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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
  • C12N15/86—Viral vectors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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  • C12N2740/00—Reverse transcribing RNA viruses
  • C12N2740/00011—Details
  • C12N2740/10011—Retroviridae
  • C12N2740/10041—Use of virus, viral particle or viral elements as a vector
  • C12N2740/10043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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  • C12N2800/00—Nucleic acids vectors
  • C12N2800/10—Plasmid DNA
  • C12N2800/108—Plasmid DNA episomal vectors
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  • C12N2810/00—Vectors comprising a targeting moiety
  • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
  • C12N2810/60—Vectors comprising as targeting moiety peptide derived from defined protein from viruses
  • C12N2810/6072—Vectors comprising as targeting moiety peptide derived from defined protein from viruses negative strand RNA viruses
  • C12N2810/6081—Vectors comprising as targeting moiety peptide derived from defined protein from viruses negative strand RNA viruses rhabdoviridae, e.g. VSV
• • C—CHEMISTRY; METALLURGY
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  • C12N2830/00—Vector systems having a special element relevant for transcription
  • C12N2830/38—Vector systems having a special element relevant for transcription being a stuffer
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N2830/00—Vector systems having a special element relevant for transcription
  • C12N2830/42—Vector systems having a special element relevant for transcription being an intron or intervening sequence for splicing and/or stability of RNA
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N2840/00—Vectors comprising a special translation-regulating system
  • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron

Definitions

• This invention relates to lentiviral vectors useful in polynucleotide delivery, and more specifically to caprine arthritis encephalitis virus-based vectors useful in polynucleotide delivery to non-dividing and dividing cells.
• Lenti viruses a re a s ubgroup o f r etroviruses t hat are c apable o f i nfecting n on- dividing, as well as dividing cells.
• Vectors derived from lentiviruses are ideal tools for delivering exogenous genes to target cells because of their ability to stably integrate into the genome of dividing and non-dividing cells and to mediate long- term gene expression (Gilbert and Wong-Staal, 2001; Mitrophanous et al., 1999; Naldini et al., 1996; Sauter and Gasmi, 2001).
• Lentiviruses have been isolated from many vertebrate species including primates, e.g., human and simian immunodeficiency viruses (HIV-I, HIV-2, SIV), as well as non-primates, e.g., feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), equine infectious virus (EIAV), caprine arthritis encephalitis virus (CAEV) and the visna virus.
• FV feline immunodeficiency virus
• BIV bovine immunodeficiency virus
• EIAV equine infectious virus
• CAEV caprine arthritis encephalitis virus
• visna virus e.g., HIV and SIV are presently best understood.
• use of such systems in humans raises serious safety concerns, due to the possibility of recombination by the vector into a virulent and disease-causing form. Accordingly, non-primate lentiviruses are preferred for use in gene therapy.
• CAEV like all lentiviruses, can infect and replicate in dividing cells as well as in terminally differentiated and non-dividing cells.
• FFV Fluoro-Fi Protected virus
• EIAV EIAV [US 2001/0044149]
• CAEV like all lentiviruses, can infect and replicate in dividing cells as well as in terminally differentiated and non-dividing cells.
• Several features of CAEV biology make this virus an attractive candidate to develop into a gene transfer/therapy vector.
• the normal host of CAEV is goats, and there are no reported cases of human infection by CAEV.
• the CAEV genome is phylogenetically most distant from HIV-I among lentiviruses.
• the genome organization of the CAEV is relatively simple compared with other lentiviruses.
• the CAEV genome contains three structural g enes (gag, pol, env) and three regulatory/accessory g enes (vif, tat and rev).
• the present invention is broadly directed to the production of CAEV-based lentiviral vector particles useful for delivering exogenous polynucleotides into target cells. These vector particles find use in anti-viral, anti-tumor and/or gene therapies.
• the present invention provides in one aspect a transfer vector for use in a CAEV-based vector production system described herein, the transfer vector comprises (a) a CAEV packaging sequence consisting essentially of (i) the untranslated region between the CAEV 5' LTR and the CAEV g ⁇ g-encoding sequence, and (ii) nucleotides 1 to X of the CAEV g ⁇ g-encoding sequence linked to the 3' end of said untranslated region, wherein X is less than 613, and (b) cz ' s-acting elements required for polyadenylation, RNA transport, reverse transcription, and integration, in operable association with said packaging sequence.
• a CAEV packaging sequence consisting essentially of (i) the untranslated region between the CAEV 5' LTR and the CAEV g ⁇ g-encoding sequence, and (ii) nucleotides 1 to X of the CAEV g ⁇ g-encoding sequence linked to the 3' end of said untranslated region, wherein X is less than 6
• X is selected from the group consisting of: 60, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575 and 600.
• X is selected from the group consisting of:
• X is greater than 25 and less than 300, (e) X is greater than 25 and less than 200,
• X is greater than 50 and less than 500
• X is greater than 50 and less than 400
• X is greater than 50 and less than 300
• x is greater than 50 and less than 200
• (k) X is greater than 75 and less than 600
• (s) X is greater than 100 and less than 300, (t) X is greater than 100 and less than 200,
• X is greater than 125 and less than 500
• w is greater than 125 and less than 400
• x is greater than 125 and less than 300
• y is greater than 125 and less than 200
• z is greater than 150 and less than 600
• aa is greater than 150 and less than 500
• X is greater than 150 and less than 400, (cc) X is greater than 150 and less than 300, (dd) X is greater than 150 and less than 200, (ee) X is greater than 200 and less than 600, (ff) X is greater than 200 and less than 500,
• (gg) X is greater than 200 and less than 400, (hh) X is greater than 200 and less than 300, (ii) X is greater than 200 and less than 200, (jj) X is greater than 250 and less than 600, (kk) X is greater than 250 and less than 500,
• X is greater than 250 and less than 400, and (mm) X is greater than 250 and less than 300. In another embodiment, X is greater than 40 and less than 613. In another embodiment, X is greater than 57 and less than 613. In yet another embodiment, X is about 327.
• the start codon of the gag-encoding sequence is mutated to prevent translation of gag protein. In a further embodiment, the start codon is mutated to TAG.
• the ATG codon of the g ⁇ g-encoding sequence is located X base pairs downstream of the start codon ATG, wherein start codon is mutated to prevent translation of gag protein, and wherein X is less than 30. In a further embodiment X is about 21.
• the transfer vector of the invention may further comprise an RRE region.
• the transfer vector comprises the CAEV 3 ' LTR wherein the U3 region is deleted.
• the transfer vector of the present invention may further comprise a heterologous promoter.
• the heterologous promoter is the human cytomegalovirus major immediate early promoter (HCMV MIEP).
• the transfer vector is pCAH/SINdl (SEQ ID NO: 68).
• the transfer vector of the present invention may further comprise a transcription cassette comprising a heterologous polynucleotide of interest operably linked to a heterologous promoter (e.g., human cytomegalovirus major immediate-early promoter HCMV MIEP, or murine cytomegalovirus major immediate-early promoter
• a heterologous promoter e.g., human cytomegalovirus major immediate-early promoter HCMV MIEP, or murine cytomegalovirus major immediate-early promoter
• Such a transfer vector permits the incorporation of the polynucleotide of interest into virus particles, thereby providing a means for amplifying the number of infected host cells containing the polynucleotide therein.
• the present invention also provides a CAEV-based lentiviral vector system for producing CAEV-based, replication-defective vector particles useful in delivering exogenous polynucleotides into mammalian cells.
• the vector particles are capable of infecting and transducing mammalian cells.
• the vector system comprises the transfer vector described above, and a packaging vector system, wherein said packaging vector system comprises: a first polynucleotide comprising a CAEV gag- po/-encoding sequence and an RRE, and a second polynucleotide comprising a viral envelope encoding sequence.
• the second polynucleotide comprises a non-CAEV env- encoding sequence. In one embodiment the second polynucleotide comprises a VSV-G- or GaLV-encoding sequence.
• the CAEV vector system comprises a third polynucleotide sequence comprising a rev-encoding sequence.
• the CAEV vector system comprises a fourth polynucleotide sequence comprising a vz/-encoding sequence.
• the first polynucleotide of each of the CAEV vector systems described above further comprises a heterologous regulatory sequence operably linked to the CAEV gag-pol-encoding sequence.
• the second polynucleotide of the above- described CAEV vector systems further comprises a heterologous regulatory sequence operable linked to said viral envelope-encoding sequence.
• the third polynucleotide further comprises a heterologous regulatory sequence operably linked to the rev-encoding sequence.
• the fourth polynucleotide further comprises a heterologous regulatory sequence operably linked to the vz/-encoding sequence.
• the CAEV vector system comprises a packaging vector system which is devoid of a competent CAEV packaging sequence.
• the packaging vector system is devoid of the 5 ' end of the CAEV genome between the splice donor site and the gag start codon.
• the CAEV vector system comprises a first vector comprising the first polynucleotide and a second vector comprising the second polynucleotide.
• the vector system comprises a first vector comprising the first polynucleotide, a second vector comprising the second polynucleotide, and a third vector comprising the third polynucleotide.
• the vector system comprises a first vector comprising the first polynucleotide, a second vector comprising the second polynucleotide, a third vector comprising the third polynucleotide, and a fourth vector comprising the fourth polynucleotide.
• the third vector may be pHYK/rev (SEQ ID NO: 75), and the fourth vector may be pHYK/vif (SEQ ID NO: 76).
• the vector system comprises a first vector comprising the first polynucleotide, the third polynucleotide and the fourth polynucleotide, and a second vector comprising the second polynucleotide.
• t he first v ector o f t he C AEV v ector s ystem comprises a CAEV g ⁇ g-encoding sequence and an RRE operable linked to a heterologous promoter.
• the promoter may be an MCMV MIEP.
• the CAEV vector system comprises the first vector pMGP/RRE (SEQ ID NO: 77).
• the second vector of the CAEV vector system is a VSV-G- encoding sequence operably linked to a heterologous promoter.
• the promoter may be an HCMV MIEP.
• the second vector may further comprise a beta globin intron.
• the CAEV vector system comprises the second vector pHGVSV-G (SEQ ID NO: 74).
• the second vector of the CAEV vector system is a GaLV ewv-encoding sequence operably linked to a heterologous promoter.
• the promoter may be an M CMV M IEP.
• T he s econd v ector may further c omprise a eukaryotic elongation factor- 1 alpha intron.
• the CAEV vector system comprises the second vector pMYKEF-1/env (SEQ ID NO: 72).
• Another aspect of the invention is a method of producing a CAEV-based - lentiviral vector particle useful for infecting mammalian cells.
• the method comprises (a) transfecting a cell with the vector system described supra, under conditions suitable for production of CAEV-based particles, where the vector particle is infection- and transduction- competent, and replication-defective, and (b) recovering the vector particle.
• the present invention also provides a composition comprising a CAEV-based lentiviral vector particle and optionally a carrier, where the vector particle is produced by the methods described supra.
• the present invention also provides a kit comprising the transfer vector or the
• the present invention also provides a packaging cell comprising a CAEV gag- po/-encoding sequence and RRE, and optionally a viral env-encoding sequence.
• the packaging cell may further comprise a rev-encoding and/or a vz/-encoding sequence.
• the cell is useful for packaging the RNA form of the transfer vector into an infection- and transduction-competent vector particle, which is replication-defective.
