such as CMV or LTR and showed production of mature miRNA. In replication-defective retrovirus and lentiviral vectors, the introduction of type III RNA polymerase III promoters such as U6 and H1 promoters express functional small interfering RNA (siRNA) that produces short hairpin RNA. (Bromberg-White et al., 2004 J Virol 78: 9, 4914-4916; Sliva et al., 2006 Virology 351, 218-225; Haga et al., 2006, Transplant Proc 38 (10 ): 3184-8). The loop sequence is cleaved by Dicer to produce a mature siRNA 21-22 nucleotides long. shRNA can be stably expressed to down-regulate target gene expression. SEQ ID NOs: 33 and 34 contain pre-miR-128 linked to the H1 promoter.

  In other embodiments, an optimized IRES comprising 6A's in the A bulge can be used in combination with a core promoter, wherein the optimized IRES is operably linked to a first heterologous coding sequence and is a core promoter or mini-promoter Is linked to a second heterologous coding sequence or siRNA, miRNA, or shRNA sequence (see, eg, WO 2014/066700 incorporated by reference).

  As used herein, “core promoter” refers to the smallest promoter comprising about 50-100 bp and lacking an enhancer element. Such core promoters include, but are not limited to, SCP1, AdML and CMV core promoters. More specifically, when a core promoter cassette is present, there is a second cassette (eg, a second mini-promoter cassette, polIII promoter cassette or IRES cassette). In some embodiments, a vector comprising a cassette with a core promoter specifically excludes the use of SCP1, AdML and CMV core promoters, and instead, the designers described further herein and below. Use the core promoter.

  A specific viral promoter is mentioned as a core promoter. A viral promoter, as used herein, is a promoter that has a core sequence and usually also has several other auxiliary elements. For example, the early promoter of SV40 contains three elements: TATA box, start site and 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 of the transcription start site. The GC repeat region is a 21 base pair repeat containing 6 GC boxes and is the site that determines the direction of transcription. This core promoter sequence is approximately 100 bp. Adding an additional 72 base pair repeats, thus making it a “minipromoter”, is useful as a transcription enhancer, which increases promoter functionality approximately 10-fold. When the SP1 protein interacts with a 21 bp repeat, the SP1 protein binds to either the first or last three GC boxes. Binding to the first three initiates early expression, and binding to the last three initiates late expression. The function of the 72 bp repeat is to increase the amount of stable RNA and increase the rate of synthesis. This is done by binding (dimerization) with AP1 (activator protein 1) to produce a 3 ′ polyadenylated, 5 ′ capped primary transcript. Other viral promoters such as Rous sarcoma virus (RSV), HBV X gene promoter, and herpes thymidine kinase core promoter can also be used as a basis for selecting the desired function.

  The core promoter typically includes -40 to +40 relative to the +1 transcription start site that defines the location where transcription is initiated by the RNA polymerase II mechanism (Juven-Gershon and Kadonaga, Dev. Biol. 339 : 225-229, 2010). Typically, RNA polymerase II interacts with a number of transcription factors that bind to a DNA motif within the promoter. These factors are generally known as “general” or “basic” transcription factors, including but not limited to TFIIA (a transcription factor for RNA polymerase IIA), TFIIB, TFIID, TFIIE, TFIIF, and TFIIH. These factors work in a “generic” manner with all core promoters and are therefore often referred to as “basic” transcription factors.

  Juven-Gershon et al., 2006 (Nat. Methods, 3 (11): 917-922, 2006) describe the elements of the core promoter. For example, the pRC / CMV core promoter consists of a TATA box and is 81 bp long. The CMV core promoter consists of a TATA box and a start site. On the other hand, the SCP synthetic core promoter (SCP1 and SCP2) consists of a TATA box, Inr (initiation factor), MTE site (motif 10 element), and 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.

  As used herein, “minipromoter” or “small promoter” refers to a regulatory domain that facilitates transcription of an operably linked gene or encoding nucleic acid sequence. A minipromoter, as indicated by its name, contains the minimum amount of elements necessary for effective transcription and / or translation of an operably linked coding sequence. Mini-promoters may include a “core promoter” or “modified core promoter” in combination with additional regulatory elements. Typically, the mini-promoter or modified core promoter will be about 100-600 bp in length, and the core promoter is typically less than about 100 bp (eg, about 70-80 bp). In other embodiments, where a core promoter is present, the cassette typically expresses operably linked coding sequences either upstream or downstream of the core promoter sequence, above the level of expression by the core promoter alone. Contains enhancer elements or other elements that facilitate.

  Thus, the present disclosure generally allows for ubiquitous expression at significant levels in target cells and is useful for stable integration into a vector as a “core promoter” element (<100 bp, <200 bp, <400 bp or Provided are mini-promoters (eg, modified core promoters) derived from cellular elements as defined for <600 bp), and in particular replicating retroviral vectors to allow efficient expression of the transgene. In addition to the core promoter, it contains minimal enhancer sequences and / or Kozak sequences to allow better gene expression compared to core promoter sequences lacking enhancer sequences and / or Kozak sequences. Mini-promoters below 200 bp, 400 bp or 600 bp are also provided. Such 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 promoters derived from mouse and human cell cycle-dependent ASK genes (63-380 bp; Yamada et al., J. Biol. Chem., 277: 27668-27681, 2002). Small promoters that are ubiquitously expressed include viral promoters such as the SV40 early and late promoters (approximately 340 bp), the RSV LTR promoter (approximately 270 bp), and the standard “TATTAA box”, 13 bp HBV X gene promoter (about 180 bp) (for example, R Anish et al., PLoS one, 4: 5103, 2009) having a core sequence 5′-CCCCGTTGCCCGG-3 ′ (SEQ ID NO: 43). In yet other embodiments, the therapeutic cassette comprising at least one mini-promoter cassette exceeds, is approximately equivalent to, or is about 1-2.5 times lower than the expression level of the IRES cassette present in the RRV. Will have an expression level.

Transcription from the core promoter or mini-promoter occurs by the interaction of various elements. In centralized transcription, for example, there is either a single major transcription start site or several start sites within a narrow region of several nucleotides. Centralized transcription is the dominant mode of transcription in simpler organisms. In distributed transcription, there are several weak transcription initiation sites over a broad region of about 50-100 nucleotides. Distributed transcription is the most common transcription mode in vertebrates. For example, distributed transcription is observed in about two thirds of human genes. In vertebrates, concentrated transcription tends to be associated with regulated promoters, whereas distributed transcription is generally observed on CpG islands in constitutive promoters.

Table 2 shows the oligonucleotides that can be used to construct and clone enhancer elements into the core promoter region. As described above, the modified / optimized core promoter of the present disclosure may include a core sequence that includes the addition of the elements of Table 1, and may further include the cloned enhancer described in Table 2. By doing so, the size of the mini-promoter can be increased. However, the final minipromoter should not exceed 600 bp and will generally be about 100 bp, 200 bp, 300 bp, 400 bp, 500 bp and any integer in between.

  In one embodiment, the present disclosure provides a recombinant replicating competent retrovirus capable of infecting non-dividing host cells, host dividing cells, or host cells with cell proliferative disorders. Recombinant replication competent retroviruses of the present disclosure comprise an optimized internal ribosome entry site having a polynucleotide sequence encoding viral GAG, viral POL, viral ENV, 6A's in the A-bulge of IRES encapsulated in virions ( Heterologous polynucleotides following IRES).

In general, the recombinant vectors of the present disclosure can transfer nucleic acid sequences to target cells. The term “non-dividing” cell means a cell that has not undergone mitosis. Non-dividing cells can be blocked at any point in the cell cycle (eg, G ₀ / G ₁ , G _{1 / S} , G _{2 / M} ) as long as the cells are not actively dividing. In exvivo infection, dividing cells can be treated to prevent cell division by standard techniques used by those skilled in the art, such as irradiation, aphidocolin treatment, serum starvation, and contact inhibition. However, it should be understood that ex vivo infection is performed without blocking cells, since many cells are already arrested (eg, stem cells). For example, a recombinant lentiviral vector can infect non-dividing cells. Examples of existing non-dividing cells in the body include nerve, muscle, liver, skin, heart, lung, and bone marrow cells and their derived cells. An oncoretrovirus vector can be used for dividing cells.

  A “dividing” cell means a cell that has undergone active mitosis or meiosis. Such dividing cells include stem cells, skin cells (eg, fibroblasts and keratinocytes), germ cells and other dividing cells known in the art. Of particular interest and included in the term dividing cell are cells having a cell proliferative disorder, such as neoplastic cells. The term “cell proliferative disorder” means a condition characterized by an abnormal number of cells. This condition can be hypertrophic (continuous growth of cells, resulting in abnormal growth of a population of cells in the tissue) and underdeveloped (deletion or depletion of cells in the tissue). It may include excessive influx or metastasis of cells into the area. The cell population is not necessarily a cancerous, tumorigenic or malignant cell, but can include normal cells as well. Cell proliferative disorders include disorders associated with abnormal growth of connective tissues such as scleroderma, arthritis, and various fibrosis including cirrhosis. Cell proliferative disorders include neoplastic diseases such as head and neck cell tumors. Head and neck cell tumors include, for example, oral cavity, esophagus, pharynx, larynx, thyroid, tongue, lips, salivary glands, nose, paranasal sinuses, nasopharyngeal cell tumors, tumors of the superior nasal vault and sinus, sensory nerves It includes blastoma, squamous cell carcinoma, malignant melanoma, sinonasal anaplastic carcinoma (SNUC) or hematologic neoplasm. Also included are carcinomas of local lymph nodes such as cervical, pre-laryngeal, paraesophageal and submandibular lymph nodes (Harrison's Principles of Internal Medicine (eds., Isselbacher, et al., McGraw-Hill , Inc., 13th Edition, ppl850-1853, 1994. Other cancer principally, but not limited to, lung cancer, colorectal cancer, breast cancer, prostate cancer, urinary tract cancer, uterine cancer lymphoma, oral cancer, pancreatic cancer , Leukemia, melanoma, gastric cancer, skin cancer, and ovarian cancer.Cell proliferative diseases are also caused by indirect rheumatism (O'Dell NEJM 350: 2591 2004) and often by inappropriate cell proliferation of cells of the immune system. Including other autoimmune diseases that are characterized (Mackay et al., NEJM 345: 340 2001).

  In other embodiments, host cells transfected with a replication competent retroviral vector of the present disclosure are provided. Examples of host cells include eukaryotic cells such as yeast cells, insect cells, and animal cells. Host cells also include prokaryotic cells such as bacterial cells. In other embodiments, the host cells have been modified or selected to grow continuously in serum-free suspension (see, eg, US Patent Publication No. 2012 / 0087894-A1, incorporated herein by reference). Please refer.)

  Also provided are engineered host cells that have been transduced (transformed or transfected) with the vectors provided herein (eg, replication competent retroviral vectors). Engineered host cells can be cultured in conventional nutrient media modified as appropriate for promoter activation, transformant selection, or amplification of the encoded polynucleotide. Culture conditions such as temperature and pH are those previously used by the host cell selected for expression and will be apparent to those skilled in the art, for example, Sambrook, Ausubel and Berger, and, for example, Freshney (1994) Culture It is evident in the literature cited herein as well as the literature cited therein, including the Animal Cells: A Manual of Basic Technique, 3rd ed. (Wiley-Liss, New York).

  Examples of suitable expression hosts include mammalian cells such as CHO, COS, BHK, HEK293 or Bowes melanoma. Typically human cells or cell lines will be used. However, it may be desirable to clone the vectors and polynucleotides of the present disclosure into non-human host cells for sequencing, amplification and cloning purposes.

  In other embodiments, the targeting polynucleotide sequence is included as part of a recombinant retroviral vector of the present disclosure. The targeting polynucleotide sequence may be a targeting ligand (eg, a peptide hormone such as heregulin, a single chain antibody, a receptor or receptor ligand), a tissue specific or cell type specific regulatory element (eg, a tissue specific or cell type specific promoter). Or a combination of a targeting ligand and a tissue / cell type specific regulatory element. The targeting ligand is operably linked to the retroviral env protein to form a chimeric retroviral env protein. Viral GAG, viral POL and viral ENV proteins may be from any suitable retrovirus (eg, from MLV or lentivirus). In other embodiments, the viral ENV protein is derived from a non-retrovirus (eg, CMV or VSV).

  In one embodiment, a retroviral vector is targeted to a cell by binding to a cell that has a molecule on the outer surface of the cell. This retroviral targeting method involves targeting ligands on the outer shell of the retrovirus to help target the virus to cells or tissues that have receptors or binding molecules that interact with the targeting ligand on the surface of the retrovirus. Utilize expression. After infection of the cell with the virus, the virus injects the nucleic acid into the cell, and the retroviral genetic material can be incorporated into the genome of the host cell.

  Accordingly, in one embodiment, the disclosure includes a chimeric env protein comprising a retroviral ENV protein operably linked to a targeting polypeptide. A targeting polypeptide may bind or interact with a cell-specific receptor molecule, a cell-specific receptor ligand, an antibody or antibody fragment against a cell-specific antigenic epitope, or a target cell that can be readily identified in the art. It can be any other ligand. Examples of targeting polypeptides or molecules include bivalent antibodies using biotin-streptavidin as a linker (Etienne-Julan et al., J. Of General Virol., 73, 3251-3255, 1992; Roux et al., Proc. Natl. Acad Sci USA 86, 9079-9083, 1989), a recombinant virus containing a sequence encoding the variable region of a single chain antibody against a hapten in the envelope (Russell et al., Nucleic Acids Research, 21, 1081-1085 (1993) ), A peptide hormone ligand cloned into the retroviral envelope (Kasahara et al., Science, 266, 1373-1376, 1994), chimeric EPO / env construct (Kasahara et al., 1994), low-density lipoprotein (LDL) receptor A single-chain antibody against the LDL receptor of narrow-host MLV that results in specific infection of somatically expressed HeLa cells (Somia et al., Proc. Natl. Acad. Sci USA, 92, 7570-7574 (1995)) Similarly, the host range of ALV is It can be altered by the introduction of phospholigands, allowing the virus to specifically infect rat glioblastoma cells across species (Valsesia-Wittmann et al., J. Virol. 68, 4609-4619 ( 1994)), Dornberg and collaborators (Chu and Dornburg, J. Virol 69, 2659-2663 (1995); M. Engelstadter et al., Gene Therapy 8,1202-1206 (2001)) are single-chain antibodies against tumor markers. We have reported tissue-specific targeting of spleen necrosis virus (SNV), avian retrovirus using an envelope containing.

  In one embodiment, the recombinant retroviruses of the present disclosure are those in which the virus is of a specific cell type (eg, smooth muscle cells, hepatocytes, kidney cells, fibroblasts, keratinocytes, mesenchymal stem cells, bone marrow cells, cartilage Cells, epithelial cells, intestinal cells, mammary cells, neoplastic cells, glioma cells, neuronal cells and other cells known to those skilled in the art, so that the retroviral vector The recombinant genome is delivered to target non-dividing cells, target dividing cells, or target cells with cell proliferative disorders.

  In other embodiments, targeting uses cell or tissue specific regulatory elements to promote viral genome expression and transcription in target cells that actively utilize the regulatory elements, as described more fully below. The transferred retroviral genetic material is then transcribed in the host cell and translated into protein. Targeting regulatory elements are typically linked to the 5 ′ and / or 3 ′ LTR to form a chimeric LTR.

  In one embodiment, the present disclosure provides a replication competent retrovirus that does not require helper virus or additional nucleic acid sequences or proteins to grow and produce virions. For example, as described above, the retroviral nucleic acid sequences of the present disclosure encode group specific antigens and reverse transcriptases (and integrase and protease enzymes required for maturation and reverse transcription), respectively. The viruses gag and pol can be derived from lentiviruses such as HIV or oncoviruses or gamma retroviruses such as MoMLV. In addition, the retroviral nucleic acid genome of the present disclosure includes a sequence encoding a viral envelope (ENV) protein. The env gene can be derived from any retrovirus. env can be a broad host envelope protein that allows transduction into cells of humans and other species, or can be a narrow host envelope protein that can only transduce mouse and rat cells. Furthermore, it may be desirable to target the recombinant virus to a specific cell type receptor by linking the envelope protein to an antibody or a specific ligand. As described above, retroviral vectors can be made target specific by inserting, for example, glycolipids or proteins. Targeting is often accomplished using antibodies that target retroviral vectors to antigens of specific cell types (eg, cell types found in certain tissues or cancer cell types). One of ordinary skill in the art knows or can easily ascertain specific methods for achieving delivery of a retroviral vector to a specific target without undue experimentation. In one embodiment, the env gene is derived from a non-retrovirus (eg, CMV or VSV). Examples of retrovirus-derived env genes include, but are not limited to: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), gibbon leukemia virus (GaLV), human immunity Deficiency virus (HIV) and Rous sarcoma virus (RSV). Other env genes such as vesicular stomatitis virus (VSV) (Protein G), cytomegalovirus envelope (CMV), or influenza virus hemagglutinin (HA) can also be used.

  In one embodiment, the retroviral genome is derived from an oncoretrovirus, more specifically a mammalian oncoretrovirus. In a further embodiment, the retroviral genome is derived from a gamma retrovirus, more specifically a mammalian gamma retrovirus. “Derived” means that the parent polynucleotide sequence differs in insertion or removal of native sequence (eg, insertion of an IRES, insertion of a heterologous polynucleotide encoding a polypeptide of interest or inhibitory nucleic acid, wild-type promoter). It means a wild-type oncovirus that has been modified by retrovirus or a more efficient promoter replacement from a virus.

  Unlike recombinant retroviruses produced by standard methods in the art, which are incomplete and require assistance to produce infectious viral particles, the present disclosure relates to a replication competent retrovirus. Provide virus.

  In other embodiments, the present disclosure provides retroviral vectors that are targeted using regulatory sequences. Cell or tissue specific regulatory sequences (eg, promoters) can be used to target expression of gene sequences in specific cell populations. Suitable mammalian and viral promoters for this disclosure are described elsewhere herein. Accordingly, in one embodiment, the present disclosure provides a retrovirus having a tissue-specific promoter element at the 5 ′ end of the retroviral genome. Typically, tissue-specific regulatory elements / including, for example, cell or tissue-specific promoters and enhancers for neoplastic cells (eg, tumor cell-specific enhancers and promoters) and inducible promoters (eg, tetracycline) The sequence is present in the U3 region of the LTR of the retroviral genome.

  In some cases it may be desirable to modulate expression. For example, various viral promoters with different strengths of activity can be utilized depending on the level of expression desired. In mammalian cells, the CMV immediate early promoter is often used to provide strong transcriptional activity. Modified versions of the CMV promoter that are not very strong are also used where reduced levels of expression of the transgene are desired. If expression of the transgene in hematopoietic cells is desired, a retroviral promoter such as MTV or LTR derived from MMTV can be used. Other viral promoters that can be used include SV40, RSV LTR, HIV-1 and HIV-2 LTR, adenoviral promoters (eg, from E1A, E2A, or MLP regions), AAV LTR, cauliflower mosaic virus, HSV-TK, and Avian sarcoma virus.

  Similarly, tissue specific or selective promoters can be used to effect transcription in specific tissues or cells in order to suppress potential toxicity or undesirable effects on non-target tissues. For example, promoters such as PSA, probasin, prostate acid phosphatase or prostate-specific glandular kallikrein (hK2) can be used to target gene expression in the prostate. Whey accessory protein (WAP) can be used for expression in milk tissue (Andres et al., PNAS 84: 1299-1303, 1987). Other promoter / regulatory domains that can be used are listed in Table 3.

  A “tissue-specific regulatory element” is a regulatory element (eg, a promoter) that can drive transcription of a gene in one tissue while remaining largely “silent” in other tissue types. However, it will be appreciated that tissue-specific promoters may have a detectable amount of “background” or “basal” activity in the tissue in which they are silenced. The degree to which the promoter is selectively activated in the target tissue can be expressed as a selection ratio (activity in the target tissue / activity in the control tissue). In this regard, tissue specific promoters useful in the practice of the present disclosure typically have a selection ratio greater than about 5. Preferably, the selection ratio is greater than about 15.

  In some instructions, it may be desirable to activate transcription after a specific time after administration of the recombinant replication competent retrovirus (RRCR) of the present disclosure. This can be done by a promoter regulatable by hormones or cytokines. For example, in therapeutic applications where the specific steroid is produced or delivered by the gonadal tissue, the use of promoters regulated by androgens or estrogens may be advantageous. Such promoters that can be regulated by hormones include MMTV, MT-1, ecdysone and rubisco. Other hormone-regulated promoters may be used, such as those that are responsive to thymus, pituitary and adrenal hormones. Cytokine and inflammatory protein-responsive promoters that can be used include K and T kininogen (Kageyama et al., 1987), c-fos, TNF-alpha, C-reactive protein (Arcone et al., 1988), haptoglobin (Oliviero et al., 1987). ), Serum amyloid A2, C / EBP alpha, IL-1, IL-6 (Poli and Cortese, 1989), complement C3 (Wilson et al., 1990), IL-8, α1 acidic glycoprotein (Prowse and Baumann, 1988) ), Α1 antitrypsin, lipoprotein lipase (Zechner et al., 1988), angiotensinogen (Ron et al., 1990), fibrinogen, c-jun (phorbol ester, TNF-α, UV irradiation, retinoic acid and hydrogen peroxide) Collagenase (derived by phorbol esters and retinoic acid), metallothionein (derived by heavy metals and glucocorticoids), stromelysin (phorbol esters, interleukin 1) Is the induced fine EGF), include α2 macroglobulin and antichymotrypsin. Tumor-specific promoters such as osteocalcin, hypoxia-responsive element (HRE), MAGE-4, CEA, α-fetoprotein, GRP78 / BiP and tyrosinase also regulate gene expression in tumor cells Can be used.