• the vector system comprises a cell comprising the first polynucleotide described supra.
• the vector system may further comprise the third and/or the fourth polynucleotide described supra.
• the vector system comprises a cell comprising the first polynucleotide and second polynucleotides described supra.
• the vector system may further comprise the third and/or the fourth polynucleotide described supra.
• the vector system comprises a cell comprising a first vector that comprises a CAEV g ⁇ g-po/-encoding sequence and an RRE.
• the first vector may further comprise a rev-encoding and/or a vz/-encoding sequence.
• the cell may comprise a first vector comprising a CAEV gag-pol- encoding sequence and an RRE, a second vector comprising a rev-encoding sequence and/or a third vector comprising a vz/-encoding sequence.
• the vector system comprises a cell comprising a first vector that comprises a CAEV g ⁇ g-/?o/-encoding sequence and an RRE, and a second vector that comprises a viral e «v-encoding sequence.
• the first vector may further comprise a rev-encoding and/or a vj/-encoding sequence.
• the cell may comprise a first vector comprising the CAEV g ⁇ g-;?o/-encoding sequence and an RRE, a second vector comprising a viral e nv-encoding sequence, and optionally a third vector comprising a rev-encoding sequence and/or a fourth vector comprising a vz/-encoding sequence.
• Another aspect of the present invention is a method of delivering a polynucleotide or polypeptide into a mammalian cell or replicating a polynucleotide molecule encoding said polypeptide, comprising contacting a mammalian cell with the vector particle described supra under conditions which may allow for integration of said polynucleotide into the genome of said cell and optionally under conditions allowing expansion of said polypeptide encoded by said polynucleotide.
• the mammalian cell may be a dividing cell, a non-dividing cell or a CD34+ stem cell.
• the method of delivering a polynucleotide or a polypeptide into a mammalian cell or replicating a polynucleotide molecule encoding said polypeptide may further comprise isolating the cell from a mammal prior to contacting the cell with the vector particle.
• the method may further comprise expanding said cell in culture after contacting it with the vector particle.
• the method may further comprise reintroducing the cell into a mammal before or after expanding the contacted cell.
• the p resent i nvention further p rovides a m ethod for d elivering a p olypeptide into a vertebrate, comprising administering to the vertebrate a CAEV -based lentiviral vector particle comprising a heterologous polynucleotide of interest, where the vector particle is produced by the method described supra, such that the polypeptide encoded by the delivered polynucleotide is expressed in the vertebrate, in an amount sufficient to be detectable or to elicit a biological response in the vertebrate.
• the present invention further provides a vector comprising a CAEV packaging sequence consisting essentially of (a) the untranslated region between the CAEV 5' LTR and the CAEV g ⁇ g-encoding sequence, and (b) nucleotides 1 to X of the CAEV g ⁇ g-encoding sequence linked to the 3' end of the untranslated region, wherein X is less than 613.
• a CAEV packaging sequence consisting essentially of (a) the untranslated region between the CAEV 5' LTR and the CAEV g ⁇ g-encoding sequence, and (b) nucleotides 1 to X of the CAEV g ⁇ g-encoding sequence linked to the 3' end of the untranslated region, wherein X is less than 613.
• the enhanced efficiency is achieved through the discovery of the optimal length of the untranslated region between the 5'LTR and the gag start codon and the g ⁇ g-encoding region, which serves as an efficient packaging sequence by allowing efficient encapsidation, which then results in increased viral titers.
• Viral titer is also improved by using a strong heterologous promoter in the d esign of the packaging plasmids.
• the enhanced safety is achieved through the construction of a tot-independent transfer vector and a plasmid-based packaging system.
• FIGURE 1 is a schematic illustration of the CAEV proviral genomic organization.
• FIGURE 2A is a schematic illustration of the plasmid pMGP/RRE (SEQ ID NO: 77).
• pMGP/RRE (SEQ ID NO: 77) is a 9,446 bp plasmid which contains an MCMV MIEP region (located at bp 1-660) located upstream of the CAEV gag-pol coding region (bp 709-5,243), the RRE region (5,426-5,627 or bp 5,368-5,669), and the bovine growth hormone (BGH) polyadenylation signal (bp 5,751-5,984).
• BGH bovine growth hormone
• the vector also contains a neomycin resistance gene coding region (bp 8,151-7,155), a SV40 origin of replication (bp 8,509-8,152), a Col El origin of replication (bp 6,115- 6,698), and an ampicillin resistance gene region (bp 9,362-8,528).
• FIGURE 2B is a schematic illustration of the plasmid pMGP/REV/RRE.
• pMGP/REV/RRE is a 9,924 bp plasmid which contains an MCMV MIEP region (located at bp 1-660) and the major splicing donor of CAEV (bp 688-704) located upstream of the CAEV gag-pol coding region (bp 726-5,258), the first exon rev coding region (bp 5,383-5,494), the RRE region (bp 5,540-5,841), the second exon rev coding region(bp 5,888-6,177), and the bovine growth hormone (BGH) polyadenylation signal (bp 6,229-6,462).
• BGH bovine growth hormone
• the vector also contains a neomycin resistance gene coding region (bp 7,633-8,629), a SV40 origin of replication (bp 8,987-8,630), a Col El origin of replication (bp 6,593-7,176), and an ampicillin resistance gene region (bp 9,840-9,006).
• FIGURE 3A is a schematic illustration of the plasmid pCAH/SINd (SEQ ID NO: 73).
• pCAH/SINd (SEQ ID NO: 73) is a 3,566 bp plasmid which contains the HCMV MIEP (bp 1-588), the R-U5 sequence regions in the CAEV 5'LTR (bp 611- 772), the RRE region (bp 796-1,154), and the U3-deleted CAEV 3'LTR region (bp 1,275-1,458).
• the vector also contains a Col El origin of replication (bp 1,863- 2,466), and a kanamycin resistance gene coding region (bp 2,698-3,510).
• FIGURE 3B is a schematic illustration of the plasmid pCAH/SINdO (SEQ ED NO: 67).
• pCAH/SINdO (SEQ ID NO: 67) is a 3,911 bp plasmid which contains the HCMV MIEP (bp 1-588), the R-U5 sequence regions in CAEV 5'LTR (bp 611-772), the residual untranslated sequences containing the primer binding site (PBS) (bp 773- 789), the RRE region (bp 1,141-1,499), and the U3-deleted CAEV 3'LTR region (bp 1,620-1,803).
• the vector also contains a Col El origin of replication (bp 2,208- 2,791) and a kanamycin resistance gene coding region (bp 3,043-3,855).
• FIGURE 3 C is a schematic illustration of the plasmid pCAH/SINdl (SEQ ID NO: 68).
• pCAH/SINdl (SEQ ID NO: 68) is a 4,238 bp plasmid which contains the HCMV MIEP (bp 1-588) promoter, the R-U5 sequence regions in the CAEV 5'LTR (bp 610-772), the residual untranslated sequences containing the PBS site (bp 773- 789), the 327 bp fragment of the gag gene (bp 1 , 121 - 1 ,448) with ATG to TAG point mutations at the start ATG codon (bpl 121-1123) and the ATG codon (bpl 142-1144) located downstream of the start ATG codon, the RRE region (bp 1,468-1,826) and the U 3 -deleted C AEV 3 'LTR region (bp 1 ,947-2,130).
• the vector also contains
• FIGURE 3D is a schematic illustration of the plasmid pCAH/SINd2 (SEQ ID NO: 69).
• Plasmid pCAH/SINd2 (SEQ ID NO: 69) is a 4,523 bp plasmid which contains the HCMV MIEP (bp 1-588), the R-U5 sequence regions in the CAEV 5'LTR (bp 610-772), the residual untranslated sequences containing the PBS site (bp 773-789), the 612 bp fragment of the gag gene (bp 1,121-1,733), with point mutations at the start ATG codon (bp 1121-1123) and the ATG codon (bp 1142-
• the vector also contains a Col El origin of replication (bp 2,820-3,403) and a kanamycin resistance gene " coding region (bp 3,655-4,467).
• FIGURE 3E is a schematic illustration of the plasmid pCAH/SINd3 (SEQ ID NO: 70).
• pCAH/SINd3 (SEQ ID NO: 70) is a 4,819 bp plasmid which contains the HCMV MEEP (bp 1-588), the R-U5 sequence regions in CAEV 5'LTR (bp 610-772), the residual untranslated sequences containing PBS site (bp 773-789), the 908bp fragment of the gag gene (bpl, 121 -2,029) with point mutations at the start ATG codon (bp 1121-1123) and the ATG codon (bp 1142-1144) located downstream of the start ATG codon, the RRE region (bp 2,049-2,407) and the U3-deleted CAEV 3'LTR region (bp 2,549-2,711).
• the vector also contains a Col El origin of replication (bp 3,116-3,699) and a kanamycin
• FIGURE 3F is a schematic illustration of the plasmid pCAH/SINd4 (SEQ ID NO: 71).
• pCAH/SINd4 (SEQ ID NO: 71) is a 5,112 bp plasmid which contains the HCMV MIEP (bp 1-588), the R-U5 sequence regions in the CAEV 5'LTR (bp 610- 772), the residual untranslated sequences containing the PBS site (bp 773-1,120), the 1198 bp fragment of the gag gene (bp 1,121-2,319) with point mutations at the start ATG codon (bp 1121-1123) and the ATG codon (bp 1142-1144) located downstream of the start ATG codon, the RRE region (bp 2,342-2,700) and the US- deleted CAEV 3'LTR region (bp 2,842-3,004).
• the vector also contains a Col El origin of replication (bp 3,409-3,992), and a
• FIGURE 3 G is a schematic illustration of the plasmid pCAH/SINdl/hlacZ (SEQ ID NO: 79).
• pCAH/SINdl/hlacZ (SEQ ID NO: 79) is an 8,127 bp plasmid derived from the p CAH/SINdl ( SEQ ID NO: 68) that expresses the 1 acZ reporter gene.
• the vector contains two HCMV MIEP promoter regions (located at bp 1-588 and bp 1,866-2,460, respectively), the R-U5 sequence regions in the CAEV 5'LTR (bp 610-772), the residual untranslated sequences containing the PBS site (bp 773- 789), the 325 bp fragment of gag gene (bp 1,121-1,446) with point mutations at the start ATG codon (bp 1121-1123) and the ATG codon (bp 1142-1144) located downstream of the start ATG codon, the RRE region (bp 1,466-1,836), the lacZ gene coding sequence (bp 2,541-5,711), and the U3-deleted CAEV 3'LTR region (bp 5,782-6,019).
• the vector also contains a Col El origin of replication (bp 6,424- 7,007), and a kanamycin resistance gene coding region (bp 7,259-8,071).
• FIGURE 3H is a schematic illustration of the plasmid pCAH/SINd60/hlacZ (SEQ ID N O: 78).