Furthermore, this list of promoters should not be construed as exhaustive or limiting, and those skilled in the art will know other promoters that can be used in conjunction with the promoters and methods disclosed herein. Let's go.

  Furthermore, the selectivity that some promoters are not restricted to one tissue type in activity, yet may be active in one tissue group and less active or silent in other groups. It will be understood to show. Such promoters are also termed “tissue specific” and are contemplated for use with the present disclosure. For example, promoters active in various central nervous system (CNS) neurons may be useful therapeutically to protect against stroke damage that can affect any of many different regions of the brain. Thus, the tissue-specific regulatory elements used in this disclosure are not only applicable as targeting polynucleotide sequences for the retroviral vectors, but are also applicable to the regulation of heterologous proteins.

  In yet another embodiment, the present disclosure provides a plasmid comprising a recombinant retrovirus-derived construct. This plasmid can be introduced directly into target cells or cell cultures such as NIH 3T3 or other tissue culture cells. The resulting cells release the retroviral vector into the culture medium.

  In view of the above and following examples, the present disclosure, in one embodiment, provides a recombinant replication competent retrovirus (RCR) comprising an optimized IRES cassette. In one embodiment, the retroviral polynucleotide sequence comprises murine leukemia virus (MLV), Moloney murine leukemia virus (MoMLV), feline leukemia virus (FeLV), baboon endogenous retrovirus (BEV), porcine endogenous retrovirus (PERV). ), Cat-derived retrovirus RD114, squirrel monkey retrovirus, heterologous murine leukemia-related virus (XMRV), avian reticuloendotheliosis virus (REV), or gibbon leukemia virus (GALV) To do. In another embodiment, the RCR is a retroviral GAG protein; a retroviral POL protein; a retroviral envelope (which can be chimeric, narrow host and broad host); a long end at the 3 ′ end of the retroviral polynucleotide sequence In the LTR at the 5 'end of the repeat (LTR), gag, pol and env genes and optimized IRES cassette (and / or optionally additional elements such as core promoters, miRNAs and other inhibitory nucleic acids), and retroviral polynucleotides A retroviral polynucleotide comprising a promoter. In one embodiment, the 3 ′ LTR comprises a sequence that is at least 98% identical to the sequence set forth from about nucleotide 9405 to about 9988 of SEQ ID NO: 19, 22, or 42. In other embodiments, the promoter sequence at the 5 ′ end of the retroviral polynucleotide is suitable for expression in mammalian cells. In any other embodiment of the above, the promoter, gag, pol, and env domains comprise a sequence that is at least 98% identical to a sequence from about 1 to about 8323 of SEQ ID NO: 19, 22, or 42, wherein retro The viral polynucleotide lacks 70 base pairs downstream from the 3 ′ LTR to the MLV sequence compared to the vector of SEQ ID NO: 21. In yet other embodiments of any of the above, the cassette comprises a sequence that is at least 98% identical to a sequence from about 8327 to 8875 of SEQ ID NO: 19, 22, or 42, and is 6 in the A-bulge of the JK branch region. Contains an optimized internal ribosome entry site (IRES) consisting of A. In a further embodiment, the optimized IRES is operably linked to a heterologous polynucleotide, wherein the cassette is located 5 ′ of the 3 ′ LTR and 3 ′ of the env nucleic acid domain encoding the retroviral envelope and is SEQ ID NO: It lacks short repeats on either side of the cassette compared to 21 vectors (pACE-CD). In yet other embodiments of any of the above, the vector contains cis-acting sequences necessary for reverse transcription, packaging and integration in the target cell. In yet another embodiment, the RCR maintains high replication capacity after 6 passages compared to the vector comprising SEQ ID NO: 21 (pACE), wherein the heterologous polynucleotide is expressed and pAC3-yCD2 (sequence Number 22) produces a heterologous polypeptide that is expressed at least 20%, 30%, 40%, or 50% or more compared to a vector. In other embodiments, the RCR infects the target cell multiple times resulting in an average copy number / diploid genome of 5 or greater. In other embodiments, the retroviral envelope is a broad host MLV envelope. In one embodiment, the promoter comprises a CMV promoter having nucleotide 1 to about nucleotide 582 of the sequence set forth in SEQ ID NO: 19, 20, 22, or 42 and is capable of inducing and initiating transcription one or more nucleobases Modifications may be included. In other embodiments, the promoter comprises a CMV-R-U5 domain polynucleotide. In yet a further embodiment, the CMV-R-U5 domain comprises an immediate early promoter from human cytomegalovirus linked to the MLV R-U5 region. In still further embodiments, the CMV-R-U5 domain polynucleotide is from about nucleotide 1 to about nucleotide 1202 of the sequence set forth in SEQ ID NO: 19, 20, 22, or 42, or SEQ ID NO: 19, 20, 22, or 42. The polynucleotide comprises a sequence that is at least 99% identical to the described sequence and the polynucleotide facilitates transcription of a nucleic acid molecule operably linked thereto. In other embodiments, the gag nucleic acid domain comprises a sequence having from about nucleotide number 1203 to about nucleotide 2819 of SEQ ID NO: 19, 20, 22, or 42, or at least 99% or 99.8% identity thereto. In other embodiments, the pol domain of the polynucleotide is derived from a gamma retrovirus. In a further embodiment, the pol domain comprises a sequence having from about nucleotide number 2820 to about nucleotide 6358 of SEQ ID NO: 19, 22, or 42, or at least 99% or 99.9% identity thereto. In still other embodiments, the env domain comprises a sequence having from about nucleotide number 6359 to about nucleotide 8323 of SEQ ID NO: 19, 22, or 42, or at least 99% or 99.8% identity thereto. In still other embodiments, the IRES consists of the sequence set forth in SEQ ID NO: 41. In still other embodiments, the heterologous nucleic acid comprises a polynucleotide having the sequence set forth in SEQ ID NO: 3, 5, 11, 13, 15 or 17. In other embodiments, the heterologous nucleic acid encodes a polypeptide comprising the sequence set forth in SEQ ID NO: 4. In a further embodiment, the heterologous nucleic acid is human codon optimized and encodes the polypeptide set forth in SEQ ID NO: 4. In still other embodiments, the heterologous nucleic acid comprises the sequence set forth from about nucleotide number 8877 to about 9353 of SEQ ID NO: 19, 22, or 42. In other embodiments, the 3 ′ LTR comprises a U3-R-U5 domain. In still further embodiments, the 3 ′ LTR comprises the sequence set forth from about nucleotide 9405 to about 9988 of SEQ ID NO: 19, 22, or 42, or a sequence that is at least 95%, 98%, or 99.5% identical thereto. In one embodiment, the present disclosure provides a retroviral polynucleotide comprising SEQ ID NO: 42. In other embodiments, the retroviral polynucleotide of SEQ ID NO: 42 is an RNA sequence in which T is replaced with U. The retroviral RNA polynucleotide set forth in SEQ ID NO: 42 wherein T is U is encapsulated in a viral capsid. In yet other embodiments of any of the above, the retroviral polynucleotide may further comprise a miRNA, siRNA or shRNA sequence that can be delivered to the target cell. The miRNA, siRNA or shRNA can be operably linked to a polIII promoter. The miRNA can be located upstream or downstream of the optimized iRES cassette. In other embodiments, the heterologous polynucleotide can be any number of coding sequences, such as cytokines, immunostimulators, thymidine kinases, cytosine deaminases, purine nucleoside phosphorylases, receptors, antibodies, fragments, and the like.

  The disclosure also provides a method of treating a cell proliferative disorder comprising contacting a subject with a retrovirus described herein. In one embodiment, the method comprises contacting the subject with a retrovirus comprising an optimized IRES and 5-fluorocytosine under conditions where the heterologous polynucleotide linked to the optimized IRES comprises cytosine deaminase activity. In one embodiment, the retrovirus infects cells resulting in the incorporation of a polynucleotide comprising SEQ ID NO: 42. In other embodiments, the cell proliferative disorder is glioblastoma multiforme. In other embodiments, the cell proliferative disorder is from the group consisting of lung cancer, colorectal cancer, breast cancer, prostate cancer, urinary tract cancer, uterine cancer, brain cancer, head and neck cancer, pancreatic cancer, melanoma, gastric cancer and ovarian cancer. Selected. The method includes a combination therapy in which the subject to be treated is contacted with a retrovirus and further contacted with an anticancer agent or chemotherapeutic agent. For example, anticancer or chemotherapeutic agents include bevacizumab, pegaptanib, ranibizumab, sorafenib, sunitinib, AE-941, VEGF Trap, pazopanib, vanazot , 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, CEP-7055, CHIR-265 Selected from the group consisting of CT-322, CX-3542, E-7080, ENMD-1198, OSI-930, PTC-299, Sirna-027, TKI-258, Veglin, XL-184, and ZK-304709 Can be done.

In any other embodiment of the above, the retrovirus is administered at about 10 ³ to 10 ⁷ TU / g brain weight. In other embodiments, the retrovirus is administered at about 10 ⁴ to 10 ⁶ TU / g brain weight.

  The present disclosure extends from 5 ′ to 3 ′: a promoter or regulatory region useful for initiating transcription; a psi packaging signal; a nucleic acid sequence encoding gag; a nucleic acid sequence encoding pol; a nucleic acid sequence encoding env; Provided is a polynucleotide construct comprising an internal ribosome entry site nucleic acid sequence comprising 6A's in the bulge; a heterologous polynucleotide encoding a marker, therapeutic or diagnostic polypeptide; and an LTR nucleic acid sequence. As described elsewhere herein and below, various portions of a polynucleotide construct of the present disclosure (eg, a recombinant replication competent retroviral polynucleotide) can be expressed in any desired host cell, Designed in part depending on the amount and the heterologous polynucleotide. The replication competent retroviral constructs of the present disclosure can be divided into several regions, each of which can be modified by one skilled in the art.

  For example, the promoter can include a CMV promoter having nucleotide 1 to about nucleotide 582 of the sequence set forth in SEQ ID NO: 19, 20, 22, or 42, as long as the modified promoter can induce and initiate transcription, One or more modifications (eg, 2-5, 5-10, 10-20, 20-30, 30-50, or more nucleobases) may be included. In one embodiment, the promoter or regulatory region comprises a CMV-R-U5 domain polynucleotide. The CMV-R-U5 domain contains the immediate early promoter from human cytomegalovirus in the R-U5 region of MLV. In one embodiment, the CMV-R-U5 domain polynucleotide is from about nucleotide 1 to about nucleotide 1202 of the sequence set forth in SEQ ID NO: 19, 20, 22 or 42, or set forth in SEQ ID NO: 19, 20, 22 or 42. Comprising a sequence that is at least 95% identical to a sequence from about nucleotide 1 to about nucleotide 1202, wherein the polynucleotide facilitates transcription of a nucleic acid molecule operably linked thereto. The gag domain of a polynucleotide can be derived from any number of retroviruses, but is typically derived from an oncoretrovirus, and more specifically from a mammalian oncoretrovirus. In one embodiment, the gag domain is a sequence from about nucleotide number 1203 to about nucleotide 2819 of the sequence set forth in SEQ ID NO: 19, 20, 22, or 42, or at least 95%, 98%, 99% or 99.8 relative thereto. Includes sequences with% identity (rounded to the nearest tenth). The pol domain of a polynucleotide can be derived from any number of retroviruses, but is typically derived from an oncoretrovirus, and more specifically from a mammalian oncoretrovirus. In one embodiment, the pol domain is a sequence of about 2820 to about 6358 nucleotides of the sequence set forth in SEQ ID NO: 19, 20, 22 or 42, or at least 95%, 98%, 99% or 99.9% ( Includes sequences with identity (rounded to the nearest tenth). The env domain of a polynucleotide can be derived from any number of retroviruses, but typically is derived from an oncotrovirus or gamma retrovirus, and more specifically from a mammalian oncoretrovirus or gamma retrovirus. . In some embodiments, the domain encoding env comprises a broad host env domain. In one embodiment, the env domain is a sequence from about nucleotide number 6359 to about nucleotide 8323 of the sequence set forth in SEQ ID NO: 19, 20, 22, or 42, or at least 95%, 98%, 99%, or 99.8 relative thereto. Includes sequences with% identity (rounded to the nearest tenth). The optimized IRES domain of a polynucleotide can be obtained from any number of internal ribosome entry sites. In one embodiment, the optimized IRES is derived from encephalomyocarditis virus. In one embodiment, the optimized IRES domain is at least 95%, 98% relative to the sequence set forth in SEQ ID NO: 41, or as long as the domain allows ribosome entry and contains 6A's in the A-bulge. Or a sequence with 99% identity (rounded to the nearest tenth). The heterologous domain can comprise a cytosine deaminase of the present disclosure. In one embodiment, the CD polynucleotide comprises a sequence optimized for human codons. In yet another embodiment, the CD polynucleotide encodes a mutant polypeptide having cytosine deaminase, wherein the mutant contributes to increased thermal stability by increasing the melting point (Tm) by 10 ° C., and This makes it possible to maintain mechanical activity over a wide temperature range and to increase the level of accumulated protein. In another embodiment, the disclosure includes human codon optimized thymidine kinase. The heterologous domain can be followed by a polypurine rich domain. The 3 ′ LTR can be derived from any number of retroviruses, typically oncoretroviruses and preferably mammalian oncoretroviruses. In one embodiment, the 3 ′ LTR comprises a U3-R-U5 domain. In yet another embodiment, the LTR is a sequence from about nucleotide 9405 to about nucleotide 9988 of the sequence set forth in SEQ ID NO: 19, 20, 22, or 42, or at least 95%, 98%, or 99.5% (most) Includes sequences that are identical (rounded to the nearest tenth).

  This disclosure describes, from 5 ′ to 3 ′, CMV-R-U5, a fusion of an early promoter from human cytomegalovirus to the R-U5 region of MLV; PBS, a primer binding site for reverse transcriptase 5 ′ splice site; ψ packaging signal; gag, ORF for MLV group specific antigen; pol, ORF of MLV polymerase polyprotein; 3 ′ splice site; 4070A env, ORF of envelope protein of MLV4070A strain; A -Optimized IRES consisting of bulge 6A's; modified cytosine deaminase (heat stabilized and codon optimized) or human codon optimized thymidine kinase; PPT, polyprinted lactate; and U3-R-U5, MLV long terminal repeat recombination Retroviral vectors are also provided.

  The present disclosure also provides a retroviral vector comprising a sequence set forth in SEQ ID NO: 42 (or SEQ ID NO: 42 where T can be U) comprising an A-bulge optimized for expression. In one embodiment, the IRES optimized A-bulge consists of 6A's.

  Retroviral vectors can be used to treat a wide range of diseases and disorders, including numerous cell proliferative diseases and disorders (eg, US Pat. Nos. 4,405,712 and 4,650,764; Friedmann, 1989, Science, 244: 1275). Mulligan, 1993, Science, 260: 926-932, R. Crystal, 1995, Science 270: 404-410, each of which is incorporated by reference in its entirety, and The Development of Human See also Gene Therapy, Theodore Friedmann, Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1999. ISBN 0-87969-528-5, which is incorporated herein by reference in its entirety).

  The present disclosure also provides gene therapy for the treatment of cell proliferative disorders. Such therapy achieves its therapeutic effect by introducing an appropriate therapeutic polynucleotide (eg, antisense, ribozyme, suicide gene, siRNA) into the cells of a subject with a proliferative disorder. Delivery of polynucleotide constructs can be accomplished using the recombinant retroviral vectors of the present disclosure, particularly those that are capable of infecting dividing cells when based on MLV.

  Furthermore, the therapeutic methods described herein (eg, gene therapy or gene delivery methods) can be performed in vivo or ex vivo. Prior to gene therapy, it may be preferred that the majority of the tumor has been removed, eg, surgically or by radiation. In some embodiments, retroviral therapy can be done prior to or following surgical, chemical, or radiation therapy.

  Accordingly, the present disclosure provides a replicating retrovirus capable of infecting non-dividing cells, dividing cells, or neoplastic cells, wherein the recombinant retrovirus is a virus GAG; virus POL; virus ENV; A bulge 6A's A heterologous nucleic acid operably linked to an IRES; and cis-acting 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 present disclosure may further comprise at least one of VPR, VIF, NEF, VPX, TAT, REV, and VPU proteins. While not wishing to be bound by any particular theory, one or more of these gene / protein products (eg, NEF) are important in increasing the viral titer of the recombinant retrovirus produced Or may be necessary for virion infection and packaging.

  The present disclosure also provides a method for transferring a nucleic acid to a target cell that results in the expression of a particular nucleic acid (eg, a heterologous sequence). Accordingly, in other embodiments, the present disclosure provides for the introduction and expression of a heterologous nucleic acid in a target cell, including infecting the target cell with a recombinant virus of the present disclosure and expressing the heterologous nucleic acid in the target cell. Provide a way. As noted above, the target cell can be any cell type, such as dividing, non-dividing, nascent, immortalized, modified, and other cell types recognized by those skilled in the art, so long as infection with a retrovirus is possible. obtain.

  It may be desirable that the expression of the gene in the cell be altered by the introduction of a nucleic acid sequence (eg, a heterologous nucleic acid sequence) by the methods of the present disclosure, where the nucleic acid sequence results in, for example, an antisense or ribozyme molecule. The term “modification” intends to suppress the expression of a gene when the gene is overexpressed or enhance the expression when the gene is underexpressed. Where cell proliferative disorders are associated with gene expression, nucleic acid sequences that interfere with gene expression at the translational level can be used. This approach utilizes, for example, an antisense nucleic acid, a ribozyme, or an antisense nucleic acid or triplex agent, masking the mRNA with an antisense nucleic acid or triplex agent, or cleaving it with a ribozyme. Either inhibits the transcription or translation of a particular mRNA.

  It may be desirable to transfer a nucleic acid encoding a biological response modifier (eg, a cytokine) to a cell or subject. Included in this category are nucleic acids that encode a number of cytokines that are classified as immunostimulators, eg, “interleukins”. These include, for example, interleukins 1-15, as well as other response modifiers and factors described elsewhere herein. Also included in this category are interferons, and in particular gamma interferon, tumor necrosis factor (TNF) and granulocyte macrophage colony stimulating factor (GM-CSF), although not necessarily acting by the same mechanism. 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 enzyme or immune defects. Nucleic acids encoding growth factors, toxin peptides, ligands, receptors, or other physiologically important proteins can also be introduced into specific target cells.

The present disclosure can also be used to deliver heterologous polynucleotides that facilitate drug-specific targeting and effects. For example, HER2, a member of the EGF receptor family, is a target for binding of the drug trastuzumab (Herceptin ^™ , Genentech). Trastuzumab is a mediator of antibody-dependent cellular cytotoxicity (ADCC). Activity preferentially targets HER2-expressing cells that have 2+ and 3+ levels of overexpression by immunohistochemistry over 1+ and non-expressing cells (Herceptin prescription information, Crommelin 2002). Optimized ADCC triggering by increasing HER2 expression in low HER2 tumors by introducing HER2 or truncated HER2 expressing vectors (expressing only extracellular and transmembrane domains) And can overcome the resistance that occurs rapidly to Herceptin observed in clinical use.

  Replacement of yCD2 with the intracellular domain of HER2 (including about 8877-9353 of SEQ ID NO: 19) allows cell surface expression of HER2 and localization of yCD2 on the cellular substrate. The HER2 extracellular domain (ECD) and transmembrane domain (TM) (from about 2026 bp of SEQ ID NO: 23 to about 2200 at position 175) can be amplified by PCR (Yamamoto et al., Nature 319: 230-234, 1986). Chen et al., Canc. Res., 58: 1965-1971, 1998) or chemically synthesized (BioBasic Inc., Markham, Ontario, Canada), the IRES of the pAC3-yCD2 vector of SEQ ID NO: 19 And the yCD2 gene (eg, between about nucleotides 8876 and 8877 of SEQ ID NO: 19). Alternatively, the yCD gene can be excised and replaced with a polynucleotide encoding a HER2 polypeptide or fragment thereof. Further truncated HER2 (approximately 290 bp from positions 1910 to 2200) with only Herceptin binding domain IV of ECD and TM domains can be amplified or chemically constructed and used as described above (Landgraf 2007; Garrett et al., J. of Immunol., 178: 7120-7131, 2007). Further modifications of this truncated form with the native signal peptide fused to domains IV and TM (positions 175-237 to about 69 bp) can be chemically synthesized and used as described above. The resulting virus can be used in combination with trastuzumab or trastuzumab and 5-FC to treat cell proliferative disorders in a subject.