• P lasmid p CAH/SlNd ⁇ O/hlacZ SEQ ID N O: 78) i s a 7 ,856 b p which contains two promoter regions, HCMV MIEP (located at bp 1-588 and bp 1,595-2,189, respectively), the R-U5 sequence regions in the CAEV 5'LTR (bp 610- 772), the residual untranslated sequences containing the PBS site (bp773- 789 bp), the 60 bp fragment of gag gene (bp 1,121-1,181) with point mutations at the start ATG codon (bp 1121 - 1123) and the ATG codon (bp 1142- 1144) located downstream of the start ATG codon, the RRE region (bp 1,195-1,565), the lacZ gene coding sequence (b
• FIGURE 4 is a schematic illustration of the plasmid pHYK/vif (SEQ ID NO:
• pHYK/vif (SEQ ID NO: 76) is a 5,729 bp plasmid which contains the HCMV MIEP (bp 1-596), the vif gene coding region (bp 691-1,380), the BGH polyadenylation signal (bp 1,467-1,695), a Col El origin of replication (bp 1,826- 2,409), a neomycin resistance gene coding region (bp 3,862-2,866), and an ampicillin resistance gene coding region (bp 5,270-4,239).
• FIGURE 5 is a schematic illustration of the plasmid pHYK/rev (SEQ ID NO: 75).
• pHYK/rev (SEQ ID NO: 75) is a 5,419 bp plasmid which contains the HCMV MIEP (bp 1-596), the rev gene coding region (bp 672-1,073), the BGH polyadenylation signal (bp 1,157-1,385), a Col El origin of replication (bp 1,516- 2,099), a neomycin resistance gene coding region (bp 3,552-2,556), and an ampicillin resistance gene coding region (bp 4,960-3,929).
• FIGURE 6A is a schematic illustration of the plasmid pHGVSV-G (SEQ ID NO: 74).
• pHGVSV-G (SEQ ID NO: 74) is a 7,623bp plasmid which contains the HCMV MIEP (bp 1-596), the ⁇ -globin intron region (bp 714-1,599), the VSV-G coding region (bp 1,632-3,312), the BGH polyadenylation signal (bp 3,361-3,589), a Col El origin of replication (bp 3,720-4,303), a neomycin resistance gene coding region (bp 5,756-4,760), an ampicillin resistance gene coding region (bp 7,164- 6,133), and a Fl origin of replication (bp 7,165-7,621).
• FIGURE 6B is a schematic illustration of the plasmid pMYKEFl/env (SEQ ID NO: 72).
• pMYKEFl/env (SEQ ID NO: 72) is a 7,579 bp plasmid which contains the MCMV MIEP (bp 1-665), a human EFl- ⁇ intron region (bp 668-1,618), the GaLV env coding region (bp 1,699-3701), the BGH polyadenylation signal (bp 3,885- 4,118), a Col El origin of replication (bp 4,349-4,832), a neomycin resistance gene coding region (bp 6,290-5,284), and an ampicillin resistance gene coding region (bp 7,496-6,666).
• FIGURE 7 shows a photograph illustrating the relative amount of transfer vector RNA transcribed from gene transfer vectors transfected into human 293T target cells.
• FIGURE 8 shows two photographs illustrating gene transfer into human 293T target cells by CAEV (A) and MuLV (B) vectors.
• FIGURE 9 shows a photographic illustration of the relative amount of transfer vector RNA expressed in the transfected 293T cells (lanes 1, 2 and 3), and encapsidated in and released from the 293T packaging cells (lanes 4, 5 and 6).
• FIGURE 10 shows a photograph illustrating the relative amount of transfer vector RNA encapsidated in and released from human 293T packaging cells.
• FIGURE l l s hows a p hotograph i llustrating t he r elative a mount o f integrated retroviral cDNA after infection and reverse transcription of lentiviral vectors pseudotyped by VSV-G or GaLV envelope protein.
• FIGURE 12 shows a photograph illustrating the relative amount of viral vector cDNA integrated into the infected host cell chromosome.
• FIGURE 13 shows two graphs illustrating the FACS analysis of (A) the control cells, and (B) the Gl- arrested cells.
• FIGURE 14 shows two graphs illustrating (A) the number of transduced cells and (B) the relative transduction efficiencies of HIV-I-, CAEV-, and MuLV-derived viral vectors on dividing and non-dividing cells.
• the invention relates to, inter alia, CAEV-based lentiviral vector systems and methods employing said vectors to deliver polypeptides of interest into dividing and non-dividing cells.
• the wild-type CAEV virus has a dimeric RNA genome (single-stranded, positive polarity) that is replicated through a double-stranded DNA intermediates and is packaged into a spherical enveloped virion containing a nucleoprotein core.
• the genome contains three genes that encode the structural and enzymatic proteins Gag, Pol, a nd E nv, a nd 1 ong terminal r epeats ( LTR) at e ach e nd o f t he i ntegrated v iral genome.
• the genome encodes three regulatory proteins, vif, tat, and rev.
• the gag gene encodes the internal structural proteins
• the pol gene encodes viral replication enzymes
• the env gene encodes an envelope glycoprotein that mediates attachment of virus to the cell surface.
• the Vif protein is associated with viral infectivity
• the Tat protein with transactivation of the 5' LTR.
• the Rev protein and its target sequence RRE Rev responsive element are associated with the stability of viral RNA, regulation of viral RNA splicing, and transport of large RNA (unspliced and singly-spliced) from the nucleus to the cytoplasm.
• the proviral LTR sequences c ontain t he U 3 ( unique s equence e lement 1 ocated d ownstream from t he structural proteins), R (short repeat at each end of the genome), and U5 (unique sequence element immediately after the R sequence) regions.
• the U3 region of 5'LTR contains the viral promoter and enhancers.
• the 3' end of the genome contains polyadenylation signal in the 3'LTR.
• the wild-type genome of CAEV also contains several cw-acting elements, including atts (attachment site) at the end of LTRs for provirus integration); promoter elements that control transcriptional initiation of the integrated provirus at the 5'LTR; a PBS (primer binding site) located downstream of the 5'LTR; a 5'-splice donor site; a packaging sequence (herein referred to interchangeably as a packaging site or a packaging signal); a ppt (polypurine tract) site located near the 3'LTR; and polyadenylation signals at the 3'LTR.
• atts attachment site
• promoter elements that control transcriptional initiation of the integrated provirus at the 5'LTR
• PBS primary binding site
• 5'-splice donor site located downstream of the 5'LTR
• a packaging sequence herein referred to interchangeably as a packaging site or a packaging signal
• ppt polypurine tract
• cis is used in reference to the presence of genes on the same chromosome or linear portion of a nucleic acid. Therefore, the term “cis- defect” refers to a defect found on a linear sequence of a nucleic acid.
• ex ⁇ acting is used in reference to the controlling effect of a regulatory gene on a gene present on the same chromosome or linear portion of a nucleic acid. For example, promoters, which affect the synthesis of downstream mRNA are cis-acting control elements.
• NCBI National Center for Biotechnology Information
• accession numbers AY081139, AY101347, AY101348, AY047362, AF402668, AF402667, AF402666, AF402665, AF402664, AJ305042, AJ305041, and AJ305040 all provide for sequences of the gag gene from Brazilian isolates of CAEV.
• Accession numbers AF015181, L78453, L78451, L78450, L78447, and L78446 also contain the sequences of gag genes from a variety of CAEV isolates.
• Accession numbers X64828 and M63106 contain the sequences of rev genes from a variety of CAEV isolates.
• Accession numbers AFOl 5182, AJ305053, K03327, L78448, L78452 and U35814 contain pol genes from a variety of CAEV isolates.
• a sequence alignment between the NCJ)Ol 463 gag gene (SEQ ID NOs: 19, 25) and the gag genes from AF402664 (SEQ ID NOs: 20, 26), AF402665 (SEQ ID NOs: 21, 27), AF402666 (SEQ ID NOs: 22, 28), AF402667 (SEQ ID NOs: 23, 29), AF402668 (SEQ ID NOs: 24, 30) is found in TABLE 8.
• a sequence alignment between the NC_001463 gag gene (SEQ ID NOs: 31, 35) and the gag genes from AJ305040 (SEQ ID NOs: 32, 36), AJ305041 (SEQ BD NOs: 33, 37), AJ305042 (SEQ ID NOs: 34, 38) is found in TABLE 9.
• a sequence alignment between the NC 001463 gag gene (SEQ ID NOs: 39, 41) and the gag gene from AY047362 (SEQ ID NOs: 40, 42) is found in TABLE 10.
• a sequence alignment between the NC_001463 (SEQ ID NOs: 43, 45) gag gene and the gag gene from AY081139 (SEQ ID NOs: 44, 46) is found in TABLE 11.
• a sequence alignment between the NC_001463 (SEQ ID NOs: 47, 5 O) gag gene and theg ⁇ g genes from AY101347 (SEQ ID NOs: 48, 51) and AY101348 (SEQ ED NOs: 49, 52) is found in TABLE 12.
• Gap extension penalty 6.6
• Gap separation penalty range 8
• TABLE 14 is a summary of the percent identity values for the sequence alignments of gag gene sequences listed above.
• TABLE 15 is a summary of the percent identity of the full genomic alignment, and alignments of the gag, 5' LTR, pol, rev, and vif regions of NC_001463 (SEQ ID NO: 1) and AF322109 (SEQ ID NO: 2). Given that the genomic sequence of two CAEV isolates, in addition to a large number of partial sequences from a variety of CAEV isolates are known and consensus sequences can be easily discerned, it would not require undue experimentation to practice the claimed invention using a variety of CAEV sequences.
• the vectors of the present invention provide a means for replicating and expressing polynucleotides or genes independent of the host cell nucleus in a broad phylogenetic range of host cells.
• This vector-mediated incorporation of heterologous nucleic acid into a host cell is referred to as transfection or infection of the host cell, wherein infection means the use of virus particles, and transfection means the use of naked molecules of nucleic acid.
• transfection means the use of virus particles
• transfection means the use of naked molecules of nucleic acid.
• gene refers to a DNA sequence that comprises control and coding sequences necessary for the production of a polypeptide or precursor.
• nucleotide or "nucleic acid molecule”, as used interchangeably herein, refers to nucleotide polymers of any length, such as two or more, and includes both DNA and RNA.
• the nucleotides can be deoxyribonucleotides, ribonucleotides, nucleotide analogs (including modified phosphate moieties, bases, or sugars), or any substrate that can be incorporated into a polymer by a suitable enzyme, such as a DNA polymerase or an RNA polymerase.
• the polypeptide can be encoded by a full-length coding s equence o r b y any portion o f t he c oding se quence s o 1 ong a s t he d esired activity of the polypeptide is retained.
• wild-type refers to a gene or gene product which has the characteristics of that gene or gene product when isolated from a naturally occurring source. A wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designed the "normal" or "wild-type" form of the gene.
• mutant refers to a gene or gene product which displays modifications in sequence and or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product.
• Naturally- occurring mutants can be isolated, and are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product.
• the term "retrovirus” is used in reference to RNA viruses that utilize reverse transcriptase during their replication cycle.
• the retroviral genomic RNA is converted into double-stranded DNA by reverse transcriptase.
• This double- stranded DNA form of the virus is capable of being integrated into the chromosome of the infected cell; once integrated, it is referred to as a "provirus.”
• the provirus serves as a template for RNA polymerase II and directs the expression of RNA molecules which encode the structural proteins and enzymes needed to produce new viral particles.
• lentivirus refers to a group (or genus) of retroviruses that give rise to slowly developing disease. Viruses included within this group include human immunodeficiency virus (HIV); visna-maedi, which causes encephalitis (visna) or pneumonia (maedi) in sheep, caprine arthritis encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV). Diseases caused by these viruses are characterized by a long incubation period and protracted course.