  Alternatively, HER2 and the above modifications can be expressed in separate vectors containing different ENV genes or other suitable surface proteins. This vector may be replication competent (Logg et al. J. Mol Biol. 369: 1214 2007) or a non-replicating “first generation” retroviral vector encoding the envelope and the gene of interest (Emi et al. J Virol 65: 1202 1991). In the latter case, infection with an existing virus will result in complementary gag and pol, allowing replication infection of the “non-replicating” vector from any previously infected cell. Alternative ENVs and glycoproteins include xenotropic and polytropic ENVs and glycoproteins that can infect human cells, such as ENV sequences from NZB strains of MLV, and MCF, VSV, GALV and others (Palu 2000, Baum et al., Mol. Therapy, 13 (6): 1050-1063, 2006). For example, the polynucleotide may comprise a sequence, wherein the GAG and POL and yCD2 genes of SEQ ID NO: 19 are deleted, the ENV corresponding to the NZB MLV or VSV-g heterotropic ENV domain, and IRES or RSV, etc. Are operably linked to HER2, HER2 ECDTM, HER2 ECDIVTM, or HER2 SECDIVTM.

  Mixed infection of cells with VSVG pseudotyped viruses and broad-host retroviruses results in the production of progeny virions with the genome of one virus encapsidated by the other envelope protein. The same is true for other envelopes that pseudotype retrovirus particles. For example, infection with the retrovirus described above results in the production of progeny virions that can encode yCD2 and HER2 (or mutants) in the infected cell. The resulting virus can be used to treat cell proliferative disorders in a subject in combination with trastuzumab or trastuzumab and 5-FC.

  Another aspect of the development of resistance to trastuzumab relates to the interference of intracellular signaling required for the activity of trastuzumab. Resistant cells show deletion of PTEN and low expression of p27kip1 (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). For example, a polynucleotide encoding PTEN can be produced recombinantly or chemically synthesized (BioBasic Inc., Markham, Canada) immediately after the yCD2 polynucleotide in the pAC3-yCD2 vector of SEQ ID NO: 19 or 22. Or can be operably inserted with the above linker sequences or as a substitution for yCD2. In a further example, a PTEN-encoding polynucleotide (SEQ ID NO: 25) can be synthesized as described above and inserted as described above between the IRES and yCD2 sequences or with a linker.

  Alternatively, PTEN can be expressed in separate vectors containing different ENV genes or other suitable surface proteins. This vector may be replication competent (Logg et al. J. Mol Biol. 369: 1214 2007) or a non-replicating “first generation” retroviral vector encoding the envelope and the gene of interest (Emi et al. J Virol 65: 1202 1991). In the latter case, infection with an existing virus will result in complementary gag and pol, allowing replication infection of the “non-replicating” vector from any previously infected cell. Alternative ENVs and glycoproteins include xenotropic and polytropic ENVs and glycoproteins that can infect human cells, such as ENV sequences from NZB strains of MLV, and MCF, VSV, GALV and others (Palu 2000, Baum et al., Mol. Therapy, 13 (6): 1050-1063, 2006). For example, the polynucleotide may comprise a sequence, wherein the GAG and POL and yCD2 genes of SEQ ID NO: 19 are deleted, the ENV corresponding to the NZB MLV or VSV-g heterotropic ENV domain, and IRES or RSV, etc. Are operably linked to PTEN.

  Mixed infection of cells with VSVG pseudotyped virus and broad-host retrovirus results in the production of progeny virions with the genome of one virus encapsidated by the other envelope protein (Emi 1991). The same is true for other envelopes that pseudotype retrovirus particles. For example, infection with the retroviruses described above results in the production of progeny virions that can encode yCD2 and PTEN (or mutants) or PTEN alone in infected cells. The resulting virus can be used to treat cell proliferative disorders in a subject in combination with trastuzumab or trastuzumab and 5-FC.

  Similarly, a polynucleotide encoding p27kip1 (SEQ ID NOs: 27 and 28) was chemically synthesized (BioBasic Inc., Markham, Canada) and immediately after the yCD2 gene in the pAC3-yCD2 vector of SEQ ID NO: 19 or SEQ ID NO: 42 Alternatively, it can be operably inserted with a linker sequence. In a further example, a p27kip1-encoding polynucleotide is synthesized as described above and inserted between the IRES and yCD2 sequences consisting of A bulge 6A's, or as described above, with a linker, or in place of the yCD2 gene.

  Alternatively, p27kip1 can be expressed in separate vectors containing different ENV genes or other suitable surface proteins. This vector may be replication competent (Logg et al. J. Mol Biol. 369: 1214 2007) or a non-replicating “first generation” retroviral vector encoding the envelope and the gene of interest (Emi et al. J Virol 65: 1202 1991). In the latter case, infection with an existing virus will result in complementary gag and pol, allowing replication infection of the “non-replicating” vector from any previously infected cell. Alternative ENVs and glycoproteins include xenotropic and polytropic ENVs and glycoproteins that can infect human cells, such as ENV sequences from NZB strains of MLV, and MCF, VSV, GALV and others Glycoproteins from these viruses (Palu 2000, Baum et al, supra). For example, the polynucleotide may comprise a sequence in which the GAG and POL and yCD2 genes of SEQ ID NO: 19 are deleted, the ENV corresponding to the NZB MLV or VSV-g heterotropic ENV domain, and A bulge 6A's A promoter such as IRES or RSV is directly operably linked to p27kip1.

  Mixed infection of cells with VSVG pseudotyped virus and broad-host retrovirus results in the production of progeny virions with the genome of one virus encapsidated by the other envelope protein (Emi 1991). The same is true for other envelopes that pseudotype retrovirus particles. For example, infection with the above retroviruses derived from any of SEQ ID NOs: 19, 22, and 42 results in the production of progeny virions that can encode yCD2 and p27kip1 (or mutants) in infected cells. The resulting virus can be used to treat cell proliferative disorders in a subject in combination with trastuzumab or trastuzumab and 5-FC.

In another example, CD20 is the target for binding of the drug rituximab (Rituxan ^™ , Genetech). Rituximab is a mediator of complement-dependent cytotoxicity (CDC) and ADCC. Cells with high mean fluorescence intensity by flow cytometry show increased sensitivity to rituximab (van Meerten et al., Clin Cancer Res 2006; 12 (13): 4027-4035, 2006). Improvement of CD20 expression by introduction of a CD20-expressing vector in CD20 low B cells may facilitate optimal triggering of ADCC.

  For example, a polynucleotide encoding CD20 (SEQ ID NOs: 29 and 30) is chemically synthesized (BioBasic Inc., Markham, Canada) and yCD2 in the pAC3-yCD2 (-2) vector of SEQ ID NO: 19, 22 or 42. It can be operably inserted immediately after the gene with a linker sequence as described above or as a replacement for the yCD2 gene. In a further example, a CD20-encoding polynucleotide is synthesized as described above and inserted between the IRES and yCD2 sequences consisting of 6A's of A bulge or with a linker as described above. As a further alternative, the CD20 sequence can be inserted into the pAC3-yCD2 vector after excision of the CD gene by Psi1 and Not1 digestion.

  In still further examples, a polynucleotide encoding CD20 (SEQ ID NOs: 29 and 30) is chemically synthesized (BioBasic Inc., Markham, Canada) and is a non-broad host ENV gene or other suitable surface protein ( Tedder et al., PNAS, 85: 208-212, 1988). Alternative ENVs and glycoproteins include xenotropic and polytropic ENVs and glycoproteins that can infect human cells, such as ENV sequences from NZB strains of MLV, and MCF, VSV, GALV and others Glycoproteins derived from viruses (Palu 2000, Baum 2006) For example, the polynucleotide may comprise a sequence in which the GAG and POL and yCD2 genes of SEQ ID NO: 19 are deleted, the ENV corresponding to the NZB MLV or VSV-g heterotropic ENV domain, and A bulge 6A's A promoter such as IRES or RSV is directly operably linked to CD20.

  Mixed infection of cells with VSVG pseudotyped virus and broad-host retrovirus results in the production of progeny virions with the genome of one virus encapsidated by the other envelope protein (Emi 1991). The same is true for other envelopes that pseudotype retrovirus particles. For example, infection with a retrovirus as described above, also derived from SEQ ID NO: 19, 22, or 42, results in the production of progeny virions that can encode yCD2 and CD20 in infected cells. The resulting virus can be used in combination with Rituxan and / or 5-FC to treat a cell proliferative disorder in a subject. Similarly, infection of a tumor with a vector encoding only the CD20 marker can make the tumor treatable by use of Rituxan.

  Enzymes and cofactors involved in pyrimidine assimilation may be limited. OPRT, thymidine kinase (TK), uridine monophosphate kinase, and pyrimidine nucleoside phosphorylase expression are lower in 5-FU resistant cancer cells compared to susceptible strains (Wang et al., Cancer Res., 64: 8167-8176, 2004). ). Many population analyzes have shown a correlation between enzyme levels and disease outcome (Fukui et al., Int'l. J. OF Mol. Med., 22: 709-716, 2008). Co-expression of CD and other pyrimidine anabolic enzymes (PAE) can be utilized to increase the activity and thus the therapeutic index of fluoropyrimidine drugs.

  The present disclosure provides a method for treating cell proliferative disorders, such as cancer and neoplasia, comprising administering an RCR vector of the present disclosure followed by treatment with a chemotherapeutic agent or an anti-cancer agent. In one aspect, the RCR vector is administered to the subject for a period of time allowing RCR infection and replication prior to administration of chemotherapy or anti-cancer agents. The subject is then treated with a chemotherapeutic or anticancer agent for a dosage and duration that inhibits growth or kills cancer cells. In one embodiment, if treatment with chemotherapy or an anti-cancer agent shrinks the cancer / tumor but cannot be killed (e.g., partial or temporary remission), then the subject will then have an RCR-derived cytotoxic gene (e.g., cytosine deaminase) Can be treated with a non-toxic therapeutic agent (eg, 5-FC) that is converted to a toxic therapeutic agent in cells expressing.

  Using such methods, the RCR vectors of the present disclosure spread during the tumor cell replication process, after which such cells are killed by treatment with anti-cancer or chemotherapeutic agents, and further killing is described herein. Can occur using the RCR treatment process.

  In yet other embodiments of the present disclosure, the heterologous gene can include a coding sequence for a target antigen (eg, a cancer antigen). In this embodiment, cells containing a cell proliferative disorder are infected with an RCR comprising a heterologous polynucleotide encoding the target antigen that results in expression of the target antigen (eg, overexpression of a cancer antigen). Thereafter, an anti-cancer agent comprising a targeting cognate moiety that specifically interacts with the target antigen is administered to the subject. The targeting cognate moiety can be operably linked to a cytotoxic agent or can itself be an anticancer agent. Thus, cancer cells infected with an RCR containing a target antigen coding sequence have increased target expression in the cancer cells, thereby resulting in increased efficiency / effect of cytotoxic targeting.

  In still other embodiments, the RCRs of the present disclosure may include a coding sequence that includes a binding domain (eg, antibody, antibody fragment, antibody domain or receptor ligand) that specifically interacts with a cognate antigen or ligand. An RCR comprising a binding domain coding sequence can be used to infect cells in a subject comprising a cell proliferative disorder, such as a cancer or neoplastic cell. The infected cells will then express the binding domain or antibody. Subsequently, an antigen or cognate that is operably linked to the cytotoxic agent or is itself cytotoxic can be administered to the subject. The cytotoxic cognate will then kill infected cells that selectively express the binding domain. Alternatively, the binding domain itself can be an anticancer agent.

The term “antibody” as used herein refers to a protein comprising at least one immunoglobulin variable domain or immunoglobulin variable domain sequence. For example, an antibody can comprise a heavy (H) chain variable region (abbreviated herein as VH) and a light (L) chain variable region (abbreviated herein as VL). In other examples, the antibody comprises two heavy (H) chain variable regions and two light (L) chain variable regions. The term “antibody” includes antigen-binding fragments of antibodies (eg, single chain antibodies, Fab fragments, F (ab ′) ₂ , Fd fragments, Fv fragments, and dAb fragments), and intact antibodies.

  The present disclosure provides a method for treating a subject having a cell proliferative disorder. The subject may be any mammal, preferably a human. The subject is contacted with a recombinant replication competent retroviral vector of the present disclosure. Contact can be in vivo or ex vivo. Methods for administering the retroviral vectors of the present invention are known to those skilled in the art, such as systemic administration, local administration, intraperitoneal administration, intramuscular administration, intracranial administration, cerebrospinal administration, and tumor or cell proliferative disorders. Includes direct administration to the site. Other routes of administration are also known in the art.

  Accordingly, the present disclosure includes various pharmaceutical compositions useful for treating cell proliferative disorders. A pharmaceutical composition according to the present disclosure comprises a retroviral vector comprising a heterologous polynucleotide sequence useful for treating or modulating a cell proliferative disorder according to the present disclosure, using carriers, excipients and additives or adjuvants. Prepared in a form suitable for administration to a subject. Commonly used simple substances or adjuvants include magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, milk protein, gelatin, starch, vitamins, cellulose and derivatives thereof, animal and vegetable oils, polyethylene glycol and sterile water, Solvents such as alcohol, glycerol and polyhydric alcohol can be mentioned. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antibacterial agents, antioxidants, chelating agents and inert gases. Other pharmaceutically usable carriers include, for example, Remington's Pharmaceutical Sciences, 15th ed. Easton: Mack Publishing Co., 1405-1412, 1461-1487 (1975) and The National, the contents of which are incorporated herein by reference. Examples include aqueous solutions, non-toxic excipients such as salts, preservatives, and buffers described in Formulary XIV., 14th ed. Washington: American Pharmaceutical Association (1975). The pH and exact concentration of the various components of the pharmaceutical composition are adjusted according to routine techniques in the art. See Goodman and Gilman's The Pharmacological Basis for Therapeutics (7th ed.).

  For example, but not limited to, retroviral vectors useful for the treatment of cell proliferative disorders include broad-host ENV proteins, GAG and POL proteins, promoter sequences in the U3 region of the retroviral genome, and replication, packaging And includes all cis-acting sequences necessary for integration of retroviral genomes into target cells.

  The following examples are intended to illustrate the present disclosure and are not intended to limit the present disclosure. Such an embodiment is exemplary that can be used, and other procedures known to those skilled in the art can be used instead.

  Expression level of yCD2 and conversion of 5-FC to 5-FU by yCD2 is efficient both in vivo and in vitro when cells are completely infected with Toca511 (pAC3-yCD2, SEQ ID NO: 22) And was shown to be stable. However, long-term (about 180 days) in vivo pilot studies show that infected Balb / c integrated proviruses from several tissues have extended or contracted oligo A sequences in the JK branch loop. It was. In tissues from 4 mice in biolocalization studies analyzed by molecular PCR cloning, heterogeneous 7 A to 8 A, 9 A, 10 A, 11 A, And 12 A stretches and 7 A to 6 A contractions were observed. This observation, and the 7 A in pEMCF versus the 6 A of ECMV IRES described first, indicate the effect of yCD2 expression mediated by IRES with varying numbers of A in the A bulge, and in particular the context of RRV Led to the study of the effects on protein translation. Therefore, a series of deletion and insertion mutants specific for the branch region A bulge were generated. Data show that neither deletion nor insertion of A bulge oligo A sequence affects RRV production, 6 A results in maximum CD and green fluorescent protein (GFP) expression, and 6 A A small change in the number of A's has a moderate effect, however, a larger change has a dramatic effect on the efficiency of IRES-mediated translation of mRNA from the transgene.

Construction of RRV containing various numbers of A's in A bulge in JK branch region
A RRV was prepared that had 4, 5, 6, 7, 8, 10, or 12 A in the A-bulge of the JK branch region, contained EMCV IRES, and encoded CD or GFP. For direct replacement of equivalent cassettes in the RRV backbone (Figure 1B), each construct was made by DNA synthesis of the entire IRES cassette with Mlu I at the 5 'end and Psi I at the 3' end, respectively. . All DNA fragments were confirmed by sequence analysis before and after cloning into the RRV backbone. RRV constructs containing the yCD2 transgene were named using the name of the transgene followed by the number of A's in the A bulge (eg, yCD2-4A adds 4 A to the yCD2 transgene and the IRES A bulge. Including).

RRV containing various numbers of A's in A bulge produced viral stocks that yielded similar titers by transient transfection of 293T cells using the calcium phosphate precipitation method. Viral supernatant was collected 42 hours after transfection. Viral infection was performed to determine the titer. Subsequently, HT1080 cells were infected with the virus supernatant of each vector to prepare RRV-producing cells. Prior to infecting naive U87-MG cells, the titer of the resulting virus was measured. Figure 1C shows that HT1080 cells infected with RRV containing various numbers of A produced similar levels of virus, suggesting that the number of A in the branch loop does not affect virus replication. doing.

RRV containing various numbers of A's in the JK branch region express equivalent levels of transcripts, but express different levels of protein, and then virus supernatants from HT1080 cells, with an infection efficiency (MOI) of 0.1 Was used to infect naive U87-MG cells. Ten days after infection when the cells were completely infected, the viral RNA levels of the cells were measured by quantitative real-time polymerase chain reaction (qRT-PCR), and the expression level of yCD2 protein was examined by immunoblotting (Perez et al. , 2012). Viral RNA expression levels of the cells are respectively determined in the env region (5'Env2: 5'-ACCCTCAACCTCCCCTACAAGT-3 ', 3'Env2: 5'-GTTAAGCGCCTGATAGGCTC-3', probe: 5'FAM-AGCCACCCCCAGGAACTGGAGA TAGA-3'BHQ), And two different primer sets located in the yCD2 region (5'yCD2: 5'-ATCATCATGTACGGCATCCCTAG-3 ', 3'yCD2: 5'-TGAA CTGCTTCATCA GCTTCTTAC-3', probe: 5'FAM-TCATCGTCAACAACCACCACCTCGT-3'BHQ) (Fig. 2A). The relative level of RNA from each vector was calculated using the 2- ^{ΔΔ (Ct)} method compared to a vector containing 6 A's. Cellular viral RNA level ratios ranged from 0.8 to 1.1 (FIG. 2B), indicating that there is no significant difference in viral RNA transcripts due to IRES modification. Western blot analysis of yCD2 protein expression levels of these vectors identified that the expression level of yCD2 protein for vectors containing 5 and 7 A was 69% and 77% of the expression level of yCD2-6A vector It was done. In contrast, a substantial reduction in yCD2 protein expression was observed in vectors containing 4, 8, 10 and 12 A. The CD protein expression level of these vectors is in the range of 4-25% of the expression level of the yCD2-6A vector (FIG. 2B). Equivalent cellular viral RNA levels and a dramatic decrease in yCD2 protein expression suggested that the length of oligo A in the IRES branch region could have a significant effect on gene expression at the post-transcriptional level. Relative intracellular CD enzyme activity was also measured by adding 5 FC to the culture and measuring 5-FU after 1 hour. The difference in activity was located similar to the Western blot data, but was not significant. This may be due to the low availability of intracellular 5-FC is less than K _m of the enzyme in an assay of cell-based assays limits, and are utilized. Therefore, the effect of the number of A's in the loop was analyzed with other transgenes for which protein expression assays were well defined. Also, using different transgenes will allow to determine whether changes in yCD2 protein expression with changes in the number of A's in A bulge are transgene specific.

  An equivalent set of RRV encoding GFP was made. The viral titers of these vectors were also comparable to the other, and this data was found to be very similar to the yCD2 transgene data (FIG. 1C). GFDP expression levels were measured by gating GFP positive cells using flow cytometry. The mean fluorescence intensity (MFI) of each vector was normalized to the viral RNA level of the cells and calculated for the GFP-6A vector. The results from this set of vectors (Figure 2C) are consistent with those observed in the yCD2 vector (Figure 2B), with the vector containing 6 A being the highest from the transgene in both sets of vectors Expresses level of protein. Furthermore, due to the sensitivity of the detection method, significant differences in GFP expression levels were observed, with GFP expression by vectors containing 10 A and 12 A showing a decrease of about 96% and 99%, respectively. It is. In both sets of vectors, RRV with 7 A showed about 30% reduction in protein expression. Consistent with the findings reported by Hoffman et al, RRV with 4 A and 5 A is significantly reduced compared to RRV with 6 A described by Hoffman et al. Phenotypes similar to 868Δ4, each with high protein translation efficiency.