• HCV human immunodeficiency virus
• visna-maedi which causes encephalitis (visna) or pneumonia (maedi) in sheep, caprine arthritis encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immune de
• the viruses latently infect monocytes and macrophages, from which they spread to other cells.
• the term "vector” is used in reference to nucleic acid molecules that transfer polynucleotide (e.g. DNA) segments from one cell to another.
• the term “vehicle” is sometimes used interchangeably with “vector.” It is intended that any form of vehicle or vector be encompassed within this definition.
• vectors include, but are not limited to viral particles, plasmids, transposons, etc. Standard techniques for the construction of the vectors of the present invention are well-known to those of ordinary skill in the art and can be found in such references as Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd Ed. (Cold Spring Harbor, N. Y., 1989). A variety of strategies are available for ligating fragments of DNA, the choice of which depends on the nature of the termini of the DNA fragments and which choices can be readily made by the skilled artisan.
• Suitable polyadenylation sequences of the present invention include, but are not limited t o t he b ovine growth h ormone ( BGH) p olyadenylation s ignal (ffle e t al.,
• a promoter of the present invention may comprise a promoter of mammalian or viral origin, and will be sufficient to direct the transcription of a distally located sequence (i.e. a sequence linked to the 5' end of the promoter sequence) in a cell.
• the promoter region may also include control elements for the enhancement or repression of transcription.
• Suitable promoters include, but are not limited to, the human or murine cytomegalovirus immediate-early promoter (HCMV MIEP or MCMV MIEP), elongation factor 1 alpha (ef-l ⁇ ), and Rous Sarcoma virus long terminal repeat promoter (pRSV). Intron sequences may also be combined with a promoter.
• Intron sequences include, but are not limited to ef-l ⁇ intron and ⁇ -globin intron.
• Inducible expression systems may also be used. Examples of inducible systems include, but are not limited to ecdysone-inducible mammalian expression system (Invitrogen, CA, USA) and Tet-On and Tet-Off gene expression systems (Clontech, CA, USA). Cell or tissue specific promoters can be utilized to target expression of gene sequences in specific cell populations.
• Enhancer sequences upstream from the promoter or terminator sequences and downstream of the coding region may be optionally included in the vectors of the present invention to facilitate expression.
• Vectors of the present invention may also contain additional nucleic acid sequences, such as an intron sequence, a localization sequence, or a signal sequence, sufficient to permit a cell to efficiently and effectively process the protein expressed by the nucleic acid of the vector.
• intron sequences include the ⁇ -globin intron (Kim et al., 2002) and the human EF- loc intron (Kim et al., 2002).
• Such additional sequences are inserted into the vector such that they are operably linked with the promoter sequence, if transcription is desired, or additionally with the initiation and processing sequence if translation and processing are desired. Alternatively, the inserted sequences may be placed at any position in the vector.
• operably linked is used to describe a linkage between a gene sequence and a promoter or other regulatory or p rocessing sequence such that the transcription of the gene sequence is directed by an operably linked promoter sequence, the translation of the gene sequence is directed by an operably linked translational regulatory sequence, and the post-translational processing of the gene sequence is directed by an operably linked processing sequence.
• SI vector refers to the self- inactivating vector that has a truncated
• the packaging sequence of the transfer vector consists essentially of (i) the untranslated region between the CAEV 5' LTR and the CAEV g ⁇ g-encoding sequence, and (ii) nucleotides 1 to X of the CAEV g ⁇ g-encoding sequence linked to the 3' end of said untranslated region, wherein X is less than 613.
• X is selected from the group consisting of: 60, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575 and 600.
• X is selected from the group consisting of:
• X is greater than 25 and less than 500, (c) X is greater than 25 and less than 400,
• (j) X is greater than 50 and less than 200, (k) X is greater than 75 and less than 600,
• X is greater than 75 and less than 500
• m is greater than 75 and less than 400
• n is greater than 75 and less than 300
• o is greater than 75 and less than 200
• (s) X is greater than 100 and less than 300, (t) X is greater than 100 and less than 200,
• (w) X is greater than 125 and less than 400
• (x) X is greater than 125 and less than 300, (y) X is greater than 125 and less than 200,
• (aa) X is greater than 150 and less than 500
• (cc) X is greater than 150 and less than 300, (dd) X is greater than 150 and less than 200,
• (ee) X is greater than 200 and less than 600
• (ff) X is greater than 200 and less than 500
• (kk) X is greater than 250 and less than 500
• (II) X is greater than 250 and less than 400, and (mm) X is greater than 250 and less than 300.
• X is greater than 40 and less than 613.
• X is about 327.
• the codon which initiates gag translation has been mutated (e.g. ATG changed to TAG, TTG, CTG, or ATT) or deleted.
• the term "codon” refers to a sequence of three nucleotides in a DNA or messenger RNA molecule that represents the instruction for incorporation of a specific amino acid into a growing polypeptide chain.
• the transfer vector further comprises a heterologous promoter and one or more cw-acting sequences.
• the term "packaging signal” or "packaging sequence” refers to sequences located adjacent to the 5' LTR of the CAEV genome which are required for encapsidation of the viral RNA into the viral capsid or particle.
• Several retroviral vectors use the minimal packaging signal (also referred to as the psi [ ⁇ ] sequence) needed for encapsidation of the viral genome.
• the terms "packaging sequence”, “packaging signal”, “psi”, and the symbol “ ⁇ ” are used in reference to the non-coding sequence required for encapsidation of CAEV RNA strands during viral particle formation.
• the transfer vector further comprises a transcription cassette.
• transcription cassette refers to a fragment or segment of nucleic acid containing a particular grouping of genetic elements, generally a polynucleotide which expresses a polypeptide of interest, operably linked to a heterologous promoter.
• the cassette can be removed and inserted into a vector or plasmid as a single unit.
• An illustrative example of a transfer vector of the present invention is shown in
• FIGURE 3C illustrates the plasmid pCAH/SINdl (SEQ ID NO: 68).
• PCAH/SINdl (SEQ ID NO: 68) is a 4,238 bp plasmid that contains the HCMV MIEP promoter, the R-U5 sequence regions in the CAEV 5'LTR, the residual untranslated sequences containing a PBS site, the 327 bp fragment of the gag gene with t he A TG ⁇ TAG d ouble p oint m utations, t he R RE r egion a nd t he U 3-deleted CAEV 3'LTR region.
• the vector also contains a Col El origin of replication (bp 2535-3118) and a kanamycin resistance gene region (bp 3370-4182). The other illustrative examples of transfer vectors are shown in FIGURE 3A-3H.
• the invention provides a CAEV vector system comprising the above- described transfer vector and a packaging vector system.
• the packaging vector system comprises a first and second polynucleotide vector sequence.
• the first polynucleotide sequence comprises CAEV gag-pol and RRE-encoding sequence and the second polynucleotide comprises a viral envelope encoding sequence.
• the second polynucleotide encodes a non-CAEV envelope.
• structural gene refers to the polynucleotide sequence encode proteins which are required for encapsidation (e.g., packaging) of the viral genome, and include gag, pol and env.
• FIGURE 2A An illustrative example of a first packaging vector of the present invention is shown in FIGURE 2A.
• FIGURE 2A illustrates the plasmid pMGP/RRE ( SEQ ID NO: 77).
• the plasmid contains 9,446 base pairs and includes a MCMV MIEP region, the CAEV gag-pol coding region, the RRE region, and the bovine growth hormone (BGH) polyadenylation signal.
• the vector also contains a neomycin resistant gene coding region, a SV40 origin of replication, a Col El origin of replication, and an ampicillin resistance gene region.
• retro viral-derived env gene examples include, but are not limited to: the G-protein of vesicular- stomatitis virus (VSV-G), gibbon ape leukemia virus (GaLV), rous sarcoma virus (RSV), moloney murine leukemia virus (MoMuLV), mouse mammary tumor virus (MMTV), and human immunodeficiency virus (HIV).
• VSV-G the G-protein of vesicular- stomatitis virus
• GaLV gibbon ape leukemia virus
• RSV rous sarcoma virus
• MoMuLV moloney murine leukemia virus
• MMTV mouse mammary tumor virus
• HAV human immunodeficiency virus
• pseudotype refers to a viral particle that contains nucleic acid of one virus but the envelope protein of another virus.
• VSV-G or GaLV pseudotyped vectors have a very broad host range, and may be pelleted to titers of high concentration by ultracentrifugation (Burns et al., 1993), while still retaining high levels of infectivity.
• FIGURE 6A illustrates the plasmid pHGVSV-G (SEQ ID NO: 74).
• pHGVSV-G (SEQ ID NO: 74) is a 7,623 bp plasmid which contains the HCMV MIEP, the ⁇ -globin intron region, the VSV-G coding region, the BGH polyadenylation signal, a Col El origin of replication, a neomycin resistance gene coding region, an ampicillin resistance gene coding region, and an Fl origin of replication.
• FIGURE 6B illustrates the plasmid pMYKEFl/env (SEQ ID NO: 72).
• This plasmid contains 7,579 bp which includes the MCMV MIEP, a human EFl - ⁇ intron region, the GaLV env coding region, the BGH polyadenylation signal, a Col El origin of replication, a neomycin resistance gene coding region, and an ampicillin resistance gene coding region.
• the packaging vector comprises a third polynucleotide which encodes Rev.
• Rev binds to the Rev- responsive element (RRE) in viral transcripts and causes the transcription of both singly-spliced and unspliced transcripts characteristic of the viral structural proteins in the late stage of replication. Accordingly, Rev mediates temporal regulation of viral gene expression. Because mammalian cell splicing mechanisms are coupled to transport of mRNA from the site of synthesis in the nucleus to the cytoplasm, Rev also influences transport of viral transcripts containing RRE.
• RRE Rev- responsive element
• FIGURE 5 illustrates the plasmid pHYK/rev (SEQ ID NO: 75).
• pHYK/rev (SEQ ID NO: 75) is a 5,419 bp plasmid which contains HCMV MIEP, the rev gene coding region, BGH polyadenylation signal, a Col El origin of replication, a neomycin resistant gene coding region, and an ampicillin resistant gene coding region.
• the packaging vector comprises a fourth polynucleotide encoding Vif. Incorporation of Vif may be necessary for infection and packaging of virions, depending on the packaging cell line chosen.
• An illustrative example of a fourth packaging vector of the present invention is shown in FIGURE 4.
• pHYK/vif SEQ ID NO: 76
• pHYK/vif SEQ ID NO: 76
• pHYK/vif SEQ ID NO: 76
• pHYK/vif is a 5,729 bp plasmid which contains the HCMV MIEP, the vif gene coding region, the BGH polyadenylation signal, a Col El origin of replication, a neomycin resistance gene coding region, and an ampicillin resistance gene coding region.
• retroviral vector DNA When retroviral vector DNA is transfected into the cells, it may or may not become integrated into the chromosomal DNA and becomes transcribed, thereby producing full-length retroviral vector RNA that contains a ⁇ sequence. Under these conditions, only the vector RNA is packaged into the viral capsid structures. These complete, yet replication-defective, virus particles can then be used to deliver the retroviral vector to target cells with relatively high efficiency.
• replication-defective refers to a virus that is not capable of complete, effective replication such that infective virions are not produced (e.g. replication-defective lentiviral progeny).
• replication-competent refers to wild-type virus or mutant virus that is capable of replication, such that viral replication o f the virus i s c apable o f producing infective virions (e.g., replication- competent lentiviral progeny).