  The present disclosure shows that the length of the A-bulge in the JK branch region affects transgene expression downstream of IRES, possibly through an effect on translation efficiency. To date, the context around AUG11, the spacing between the polypyrimidine tract located in the 3'IRES and the first AUG in the cistron, and the placement of the cistron in the mRNA play a role in modifying protein translation It implies. The data show that the presence of 6 A results in the highest level of transgene protein expression, and changing the number of A in A bulge by contraction or extension of 2-4 nucleotides results in expression of the transgene downstream of IRES. It shows that it can significantly affect the level. Protein expression results show that the optimal IRES configuration generally has 6 A in the branching loop, but 7 A by rescue with polypyrimidine tract binding protein (PTB) previously described by Kaminiski et al. Suggests that extension of bulge A from 6 A to 7 A makes the function of IRES dependent on polypyrimidine tract binding protein (PTB). ing. Vector variants with 4, 5, 8, 10, and 12 A also require binding of PBT to the polypyrimidine tract for efficient protein translation, and these vector variants It may also distort the secondary and tertiary structure of IRES, thus impairing the binding of PBT and / or other trans-acting factors to the polypyrimidine tract and thus abrogating the rescue of PBT-mediated translational activity. Other than the EMCV IRES synthetic construct created for the bicistronic expression vector, no variation in the number of adenosine residues in the ECMV A-bulge is described. The change in the number of adenosine residues is unlikely to be driven by some sort of selective pressure, but rather appears to have occurred during 180 days of extensive RRV replication in mice due to mutated reverse transcription activity. In conclusion, in RRV including ECMV IRES, not only for increased transgene expression, but more frequent induction of oligo A number drift seems to be preferentially seen in longer bulge A However, it is preferable to use the 6A version of IRES. Thus, if the bulge begins with 6A's, there is a higher tolerance for the transgene to the acquisition of a single extra adenosine nucleotide.

An RRV construct with a minimal IRES with 6 A results in similar titers, viral transcripts, and transgene protein expression as an RRV with 6A alone
Mutants generated by progressive deletion from 5'EMCV IRES have been shown to have differential translational efficiency (Duke et al., J Virol. 66: 1602-9 1992). Various lengths designated herein as 6A-406 (eg, bases 123-544 of SEQ ID NO: 41) and 6A-466 (bases 183-544 of SEQ ID NO: 41) (see FIG. 5) Create an RRV containing the smallest IRES. Other similar constructs with other numbers of either A's and either 406 or 466 IRES sequences can also be constructed (designated 7A-406 and 7A-466 (refers to 7A etc. with minimal IRES)) and equivalent numbers Can be implemented roughly in proportion to a construct having a maximum of A's and a maximum length of IRES. For direct replacement of equivalent cassettes in the RRV backbone, each construct was made by DNA synthesis (BioBasics Inc.) of the entire IRES cassette with Mlu I at the 5 ′ end and Psi I at the 3 ′ end, respectively. To do. All DNA fragments are confirmed by sequence analysis before and after cloning into the RRV backbone. RRV constructs containing the yCD2 transgene were named using the name of the transgene followed by the number of A's in the A bulge (eg, yCD2-4A adds 4 A to the yCD2 transgene and the IRES A bulge. Including). The data show that the titers from transiently transfected 293T and maximally infected HT1080 cells are similar to that of the bulge A mutant. The protein expression of yCD2 is measured from fully infected U87-MG cells. The 6A-406 mutant expresses an equivalent level (within 2, 5, or 10 fold) of yCD2 protein compared to the 6A mutant with full-length IRES. The 6A-466 mutant with an additional deletion of 5'IRES shows expression of yCD2. In addition, replication kinetics and vector stability data from staged infection also indicate that both 6A-406 and 6A-466 are stable up to at least 10 infection cycles.

  Several embodiments of the present disclosure have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the disclosure. Accordingly, other embodiments are within the scope of the appended claims.

Claims (93)