• packaging may be inducible, as well as non- inducible.
• inducible packaging cells and packaging cell lines CAEV particles are produced in response to at least one inducer, hi preferred embodiments with inducible cell lines, the inducer is Tat.
• non-inducible packaging cell lines and packaging cells no inducer is required in order for lentiviral particle production to occur.
• Functionally equivalent sequences of the present invention also encompass various fragments of a CAEV genome that retain substantially the same function as the respective native sequence.
• Such fragments will comprise at least about 10, 15 contiguous nucleotides, at least about 20 contiguous nucleotides, at least about 24, 50, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 340, 360, 380, or up to the entire contiguous nucleotides of the specific genetic element of interest.
• Such fragments may be obtained by use of restriction enzymes to cleave the native viral genome; by synthesizing a nucleotide sequence from the native nucleotide sequence of the virus genome; or may be obtained through the use of PCR technology.
• variants of the various vector components are encompassed by the methods of the present invention. As described in more detail below, methods are available in the art for determining functional equivalence. By “variant” it is intended to include substantially similar sequences. Thus, for nucleotide sequences or amino acid sequences, variants include sequences that are functionally equivalent to the various components of the viral vector system. Variant nucleotide sequences also include synthetically derived nucleotide sequences that have been generated, for example, by site directed mutagenesis, but which still retain the function of the native sequence.
• nucleotide sequence variants or amino acid sequence variants of the invention will have at least 70%, generally 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to its respective native nucleotide sequence.
• Variants of the invention include polynucleotides (e.g., vectors) comprising, consisting essentially of, or consisting of, sequences at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequences of the vectors disclosed herein (SEQ ID NOs: 67-79).
• nucleic acid constructs disclosed yield a functionally identical construct.
• Conservative variations of a particular nucleic acid sequence refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences.
• a large number of functionally identical nucleic acids encode any given polypeptide.
• substitutions i.e., substitutions of a nucleic acid sequence which do not result in an alteration in an encoded polypeptide
• amino acid substitutions in one or a few amino acids in an amino acid sequence of a packaging or packageable construct are substituted with different amino acids with highly similar properties, are also readily identified as being highly similar to a disclosed construct.
• the codons CGU, CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine.
• the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
• Such nucleic acid variations are "silent variations,” which are one species of
• nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation.
• One of skill will recognize t hat e ach c odon i n a n ucleic a cid (except A UG, w hich i s o rdinarily t he only codon for methionine) can be modified to yield a functionally identical molecule by standard techniques. Accordingly, e ach "s ilent variation" of a nucleic acid which encodes a polypeptide is implicit in any described sequence.
• I Isoleucine
• L Leucine
• M Methionine
• V Valine
• F Phenylalanine
• Y Tyrosine
• W Tryptophan
• variants include those polypeptides that are derived from the native polypeptides by deletion (so- called truncation) or addition of one or more amino acids to the N-terminal and/or C- terminal end of the native polypeptide; deletion or addition of one or more amino acids at one or more sites in the native polypeptide; or substitution of one or more amino acids at one or more sites in the native polypeptide.
• Such variants may result from, for example, genetic polymorphism or from human manipulation. Methods for such manipulations are generally known in the art.
• a variant of a native nucleotide sequence or native polypeptide has substantial identity to the native sequence or native polypeptide.
• a variant may differ by as few as 1 to 10 amino acid residues, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.
• a variant of a nucleotide sequence may differ by as low as 1 to 30 nucleotides, such as 6 to 20, as low as 5, as few as 4, 3, 2, or even 1 nucleotide residue.
• sequence identity it is intended by "s equence identity" that the same nucleotides or amino acid residues are found within the variant sequence and a reference sequence when a specified, contiguous segment of the nucleotide sequence or amino acid sequence of the variant is aligned and compared to the nucleotide sequence or amino acid sequence of the ⁇ reference sequence. Methods for sequence alignment and for determining identity between sequences are well known in the art. With respect to optimal alignment of two nucleotide sequences, the contiguous segment of the variant nucleotide sequence may have additional nucleotides or deleted nucleotides with respect to the reference nucleotide sequence.
• the contiguous segment of the variant amino acid sequence may have additional amino acid residues or deleted amino acid residues with respect to the reference amino acid sequence.
• the contiguous segment used for comparison to the reference nucleotide sequence or reference amino acid sequence will comprise at least 20 contiguous nucleotides, or amino acid residues, and may be 30, 40, 50, 100, or more nucleotides or amino acid residues. Corrections for increased sequence identity associated with inclusion of gaps in the variant's nucleotide sequence or amino acid sequence can be made by assigning gap penalties. The determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
• percent identity of an amino acid sequence can be determined using the Smith- Waterman homology search algorithm using an aff ⁇ ne 6 gap search with a gap open penalty of 12 and a gap extension penalty of 2, BLOSUM matrix 62.
• percent identity of a nucleotide sequence is determined using the Smith- Waterman homology search algorithm using a gap open penalty of 25 and a gap extension penalty of 5.
• sequence identity can be performed using, for example, the DeCypher Hardware Accelerator from TimeLogic Version G.
• the Smith- Waterman homology search algorithm is taught in Smith and Waterman , herein incorporated by reference.
• the alignment program GCG Gap (Wisconsin Genetic Computing G roup, S uite V ersion 1 0.1) u sing t he d efault p arameters m ay b e u sed.
• the GCG Gap program applies the Needleman and Wunch algorithm and for the alignment of nucleotide sequences with an open gap penalty of 3 and an extend gap penalty of 1 may be used.
• Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (Karlin and Altschul, 1990), modified as in Karlin and Altschul (Karlin and Altschul, 1993).
• Gapped BLAST can be utilized as described in Altschul et al. (Altschul et al., 1997).
• PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al.
• nucleic acid of the invention can select a desired nucleic acid of the invention based upon the sequences provided and upon knowledge in the art regarding CAEV generally.
• the life-cycle, genomic organization, developmental regulation and associated molecular biology of lentiviruses have been the focus of over a decade of intense research.
• the specific effects of many mutations in many lentiviral genomes are known.
• the nucleic acid sequence variations of some CAEV strains are known.
• general knowledge regarding the nature of proteins and nucleic acids allows one of skill to select appropriate sequences with activity similar or equivalent to the nucleic acids and polypeptides disclosed in the sequence listings herein.
• nucleic acids are evaluated by routine screening techniques in suitable assays for the desired characteristic. For instance, changes in the immunological character of encoded polypeptides can be detected by an appropriate immunological assay. Modifications of other properties such as nucleic acid hybridization to a complementary nucleic acid, redox or thermal stability of encoded proteins, hydrophobicity, susceptibility to proteolysis, or the tendency to aggregate are all assayed according to standard techniques.
• the nucleotide sequence of the inserted polynucleotide of interest may be of any nucleotide sequence.
• the polynucleotide sequence may be a reporter gene sequence or a selectable marker gene sequence.
• a reporter gene sequence is any gene sequence which, when expressed, results in the production of a protein whose presence or activity can be monitored. Examples of suitable reporter genes include the gene for galactokinase, ⁇ -galactosidase, chloramphenicol acetyltransferase, ⁇ -lactamase, green fluorescent protein, enhanced green fluorescent protein, etc.
• the reporter gene sequence may be any gene sequence whose expression produces a gene product that affects cell physiology.
• Polynucleotide sequences of the present invention may comprise one or more gene sequences that already possess on or more promoters, initiation sequences, or processing sequences.
• a selectable marker gene sequence is any gene sequence capable of expressing a protein whose presence permits one to selectively propagate a cell which contains it.
• selectable marker genes include gene sequences capable of conferring host resistance to antibiotics (e.g., puromycin, hygromycin, neomycin, zeocin and the like), or of conferring host resistance to amino acid analogues, or of permitting the growth o f b acteria o n a dditional c arbon s ources o r u nder o therwise i mpermissible culture conditions. Reporter or selectable marker gene sequences are sufficient to permit the recognition or selection of the vector in normal cells.
• the reporter gene sequence may encode an enzyme or other protein which is normally absent from mammalian cells, and whose presence can, therefore, definitively establish the presence of the vector in such a cell.
• the transfer vectors of the present invention additionally permit the incorporation of heterologous nucleic acid, or polynucleotides, into virus particles, thereby providing a means for amplifying the number of infected host cells containing heterologous nucleic acid therein.
• the incorporation of the heterologous polynucleotide facilitates the replication of the heterologous nucleic acid within the viral particle, and the subsequent production of a heterologous protein therein.
• a heterologous protein is herein defined as a protein or fragment thereof wherein all or a portion of the protein is not expressed by the host cell.
• a nucleic acid or gene sequence is said to be heterologous if it is not naturally present in the wild-type of the viral vector used to deliver the gene into a cell.
• the term heterologous nucleic acid sequence or polynucleotide sequence, as used herein, is intended to refer to a nucleic acid molecule (preferably DNA).
• the polynucleotide sequence or heterologous polynucleotide sequence may also comprise the coding sequence of a desired product such as a suitable biologically active protein or polypeptide, immunogenic or antigenic protein or polypeptide, or a therapeutically active protein or polypeptide.
• the polypeptide may supplement deficient or nonexistent expression of an endogenous protein in a host cell.
• gene sequences may be derived from a variety of sources including DNA, cDNA, synthetic DNA, RNA or combinations thereof.
• Such gene sequences may comprise genomic DNA which may or may not include naturally occurring introns.
• genomic DNA may be obtained in association with promoter sequences or polyadenylation sequences.
• the gene sequences of the present invention are preferably cDNA. Genomic or cDNA may be obtained in any number of ways. Genomic DNA can be extracted and purified from suitable cells by means well-known in the art. Alternatively, mRNA can be isolated from a cell and used to prepare cDNA by reverse transcription, or other means.
• the polynucleotide sequence may comprise a sequence complementary to an RNA sequence, such as an antisense RNA sequence, which antisense sequence can be administered to an individual to inhibit expression of a complementary polynucleotide in the cells of the individual.
• heterologous gene may provide an immunogenic or antigenic protein or polypeptide to achieve an antibody response.
• the antibodies thus raised may be collected from an animal in a body fluid such as blood, serum or ascites.
• the heterologous gene can also be any nucleic acid of interest that can be transcribed.
• the foreign gene encodes a polypeptide.
• the polypeptide has some therapeutic benefit.
• the polypeptide may supplement deficient or nonexistent expression of an endogenous protein in a host cell.
• the polypeptide can confer new properties on the host cell, such as a chimeric signaling receptor, see U.S. Pat. No. 5,359,046.
• One of ordinary skill can determine the appropriateness of a foreign gene practicing techniques taught herein and known in the art. For example, the artisan would know whether a foreign gene is of a suitable size for encapsidation and whether the foreign gene product is expressed properly.
• heterologous protein that can be employed in the present invention is not critical thereto.
• heterologous proteins which can be employed in the present invention include dystrophin (Hoffman, Brown, and Kunkel, 1987), coagulation factor VIII (Wion et al., 1985), Cystic Fibrosis Transmembrane Regulator Protein (CFTR) (Anderson et al., 1991; Crawford, 1991), Ornithine Transcarbamylase (OTC) (Murakami et al., 1988), and ⁇ l -antitrypsin (Fagerhol and Cox, 1981).
• the genes encoding many heterologous proteins are well-known in the art, and can be cloned from genomic or cDNA libraries [Sambrook et al, supra].
• genes include the dystrophin gene(Lee et al., 1991), the Factor VIII gene (Toole et al., 1984), the CFTR gene (Rommens et al., 1989; Riordan, 1989), the OTC gene (Horwich et al., 1984), and the ⁇ l -antitrypsin gene (Lemarchand et al., 1992).
• heterologous proteins such as Rb, for the treatment of vascular proliferative disorders like atherosclerosis (Chang et al., 1995), and p53 for the treatment of cancer (Wills et al., 1994; dayman, 1995), and HIV disease (Bridges and Sarver, 1995), can be employed in the present invention.
• v ector d oes n ot a lways n eed t o c ode for a functional, h eterologous gene product, i.e., it may also code for a partial gene product which acts as an inhibitor of a eukaryotic enzyme (Warne, Viciana, and Downward, 1993; Wang, 1991).
• a gene regulating molecule in a cell by the introduction of a molecule by the method of the invention.
• modulate envisions the suppression of expression of a gene when it is over-expressed or augmentation of expression when it is under-expressed.
• nucleic acid sequences that interfere with the expression of a gene at the translational level can be used.
• the approach can utilize, for example, antisense nucleic acid, ribozymes or triplex agents to block transcription or translation of a specific mRNA, either by masking that mRNA with an antisense nucleic acid or triplex agent, or by cleaving same with a ribozyme.
• Antisense nucleic acids are DNA or RNA molecules which are complementary to at least a portion of a specific mRNA molecule . In the cell, the antisense nucleic acids hybridize to the corresponding mRNA forming a double-stranded molecule. The antisense nucleic acids interfere with the translation of the mRNA since the cell will not translate an mRNA that is double-stranded. Antisense oligomers of about 15 nucleotides or more are preferred since such are synthesized easily and are less likely to cause problems than larger molecules when introduced into the target cell. The use of antisense methods to inhibit the in vitro translation of genes is well known in the art (Marcus-Sekura, 1988).
• the antisense nucleic acid can be used to block expression of a mutant protein or a dominantly active gene product, such as amyloid precursor protein that accumulates in Alzheimer's disease. Such methods are also useful for the treatment of Huntington's disease, hereditary Parkinsonism and other diseases. Antisense nucleic acids are also useful for the inhibition of expression of proteins associated with toxicity.
• oligonucleotide to stall transcription can be by the mechanism known as the triplex strategy since the oligomer winds around double-helical DNA, forming a three-strand helix. Therefore, the triplex compounds can be designed to recognize a unique site on a chosen gene (Maher, Wold, and Dervan, 1991; Helene, 1991).
• Ribozymes are RNA molecules possessing the ability to specifically cleave other single-stranded RNA in a manner analogous to DNA restriction endonucleases. Through the modification of nucleotide sequences which encode those RNA's, it is possible to engineer molecules that recognize and cleave specific nucleotide sequences in an RNA molecule (Cech, 1988). A major advantage of that approach is only mRNA's with particular sequences are inactivated.
• nucleic acid encoding a biological response modifier.
• immunopotentiating agents including nucleic acids encoding a number of the cytokines classified as "interleukins", for example, interleukins 1 through 12.
• interferons include gamma interferon ( ⁇ -IFN), tumor necrosis factor (TNF) and granulocyte-macrophage colony stimulating factor (GM-CSF).
• ⁇ -IFN gamma interferon
• TNF tumor necrosis factor
• GM-CSF granulocyte-macrophage colony stimulating factor
• the recombinant CAEV vector system of the invention can be used to treat an HIV-infected cell (e.g., T-cell or macrophage) with an anti-HIV molecule.
• respiratory epithelium for example, can be infected with a recombinant lentivirus of the invention having a gene for cystic fibrosis transmembrane conductance regulator (CFTR) for treatment of cystic fibrosis.
• CFTR cystic fibrosis transmembrane conductance regulator
• the recombinant CAEV vector system of the invention can be used to treat many human diseases.
• human diseases include, but are limited to: Alzheimer's diseases, Parkinson's diseases, amyotrophic lateral sclerosis disease, Huntington's disease, beta-thalassemia, retinitis pigmentosa, mucopolysaccharide disease, leukodystrophy diseases, X-linked SCID, phenylketonuria, tryosinemia, hemophilia A and B, Wilson's diseases, LDL receptor deficiency, Human Immunodeficiency, and Duchenne's dystrophy.
• infectious and replication-defective CAEV vector particles may be prepared according to the methods disclosed herein in combination with techniques known to those skilled in the art.
• the method includes transfecting a lentivirus-permissive cell with the vector expression system of the present invention; producing the CAEV-derived particles in the transfected cell; and collecting the virus particles from the cell.
• transfection refers to the introduction of foreign DNA into eukaryotic cells. Transfection may be accomplished by a variety of means known to the art including but not limited to calcium phosphate-DNA co- precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection and protoplast fusion. These techniques are well known in the art.
• transduction refers to the delivery of a gene using a viral or retroviral vector particles by means of infection rather than by transfection.
• retroviral vectors are transduced.
• a "transduced gene” is a g ene t hat h as b een i ntroduced i nto t he c ell v ia 1 enti viral o r v ector i nfection a nd provirus integration.
• the CAEV viral vector particles transduce genes into "target cells" or host cells.
• the step of facilitating the production of the infectious viral particles in the cells may also be carried out using conventional techniques, such as by standard cell culture growth techniques.
• the step of collecting the infectious virus particles may also be carried out using conventional techniques.
• the infectious particles may be collected by collection of the supernatant of the cell culture, as is known in the art.
• the collected virus particles may be purified if desired. Suitable purification techniques are well known to those skilled in the art.
• CAEV stock solutions may be prepared using the vectors and methods of the present invention. Methods of preparing viral stock solutions are known in the art and are illustrated by, e.g., (Soneoka et al., 1995) and (Landau and Littman, 1992).
• lentiviral-permissive cells are transfected with the vector system of the present i nvention. T he cells are then grown under suitable cell culture conditions, and the CAEV particles are collected from the cell culture media as described above.
• Suitable permissive cell lines include, but are not limited to, the human cell lines 293, 293T, and HeLa the monkey cell line Vero ,and the goat cell lines GSM and ChIEs.
• the vectors of the present invention are also useful in preparing stable packaging cells (i.e. cells that stably express CAEV structural proteins, which cells, by t hemselves, c annot generate i nfectious v irus particles) and v irus p roducer cells (VPC).
• stable packaging cells i.e. cells that stably express CAEV structural proteins, which cells, by t hemselves, c annot generate i nfectious v irus particles
• VPC v irus p roducer cells
• a packaging cell will comprise a lentivirus-permissive host cell comprising a CAEV nucleic acid sequence from at least one CAEV packaging vector described in this invention, which nucleic acid sequence is packaging-signal defective, thus rendering the cell itself capable of producing at least one CAEV structural protein, but not capable of producing replication-competent infectious virus.
• a packaging cell may be made by transfecting a CAEV-permissive host cell (e.g., a human embryonic kidney 293 or 293T cells) with a suitable CAEV nucleic acid sequence as provided above according to known procedures.
• the resulting packaging cell is thus able to express and produce at least one CAEV structural protein.
• the packaging cell is still not able to produce recombinant CAEV virus.
• the packaging cell may then be transfected with other nucleic acid sequences, i.e., a transfer vector, which may contain heterologous genes of interest and an appropriate packaging signal. Once transfected with the additional sequence or sequences, the packaging cell may thus be used to provide stocks of CAEV viruses that contain heterologous genes, but which viruses are themselves replication-incompetent.
• the resulting virus producing cell (VPC) is thus able to produce infectious virus particles containing heterologous gene of interest.
• Target cells for therapeutic gene transfer include, but are not limited to hematopoietic stem cell, lymphocyte, vascular endothelial cell, respiratory epithelial cell, keratinocyte, skeletal and muscle cells, liver cell, neuron cell, and cancer cell .
• the gene transfer technology of the present invention may also be used in elucidating the processing of peptides and identification of the functional domains of various proteins.
• Cloned cDNA or genomic sequences for proteins can be introduced into different target cells ex vivo, or in vivo, in order to study cell-specific differences in processing and cellular fate.
• By placing the coding sequences under the control of a strong promoter a substantial amount of the desired protein can be made.
• the specific residues involved in protein processing, intracellular sorting, or biological activity can be determined by mutational change in discrete residues of the coding sequences.
• Gene transfer technology of the present invention can also be applied to provide a means to control expression of a protein and to assess its capacity to modulate cellular events.
• Some functions of proteins, such as their role in differentiation, may be studied in tissue culture, whereas others will require reintroduction into in vivo systems a t d ifferent t imes i n d evelopment i n o rder to rn onitor c hanges i n r elevant properties.
• Gene transfer provides a means to study the nucleic acid sequences and cellular factors that regulate expression of specific genes.
• One approach to such a study would be to fuse the regulatory elements to be studied to reported genes and subsequently assaying the expression of the reporter gene.
• Gene transfer also possesses substantial potential use in understanding and providing therapy for disease states.
• deficiency state diseases gene transfer could be used to bring a normal gene into affected tissues for replacement therapy, as well as to create animal models for the disease using antisense m utations.
• the methods of the present invention permit the treatment of genetic diseases.
• a disease state is treated by partially or wholly remedying the deficiency or imbalance which causes the disease or makes it more severe.
• the use of site-specific integration of nucleic sequences to cause mutations or to correct defects is also possible.
• the method of the invention may also be useful for neuronal, glial, fibroblast or mesenchymal cell transplantation, or "grafting", which involves transplantation of cells infected with the recombinant lentivirus of the invention ex vivo, or infection in vivo into the central nervous system or into the ventricular cavities or subdurally onto the surface of a host brain.
• grafting Such methods for grafting will be known to those skilled in the art and are described in Neural Grafting in the Mammalian CNS, Bjorklund & Stenevi, eds. (1985).
• gene transfer could introduce a normal gene into the affected tissues for replacement therapy, as well as to create animal models for the disease using antisense mutations.
• a Factor VIII or IX encoding nucleic acid into a CAEV particle for infection of a muscle, spleen or liver cell.
• stem cells includes but is not limited to hematopoietic stem cells, neuronal stem cells, mesenchymal (particularly muscular) stem cells, and liver stem cells. Stem cells are capable of repopulating tissues in vivo. Hematopoietic stem cells are progenitor cells derived from primitive human hematopoietic cells. Gene therapy using hematopoietic stem cells is also useful to treat a genetic abnormality in lymphoid and myeloid cells that results generally in the production of a defective protein or abnormal levels of expression of the gene.
• Hematopoietic stem cell gene therapy is beneficial for the treatment of genetic disorders of blood cells such as ⁇ - and ⁇ -thalassemia, sickle cell anemia and hemophilia A and B in which the globin gene or clotting factor genes (e.g., Factor IX and Factor X genes) are defective.
• Another good example is the treatment of severe combined immunodeficiency disease (SCIDS), in which patients lack the adenosine deaminase (ADA) enzyme which helps eliminate certain byproducts that are toxic to T and B lymphocytes and render the patients defenseless against infection.
• SCIDS severe combined immunodeficiency disease
• ADA adenosine deaminase
• Such patients are ideal candidates to receive gene therapy by introducing the ADA gene into their hematopoietic stem cells instead of the patient's lymphocytes as done in the past.