A recombinant replication competent retrovirus comprising:
Retroviral GAG protein;
Retroviral POL protein;
Retroviral envelope;
A retroviral polynucleotide, a long terminal repeat (LTR) sequence at the 3 ′ end of the retroviral polynucleotide sequence, a promoter sequence suitable for expression in mammalian cells at the 5 ′ end of the retroviral polynucleotide, a gag nucleic acid Said retroviral polynucleotide comprising a domain, a pol nucleic acid domain and an env nucleic acid domain;
A cassette containing an internal ribosome entry site (IRES) consisting of 6A's of A bulge in the IRES branch region, wherein the IRES is operably linked to a heterologous polynucleotide, and the cassette is 5 'to the 3'LTR. And a cassette located 3 ′ of the env nucleic acid domain encoding the retroviral envelope; and cis-acting sequences necessary for reverse transcription, packaging and integration in the target cell,
The above-mentioned recombinant replication competent retrovirus, which maintains high replication ability after 6 passages compared to a vector (pACE) in which the RCR contains SEQ ID NO: 21.   2. The recombinant replication competent retrovirus of claim 1, wherein the virus infects the target cell multiple times resulting in an average copy number / diploid genome of 5 or greater.   Retroviral polynucleotide sequences are murine leukemia virus (MLV), Moloney murine leukemia virus (MoMLV), feline leukemia virus (FeLV), baboon endogenous retrovirus (BEV), porcine endogenous virus (PERV), feline-derived retrovirus 2. From a virus selected from the group consisting of RD114, squirrel monkey retrovirus, heterologous murine leukemia-related virus (XMRV), avian reticuloendotheliosis virus (REV), and gibbon leukemia virus (GALV). The recombinant replication competent retrovirus described.   2. The recombinant replication competent retrovirus of claim 1, wherein the retroviral envelope is a broad host MLV envelope.   The recombinant replication competent retrovirus according to claim 1, wherein the retrovirus is a gamma retrovirus.   The recombinant replication competent retrovirus according to claim 1, wherein the target cell is a cell having a cell proliferative disorder.   The recombinant replication competent retrovirus according to claim 1, wherein the target cell is a neoplastic cell.   Cell proliferative disorders from lung cancer, colorectal cancer, breast cancer, prostate cancer, urinary tract cancer, uterine cancer, brain cancer, head and neck cancer, pancreatic cancer, melanoma, gastric cancer and ovarian cancer, rheumatoid arthritis and other autoimmune diseases The recombinant replication competent retrovirus according to claim 6, wherein the recombinant replication competent retrovirus is selected from the group consisting of:   2. A recombinant replication competent retrovirus according to claim 1, wherein the promoter sequence is associated with a growth regulatory gene.   2. The recombinant replication competent retrovirus of claim 1, wherein the promoter sequence comprises a tissue specific promoter sequence.   11. The recombinant replication competent retrovirus of claim 10, wherein the tissue specific promoter sequence comprises at least one androgen response element (ARE).   The promoter includes a CMV promoter having nucleotide 1 to about nucleotide 582 of the sequence set forth in SEQ ID NO: 19, 20, 22, or 42, and may include one or more nucleobase modifications that can induce and initiate transcription. The recombinant replication competent retrovirus according to claim 1.   2. The recombinant replication competent retrovirus of claim 1, wherein the promoter comprises a CMV-R-U5 domain polynucleotide.   14. The recombinant replication competent retrovirus of claim 13, wherein the CMV-R-U5 domain comprises an early promoter from human cytomegalovirus linked to the MLV R-U5 region.   CMV-R-U5 domain polynucleotide is at least 95% from about nucleotide 1 to about nucleotide 1202 of the sequence set forth in SEQ ID NO: 19, 20, 22 or 42, or the sequence set forth in SEQ ID NO: 19, 20, 22 or 42 15. A recombinant replication competent retrovirus according to claim 14, comprising a sequence that is identical and wherein said polynucleotide facilitates transcription of a nucleic acid molecule operably linked thereto.   2. The recombinant replication competent retrovirus of claim 1, wherein the gag polynucleotide is derived from a gamma retrovirus.   The gag nucleic acid domain comprises a sequence having from about nucleotide number 1203 to about nucleotide 2819 of SEQ ID NO: 19, 20, 22, or 42, or at least 95%, 98%, 99% or 99.8% identity thereto. Item 17. The recombinant replication competent retrovirus according to Item 16.   2. The recombinant replication competent retrovirus of claim 1, wherein the pol domain of the polynucleotide is derived from a gamma retrovirus.   The pol domain comprises a sequence having from about nucleotide number 2820 to about nucleotide 6358 of SEQ ID NO: 19, 20, 22, or 42, or at least 95%, 98%, 99% or 99.8% identity thereto. A recombinant replication competent retrovirus according to claim 18.   The env domain comprises a sequence having from about nucleotide number 6359 to about nucleotide 8323 of SEQ ID NO: 19, 20, 22 or 42, or at least 95%, 98%, 99% or 99.9% identity thereto. 2. The recombinant replication competent retrovirus according to 1.   The recombinant replication competent retrovirus according to claim 1, wherein the IRES consists of the sequence set forth in SEQ ID NO: 41.   The recombinant replicable competent retrovirus of claim 1, wherein the retroviral polynucleotide sequence comprises (i) the sequence set forth in SEQ ID NO: 42, or (ii) the sequence set forth in SEQ ID NO: 42 wherein T is U. .   The recombinant replication competent retrovirus of claim 1, wherein the heterologous nucleic acid comprises a polynucleotide having the sequence set forth in SEQ ID NO: 3, 5, 11, 13, 15, or 17.   2. The recombinant replication competent retrovirus of claim 1, wherein the heterologous nucleic acid encodes a polypeptide comprising the sequence set forth in SEQ ID NO: 4.   2. The recombinant replication competent retrovirus of claim 1, wherein the heterologous nucleic acid is human codon optimized and encodes the polypeptide set forth in SEQ ID NO: 4.   The recombinant replicable competent retrovirus of claim 1, wherein the heterologous nucleic acid comprises the sequence set forth in about nucleotide number 8877 to about 9353 of SEQ ID NO: 19 or 22.   The recombinant replication competent retrovirus according to claim 1, wherein the 3'LTR is derived from a gamma retrovirus.   28. The recombinant replication competent retrovirus of claim 27, wherein the 3'LTR comprises a U3-R-U5 domain.   The recombination of claim 28, wherein the 3'LTR comprises the sequence set forth from about nucleotide 9405 to about 9988 of SEQ ID NO: 19 or 22, or a sequence that is at least 95%, 98%, or 99.5% identical thereto. Replication competent retrovirus.   2. The recombinant replication competent retrovirus of claim 1 wherein the heterologous nucleic acid sequence encodes a biological response modifier or an immunostimulatory cytokine.   31. The recombinant replication compilation of claim 30, wherein the immunostimulatory cytokine is selected from the group consisting of interleukins 1-15, interferon, tumor necrosis factor (TNF), and granulocyte macrophage colony stimulating factor (GM-CSF). Tent retrovirus.   The recombinant replication competent retrovirus according to claim 30, wherein the immunostimulatory cytokine is interferon gamma.   2. The recombinant replication competent retrovirus of claim 1, wherein the heterologous nucleic acid encodes a polypeptide that converts a non-toxic prodrug to a toxic agent.   34. The recombinant replication competent retrovirus according to claim 33, wherein the polypeptide that converts a non-toxic prodrug to a toxic drug is thymidine kinase, purine nucleoside phosphorylase (PNP), or cytosine deaminase.   2. The recombinant replication competent retrovirus of claim 1, wherein the heterologous nucleic acid sequence encodes a receptor domain, antibody, or antibody fragment.   2. The recombinant replication competent retrovirus of claim 1, wherein the heterologous nucleic acid sequence comprises an inhibitory polynucleotide.   38. The recombinant replication competent retrovirus of claim 36, wherein the inhibitory polynucleotide comprises a miRNA, RNAi, or siRNA sequence.   A recombinant retroviral polynucleotide genome for producing the retrovirus of claim 1.   35. A cell proliferative disorder comprising contacting the subject with the recombinant replication competent retrovirus of claim 34 under conditions in which a cytosine deaminase polynucleotide is expressed, and contacting the subject with 5-fluorocytosine. How to treat.   40. The method of claim 39, wherein the cell proliferative disorder is glioblastoma multiforme.   The cell proliferative disorder is selected from the group consisting of lung cancer, colorectal cancer, breast cancer, prostate cancer, urinary tract cancer, uterine cancer, brain cancer, head and neck cancer, pancreatic cancer, melanoma, gastric cancer and ovarian cancer. 40. The method according to 39.   A vector for expressing a heterologous gene in mammalian cells from ECMV IRES having 6 A in the A bulge of the J-K branch region.   43. The vector of claim 42, wherein the vector is a viral vector.   44. The vector of claim 42 or 43, which is a retroviral replication vector.   45. The vector according to any one of claims 42 to 44, which is a retroviral replication vector derived from a gamma retrovirus.   46. The vector of claim 45, wherein the gamma retrovirus is derived from any one of murine leukemia virus, baboon endogenous virus, gibbon leukemia virus, feline leukemia virus.   47. The vector according to any one of claims 42 to 46, wherein the heterologous gene is a gene having therapeutic activity in a mammal.   48. The vector of claim 47, wherein the therapeutic activity is anticancer activity.   48. The vector of claim 47, wherein the heterologous gene is a prodrug activating gene.   50. The vector according to any one of claims 42 to 49, capable of expressing a heterologous gene in mammalian cells from ECMV IRES in the absence of PTB-1 protein.   51. A method for treating cancer, comprising administering the vector according to any one of claims 42 to 50. A recombinant replication competent retrovirus comprising:
Retroviral GAG protein;
Retroviral POL protein;
Retroviral envelope;
A retroviral polynucleotide, a long terminal repeat (LTR) sequence at the 3 ′ end of the retroviral polynucleotide sequence, a promoter sequence suitable for expression in mammalian cells at the 5 ′ end of the retroviral polynucleotide, a gag nucleic acid Said retroviral polynucleotide comprising a domain, a pol nucleic acid domain and an env nucleic acid domain;
(I) a minimal internal ribosome entry site (IRES) operably linked to a heterologous polynucleotide, (ii) a polIII promoter linked to a miRNA, or (iii) a heterologous polynucleotide preceding or following (ii) A cassette comprising a operably linked mini-promoter, located 5 ′ of the 3 ′ LTR and 3 ′ of the env nucleic acid domain encoding the retroviral envelope; and reverse transcription, packaging in the target cell Cis-acting sequences required for binding and integration;
A recombinant replication competent retrovirus comprising the above.   53. The replication competent retrovirus of claim 52, wherein the minimal IRES consists of a sequence of about bases 123 to 544 of SEQ ID NO: 41.   53. The replication competent retrovirus of claim 52, wherein the minimal IRES consists of a sequence of about bases 183 to 544 of SEQ ID NO: 41.   55. A replication competent retrovirus according to any one of claims 52 to 54, wherein the IRES has 6 A's in the A bulge.   53. The recombinant replication competent retrovirus of claim 52, wherein the virus infects the target cell multiple times resulting in an average copy number / diploid genome of 5 or greater.   Retroviral polynucleotide sequences are murine leukemia virus (MLV), Moloney murine leukemia virus (MoMLV), feline leukemia virus (FeLV), baboon endogenous retrovirus (BEV), porcine endogenous virus (PERV), feline-derived retrovirus Derived from a virus selected from the group consisting of RD114, squirrel monkey retrovirus, heterologous murine leukemia-associated virus (XMRV), avian reticuloendotheliosis virus (REV), or gibbon leukemia virus (GALV). The recombinant replication competent retrovirus described.   53. The recombinant replication competent retrovirus of claim 52, wherein the retroviral envelope is a broad host MLV envelope.   53. The recombinant replication competent retrovirus of claim 52, wherein the retrovirus is a gamma retrovirus.   53. The recombinant replication competent retrovirus according to claim 52, wherein the target cell is a cell having a cell proliferative disorder.   53. The recombinant replication competent retrovirus according to claim 52, wherein the target cell is a neoplastic cell.   Cell proliferative disorders from lung cancer, colorectal cancer, breast cancer, prostate cancer, urinary tract cancer, uterine cancer, brain cancer, head and neck cancer, pancreatic cancer, melanoma, gastric cancer and ovarian cancer, rheumatoid arthritis and other autoimmune diseases 61. The recombinant replication competent retrovirus of claim 60, selected from the group consisting of:   53. The recombinant replication competent retrovirus of claim 52, wherein the promoter sequence is associated with a growth regulatory gene.   53. The recombinant replication competent retrovirus of claim 52, wherein the promoter sequence comprises a tissue specific promoter sequence.   65. The recombinant replication competent retrovirus of claim 64, wherein the tissue specific promoter sequence comprises at least one androgen response element (ARE).   The promoter includes a CMV promoter having nucleotide 1 to about nucleotide 582 of the sequence set forth in SEQ ID NO: 19, 20, 22, or 42, and may include one or more nucleobase modifications that can induce and initiate transcription. 53. The recombinant replication competent retrovirus of claim 52.   53. The recombinant replication competent retrovirus of claim 52, wherein the promoter comprises a CMV-R-U5 domain polynucleotide.   68. The recombinant replication competent retrovirus of claim 67, wherein the CMV-R-U5 domain comprises an early promoter from human cytomegalovirus linked to the MLV R-U5 region.   CMV-R-U5 domain polynucleotide is at least 95% from about nucleotide 1 to about nucleotide 1202 of the sequence set forth in SEQ ID NO: 19, 20, 22 or 42, or the sequence set forth in SEQ ID NO: 19, 20, 22 or 42 69. The recombinant replication competent retrovirus of claim 68, comprising a sequence that is identical and wherein said polynucleotide facilitates transcription of a nucleic acid molecule operably linked thereto.   53. The recombinant replication competent retrovirus of claim 52, wherein the gag polynucleotide is derived from a gamma retrovirus.   The gag nucleic acid domain comprises a sequence having from about nucleotide number 1203 to about nucleotide 2819 of SEQ ID NO: 19, 20, 22, or 42, or at least 95%, 98%, 99% or 99.8% identity thereto. Item 71. The recombinant replication competent retrovirus according to Item 70.   53. The recombinant replication competent retrovirus of claim 52, wherein the pol domain of the polynucleotide is derived from a gamma retrovirus.   The pol domain comprises a sequence having from about nucleotide number 2820 to about nucleotide 6358 of SEQ ID NO: 19, 20, 22, or 42, or at least 95%, 98%, 99% or 99.9% identity thereto. 72. The recombinant replication competent retrovirus according to 72.   The env domain comprises 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. 53. A recombinant replication competent retrovirus according to 52.   53. The recombinant replication competent retrovirus of claim 52, wherein the heterologous nucleic acid comprises a polynucleotide having the sequence set forth in SEQ ID NO: 3, 5, 11, 13, 15, or 17.   53. The recombinant replication competent retrovirus of claim 52, wherein the heterologous nucleic acid encodes a polypeptide comprising the sequence set forth in SEQ ID NO: 4.   53. The recombinant replication competent retrovirus of claim 52, wherein the heterologous nucleic acid is human codon optimized and encodes the polypeptide set forth in SEQ ID NO: 4.   53. The recombinant replication competent retrovirus of claim 52, wherein the heterologous nucleic acid comprises the sequence set forth in about SEQ ID NO: 19 or 22 from about nucleotide number 8877 to about 9353.   53. The recombinant replication competent retrovirus of claim 52, wherein the 3'LTR is derived from a gamma retrovirus.   80. The recombinant replication competent retrovirus of claim 79, wherein the 3'LTR comprises a U3-R-U5 domain.   80. The recombinant of claim 79, wherein the 3'LTR comprises the sequence set forth from about nucleotide 9405 to about 9988 of SEQ ID NO: 19 or 22, or a sequence that is at least 95%, 98%, or 99.5% identical thereto. Replication competent retrovirus.   53. The recombinant replication competent retrovirus of claim 52, wherein the heterologous nucleic acid sequence encodes a biological response modifier or an immunostimulatory cytokine.   84. The recombinant replication compilation of claim 82, wherein the immunostimulatory cytokine is selected from the group consisting of interleukins 1-15, interferon, tumor necrosis factor (TNF), and granulocyte macrophage colony stimulating factor (GM-CSF). Tent retrovirus.   83. The recombinant replication competent retrovirus of claim 82, wherein the immunostimulatory cytokine is interferon gamma.   53. The recombinant replication competent retrovirus of claim 52, wherein the heterologous nucleic acid encodes a polypeptide that converts a non-toxic prodrug to a toxic agent.   86. The recombinant replication competent retrovirus of claim 85, wherein the polypeptide that converts a non-toxic prodrug to a toxic drug is thymidine kinase, purine nucleoside phosphorylase (PNP), or cytosine deaminase.   53. The recombinant replication competent retrovirus of claim 52, wherein the heterologous nucleic acid sequence encodes a receptor domain, antibody, or antibody fragment.   53. The recombinant replication competent retrovirus of claim 52, wherein the heterologous nucleic acid sequence comprises an inhibitory polynucleotide.   90. The recombinant replication competent retrovirus of claim 88, wherein the inhibitory polynucleotide comprises a miRNA, RNAi, or siRNA sequence.   53. A recombinant retroviral polynucleotide genome for producing the retrovirus of claim 52.   90. A cell proliferative disorder comprising contacting a subject with a recombinant replication competent retrovirus according to claim 86 under conditions in which a cytosine deaminase polynucleotide is expressed, and contacting a subject with 5-fluorocytosine. How to treat.   92. The method of claim 91, wherein the cell proliferative disorder is glioblastoma multiforme.   The cell proliferative disorder is selected from the group consisting of lung cancer, colorectal cancer, breast cancer, prostate cancer, urinary tract cancer, uterine cancer, brain cancer, head and neck cancer, pancreatic cancer, melanoma, gastric cancer and ovarian cancer. 91. The method according to 91.

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