• GDNF Glial cell line-derived neurotrophic factor
• CAEV vector can carry a gene that encodes, for example, a toxin or an apoptosis inducer effective to specifically kill the cancerous cells.
• Specific killing of tumor cells can also be accomplished by introducing a suicide gene to cancerous hematopoietic cells under conditions that only the tumor cells express the suicide gene.
• the suicide gene product confers lethal sensitivity to the cells by converting a normally nontoxic drug to a toxic derivative.
• the enzyme cytosine deaminase converts the nontoxic substance 5'-fluorocytosine to a toxic derivative, 5- fluorouracil (Mullen, Kilstrup, and Blaese, 1992).
• Tumor-specific lymphocytes can be genetically modified for example, to locally deliver gene products with anti-tumor activity to sites of the tumor to circumvent the toxicity associated with the systemic delivery of these gene products.
• a gene therapy approach can also be applied to render bone marrow cells resistant to the toxic effects of chemotherapy.
• Gene therapy can also be used to prevent or combat viral infections such as HIV and HTLV-I infection.
• hematopoietic stem cells can be genetically modified to r ender t hem r esistant t o i nfection b y HIV.
• One approach i s t o i nhibit viral gene expression specifically by using antisense RNA or by subverting existing viral regulatory pathways.
• Antisense RNAs complementary to retroviral RNAs have been s hown t o i nhibit the r eplication o f a n umber o f r etro viruses ( To, B ooth, a nd Neiman, 1986) including HIV (Rhodes and James, 1991) and HTLV-I (von Ruden and Gilboa, 1989).
• the therapeutic gene can encode, e.g., a B or T cell signaling molecule capable of reconstituting the normal apoptotic signal that results in the death and elimination of autoreactive cells.
• Ex vivo cell transformation for diagnostics, research, or for gene therapy e.g., via re-infusion of the transformed cells into the host organism
• cells are isolated from the subject organism, transfected with a vector of the invention comprising a polypeptide of interest, and re-infused back into the subject organism (e.g., patient).
• Mammalian cell systems often will be in the form of monolayers of cells, although mammalian cell suspensions are also used.
• cells can be derived from those stored in a cell bank (e.g., a blood bank).
• Illustrative examples of mammalian cell lines include the HEC-I-B cell line, VERO and HeIa cells, Chinese hamster ovary (CHO) cell lines, Wl 38, BHK, Cos-7 or MDCK cell lines (see, e.g., Freshney, supra).
• T cells or B cells are also used in some ex vivo gene transfer procedures. Several techniques are known for isolating T and B cells. The expression of surface markers facilitates identification and purification of such cells.
• the viral vectors of the present invention can be used to stably transduce either dividing or non-dividing cells, and stably express a heterologous gene.
• this vector system it is now possible to introduce into dividing or non- dividing cells, genes that encode proteins that can affect the physiology of the cells.
• the vectors of the present invention can thus be useful in gene therapy for disease states, or for experimental modification of cell physiology. Kits
• kits or drug delivery system comprising the vectors for use in the methods described herein. All the essential materials and reagents required for administration of the targeted retroviral particle may be assembled in a kit (e.g., packaging cell construct or cell line). The components of the kit may be provided in a variety of formulations. The one or more CAEV particles may be formulated with one or more agents (e.g., a chemotherapeutic agent) into a single pharmaceutically acceptable composition or separate pharmaceutically acceptable compositions. The components of these kits or drug delivery systems may also be provided in dried o r 1 yophilized forms.
• agents e.g., a chemotherapeutic agent
• kits of the invention may also comprise instructions regarding the dosage and or administration information for the targeted CAEV particle.
• kits or drug delivery systems of the present invention also will typically include a means for containing the vials in close confinement for commercial sale such as, e.g., injection or blow-molded plastic containers into which the desired vials are retained.
• Such an instrument may be an applicator, inhalant, syringe, pipette, forceps, measured spoon, eye-dropper or any such medically approved delivery vehicle.
• the following examples serve to illustrate certain embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.
• the following examples demonstrate the finding that the recombinant CAEV- based lentiviral vector system of the present invention is as effective in expression as the well-known HIV-I based lentiviral system.
• the examples show that the level of genomic RNA transcription, encapsidation, transduction, reverse transcription, and integration of the CAEV-based vector particle production system of the present invention is comparable to that of HIV-I -based lentiviral vector system, which has long been accepted as a highly efficient gene transfer system (Naldini et al., 1996).
• the parent plasmids The parent plasmids.
• the parent plasmids from which the CAEV vectors of the present invention were derived are the plasmid pWTE-BM and the plasmid pCAEV- LTR, kindly provided by Dr. Marie Suzan (Institut National de Ia Sante et de Ia mecanic M edicale " INSERM", F ranee) T he pWTE-BM p lasmid c ontains a full- length genomic CAEV cDNA except for the 0.4kb Hind III fragment which contains parts of env, rev, and U3 regions and a 1337 base pair stuffer fragment.
• the plasmid pCAEV-LTR contains the 0.4kb Hind III fragment lacking in the pWTE-BM (Saltarelli et al., 1990; Saltarelli, 1993). Neither of the vectors can generate a wild- type virus.
• CAEV gag-pol expression vectors (pMGP/RRE (SEQ ID NO: 77) and pMGP/REV/RRE).
• the pMGP/RRE (SEQ ID NO: 77) plasmid is a PWTE-BM derived gag-pol expression plasmid (shown in FIGURE 2A).
• the pMGP/RRE (SEQ ID NO: 77) plasmid contains a strong and heterologous MCMV major immediate- early promoter (MCMV MIEP), the gag-pol g ene, and the rev responsive element (RRE).
• the pMGP/RRE (SEQ ID NO: 77) plasmid also encodes the neomycin resistant gene as an antibiotic selection marker.
• the gag-pol gene fragment (nucleotide 5 12 through nucleotide 5046 o f the C AEV genome) from p WTE-BM was subcloned into the pGL2-Basic (Promega, WI, USA) cloning vector by using standard protocols for several PCR and subcloning steps.
• the MCMV MIEP fragment was excised from the plasmid pMYK (Kim et al, 2002) was then inserted upstream of the gag gene, and the RJRE region (from nucleotide 7824 to nucleotide 8183 or nucleotide 7849 to nucleotide 8150 of the CAEV genome) was inserted downstream of the pol gene.
• the pMGP/REV/RRE is another gag-pol expressing plasmid (shown in FIGURE 2B) containing the CAEV rev gene.
• the major splicing donor site of the CAEV was inserted downstream of the MCMV promoter.
• Transfer vectors pCAH/SINd series. The plasmids in the pCAH/SINd series
• FIGURES 3A-3H SEQ ID NOs: 67-71, 73, 78, and 79
• SEQ ID NOs: 67-71, 73, 78, and 79 were constructed to identify an optimal packaging sequence for the design of the transfer vectors of the present invention.
• Each of the plasmids in the series were designed to contain different lengths of the 5 'untranslated region and the beginning of the g ⁇ g-encoding region to allow for the side by side comparison of the effects of the various lengths in this region.
• SIN self-inactivation
• RNA from the transfer vectors in the absence of a trans ⁇ acting factor, t ⁇ t, the U3 region of the 5'LTR was replaced with an HCMV MEEP.
• all known cw-acting sequence elements required for polyadenylation, RNA transportation, reverse transcription, and integration were included in the transfer vector series.
• the plasmids of the pCAH/SINd series (SEQ ID NOs: 67-71, 73, 78, 79) were constructed as follows.
• pCAH/SINd PBS-deficient negative control vector
• SEQ ID NO: 73 (FIGURE 3A) was designed to contain only the 5' untranslated sequences (R and U5 region)in the 5' LTR (from nucleotide 1 to nucleotide 163 of the CAEV genome).
• pCAH/SINdO (SEQ ID NO: 67) (FIGURE 3B) was designed to contain the entire 5' untranslated region (from nucleotide 1 to nucleotide 511 of the CAEV genome).
• pCAH/SINdl (SEQ ID NO: 68) (FIGURE 3C) was designed to contain the entire 5' untranslated region and the 327 bp fragment of the gag gene (from nucleotide 1 to nucleotide 839 of the CAEV genome) with point mutations.
• pCAH/SINd2 (SEQ ID NO: 69) (FIGURE 3D) was designed to contain the entire 5' untranslated region and the 612 bp fragment of the gag gene (from nucleotide 1 to nucleotide 1124 of the CAEV genome) with point mutations.
• Plasmid pCAH/SINd3 (SEQ ID NO: 70) (FIGURE 3E) was designed to contain the entire 5' untranslated region and the 908 b p fragment o f the gag gene ( from n ucleo tide 1 to nucleotide 1420 of the CAEV genome) with point mutations.
• Plasmid ⁇ CAH/SINd4 (SEQ E) NO: 71) (FIGURE 3F) was designed to contain the entire 5' untranslated region and the 1,198 bp fragment of the gag gene (from nucleotide 1 to nucleotide 1710 of the CAEV genome) with point mutations.
• pCAH/SINdl/hlacZ (SEQ ID NO: 78) (FIGURE 3G) was constructed by inserting the expression cassette consisting of the HCMV MIEP and the lacZ gene into the pCAH/SINdl (SEQ ID NO: 68).
• the plasmid pCAH/SINd60/hlacZ (SEQ ID NO: 78) (FIGURE 3H) has the same design as the pCAH/SINdl (SEQ ID NO: 68) except for the length of the gag gene, where it contains the first 60 bp fragment of the gag gene with point mutations (from nucleotide 1 to nucleotide 569 of the CAEV genome).
• CAEV vif expression vector (pHYK/vij) (SEQ ID NO: 76).
• the vif gene (from nucleotide 5006 to nucleotide 5695 of the CAEV genome), which is known to be required for rapid and efficient virus replication, was cloned into a eukaryotic expression vector pHYK (Kim et al., 2002) (FIGURE 4).
• CAEV rev expression vector (pHYK/rev) (SEQ ID NO: 75).
• the rev gene which regulates viral gene expression at the post-transcriptional level by interacting with the RRE, c onsists of two exons (the first exon i s p ositioned from nucleotide 6,012 to 6,123, and the second exon is from nucleotide 8514 to 8803 of the CAEV genome).
• the Rev/RRE system promotes the nuclear export of unspliced RNA and is known to be essential for lentiviral replication.
• the full-length cDNA of rev g ene was synthesized by RT-PCR and subcloned into the pHYK vector (FIGURE 5).
• the envelope gene expression vector systems used herein are the plasmid pHGVSV-G (SEQ ID NO: 74) and the plasmid pMYKEFl/env (SEQ ID NO: 72) (FIGURES 6A and 6B).
• the plasmid pHGVSV-G (SEQ ID NO: 74) was designed to express the vesicular stomatitis virus G (VSV-G) glycoprotein and contains the HCMV MEEP with ⁇ -globin intron as a promoter.
• the pMYKEF-1/env (SEQ ID NO: 72) was designed to express the gibbon ape leukemia virus (GaLV) envelope protein and contains the MCMV MIEP with eukaryotic elongation factor- l ⁇ intron as a promoter.
• GaLV gibbon ape leukemia virus
• MuLV- and HIV-I -based plasmids As control vector systems, pMFG/lac/Zpuro and pHR/lacZ vectors were used in the present invention, that were lacZ-containing retrovirus vectors derived from the murine leukemia virus (MuLV) (Kim et al., 1997) and the human immunodeficiency virus type 1 (HIV-I) (Naldini et al., 1996), respectively.
• pEQPAM3 Persons et al., 1998) and pCMV ⁇ R8-2 were used, respectively.
• HIV- 1 packaging plasmid pCMV ⁇ R8-2 is identical with pCMV ⁇ R9 (Naldini et al., 1996) except for encoding a functional HIV-I vpu gene and deletion of the 1.3-kb
• Pseudotyped CAEV-based lentiviral vector particles were produced by liposome mediated transient transfection of three or more plasmids into 293T cells plated one day prior to transfection at a density of 5X10 5 cells per 6-well culture dish.
• plasmid cotransfections were performed at a 3:3:3:1 molar ratio of a gag-pol expressing plasmid, a transfer vector plasmid, an ewv-encoding plasmid, and a rev- expressing plasmid.
• Five plasmid cotransfections were performed at a 3:3:3:1:1 molar ratio of a gag-pol expressing plasmid, a transfer vector plasmid, and an env- encoding plasmid, a rev-expressing plasmid and a w/-expressing plasmid.
• the culture supernatant containing viral vector particles was harvested 48 hours later, clarified with a 0.45 ⁇ M membrane filter (Nalgene, NY, USA), and either used immediately or stored at -7O 0 C deep-freezer.
• Transduction was carried out by adding the viral vector particles onto 293T cells for 4 hours, in the presence of 8 ⁇ g/ml polybrene followed by the addition of fresh media. After 48 hours Beta-Gal expression was assayed after the cells were fixed in a solution consisting of 1% formaldehyde and 0.2% glutaraldehyde and stained for 12 hours at 37 0 C in a solution containing 300 ⁇ g of 5-bromo-4-chloro-3- indolylyly b-D-galactoside (X-GaI, Promega, WI, USA), 4mM potassium ferrocyanide, 4mM potassium ferricyanide, and 2mM Mgcl 2 . Titers can be determined by counting the number of blue foci as LacZ- forming units per ml (LFU/ml).
• RT reverse transcription
• the RT reaction was carried out in the presence of MuLV reverse transcriptase, oligo-dT primer or C -terminal specific primer, and dNTPs mix.
• PCR amplification was carried out for semi-quantitative analysis o f t emplate D NA w ith s pecific p rimers.
• P CR p roduct D NA was synthesized from the cDNA or chromosomal DNA in the presence of heat stable Ex Taq polymerase, sequence specific DNA primers, and dNTPs mix.
• Genomic DNA was prepared from cells transduced with either pseudotyped HIV-I or CAEV vector particles, and mock-transduced control cells using the DNeasy Tissue Kit(Qiagen, Germany). Ten ⁇ g of genomic DNA from the HIV-I vector transduced cells were digested with BamH I and Kpn I. Ten ⁇ g each of the genomic DNA from the CAEV vector transduced cells and the negative control cells were double digested with EcoR I and Ssp I. The digested genomic DNAs were separated by electrophoresis on 0.7% agarose gel and transferred onto positive charged nylon membrane (Roche, Germany). Dig-labeled probes were prepared by
• 293T cells were growth-arrested with aphidicolin(Sigma, USA) treatment(25 ⁇ g/ml), then transduced with CAEV viral vector particles.
• aphidicolin Sigma, USA
• cells were transduced side-by-side with either an HIV-I vector or MuLV retrovirus vector.
• Two days after transduction cells were stained with X-gal for beta-gal activity.
• aphidicolin was present before and after infection.
• the growth arrest of c ells was confirmed by F ACS analysis.
• the aphidicolin treated or untreated control cells were washed in PBS, fixed overnight in 70% ethanol at -20°C, and were followed by treatment of propidium iodide (lOO ⁇ g/ml) (Sigma, USA) and RNAse A (lOO ⁇ g/ml) (Qiagen, Germany) at RT for 1 hour.
• the cells were evaluated by FACS analysis, and the percent of total viable cells in Gl, S and G2/M phase of the cell cycle was calculated (Becton Dickinson, Sanjose, CA).
• Replication defective lentiviral vector particles were generated by transient co- transfection of human 293T cells with a minimum of three-plasmid system of a
• CAEV gag-pol expressing plasmid a CAEV env-expressing plasmid and a transfer vector plasmid.
• a CAEV rev expressing plasmid is added, and in a five-plasmid system, a CAEV vif expressing plasmid is added.
• transfer vectors were designed to contain the beginning of the gag- encoding sequence, where mutations were introduced into the start ATG codon and an ATG codon located downstream (ATG to TAG)to prevent the expression of gag proteins.
• RRE was included to boost packaging efficiency and the rev in the four- and five-plasmid systems was expressed from the vector to support the CAEV mRNA export.
• the internal HCMV-MIEP promoter-driven ⁇ -galactosidase gene in the transfer vector plasmid was inserted to serve as a reporter gene.
• the U3 region of the 5'LTR was replaced with the strong viral promoter, HCMV-MIEP, allowing the vector genome to be tat independent.
• Transfer vector RNA transcription level Transcription level of genomic RNA from a transfer vector is one of the critical factors mediating high titer production of recombinant viral vectors from packaging cells.
• HCMV enhancer/promoter element was used to construct the HCMV/CAEV hybrid LTR promoter system for safe and efficient transcription of the transfer vector RNA.
• each of the transfer vector plasmids was introduced into human T cells, together with the packaging plasmids (pMGP/RRE (SEQ ID NO: 77), pHYK/rev (SEQ ID NO: 75), pHYK/vif (SEQ ID NO: 76), pHGVSV-G (SEQ ID NO: 74) or pMYKEFl/env (SEQ ID NO: 72)), by liposome-mediated transfection.
• pMGP/RRE SEQ ID NO: 77
• pHYK/rev SEQ ID NO: 75
• pHYK/vif SEQ ID NO: 76
• pHGVSV-G SEQ ID NO: 74
• pMYKEFl/env SEQ ID NO: 72
• the PCR primer set (RRE primer set) for the CAEV transfer vectors was designed for synthesizing 348-bp PCR product coding for a part of RRE region.
• Another PCR primer set (lacZ primer set) for the HIV-I transfer vector, pHRlacZ (Naldini et al., 1996) was designed for synthesizing the 645 bp PCR product coding for a part of the lacL gene.
• the CAEV transfer vectors of the present invention produced RNA transcript at a level comparable to that of the HIV-I -based lentiviral transfer vector. Formation and release of the vector particles.
• CAEV vector particles were produced following liposome-mediated co-transfection of the pMGP/RRE (SEQ ID NO: 77) gag-pol expression plasmid, the pHGVSV-G (SEQ ID NO: 74) env expression plasmid, the pHYK/rev (SEQ ID NO: 75) rev expression plasmid, pHYK/vif ( SEQ ID NO: 76) vz/expression p lasmid, and the p CAH/SINd60/hlacZ (SEQ ID NO: 78) transfer vector plasmid into human 293T cells (DuBridge et al.,
• the culture supernatant was harvested from the transfected cells and applied to fresh human 293T cells in the presence of 8 ⁇ g/ml polybrene for infection.
• the vector particle production system of (1) the three- plasmid system (pCAH/SDSf, pMGP/RRE (SEQ ID NO: 77), pHGVSV-G (SEQ ID NO: 74) or pMYKEFl/env (SEQ ID NO: 72)), which is devoid of rev- and vif- encoding sequences, (2) the four-plasmids system (pCAH/SIN, pMGP/RRE (SEQ ID NO: 77), pHGVSV-G (SEQ ID NO: 74) or pMYKEFl/env (SEQ ID NO: 72), pHYK/rev (SEQ ID NO: 75)), which is devoid of vz/-encoding sequence and (3) the five-plasmid system (pCAH/SIN, pMGP/RRE (SEQ ID NO: 77), pHGVSV-G (SEQ ID NO: 74) or
• the plasmids of each system were transfected into 293T cells.
• the transfer vector RNA and the virion RNA were extracted from the transfected cells and the culture medium of the transfected cells, respectively, and used as RT-
• Human 293T cells were co-transfected with the pMGP/RRE (SEQ ID NO: 77) gag-pol expression plasmid, the pHGVSV-G (SEQ ID NO: 74) env expression plasmid, the pHYK/rev (SEQ ID NO: 75) rev expression plasmid, the pHYK/vif (SEQ ID NO: 76) vif expression plasmid, and the pCAH/SINd (SEQ ID NOs: 67-71, 73, 78, and 79) transfer vector series plasmid.
• a CAEV transfer vector pCAM/lacZ(L) was transfected in the absence of packaging plasmids.
• virion RNA was extracted from the culture medium of the transfected cells and used as an RT-PCR template with the RRE primer set to detect the CAEV transfer vector series RNA genome, or with the lacL primer set to detect the HIV-I transfer vector RNA genome.
• a strong PCR product signal indicating efficient release of the virus particles containing the viral RNA, was obtained from the culture medium harvested from the virus producing 293T cells transfected with pCAH/SINdl (SEQ ID NO: 68), which contained the complete 5'LTR as well as the first 327 bp of the gag region (lane 3 in FIGURE 10).
• This signal was comparable to that obtained with the positive control, the HIV-I vector, indicating that the amount of the encapsidated CAEV transfer vector RNA of the present invention is comparable to that of the HIV-I -based transfer vectors (lane 8 in FIGURE 10).
• the packaging efficiency of the CAEV transfer vectors with gag-coding region of the first 612 bp or longer was significantly reduced (lanes 4, 5, and 6).
• the PCR product signals were not detectable when the transfer vectors used were devoid of the gag-coding sequences (lane 1 and 2 in FIGURE 10).
• Negative control was transfected with a transfer vector only, and the positive control, HIV-I vector, was transfected with pCMV ⁇ R8-2, pHR'/lacZ and pHGVSV-G (SEQ ID NO: 74) (lanes 7 and 8 in FIGURE 10).
• the transfer vector RNAs were encapsidated efficiently in the packaging cells only when the transfer vectors included less than about 600 bp of the N-terminal gag-coding sequences as well as the entire untranslated region between the 5'LTR and the gag start codon.
• CAEV vector virion was cotransfected with a transfer vector plasmid and the packaging plasmids into human 293T cells.
• the CAEV vector of the present invention was pseudotyped successfully with the GaLV e nvelope ( Lane 2 in FIGURE 11).
• This pseudotyping ability of the CAEV vectors with the GaLV envelope can afford a great advantage in the development of a clinical grade lentiviral vector system.
• MuLV transfected with pEQPAM3, pMFG/lacZ/puro and pHGVSV-G (SEQ ID NO: 74)
• HIV-I transfected with pCMV ⁇ R8-2, pHR'/lacZ and pHGVSV-G (SEQ ID NO: 74) vector controls are shown in lanes 3 and 4, respectively.
• Both the pMGP/RRE (SEQ ID NO: 77) and the pHYK/rev (SEQ ID NO: 75) vectors encode a ned gene for selection in eukaryotic cells.
• another CAEV gag- pol expression vector may be constructed by replacing the ned gene with the other antibiotic resistance genes such as bacterial gpt gene.
• one packaging plasmid system encoding the gag, pol and rev g enes could be used.
• CAEV vector particles were produced by liposome-mediated co-transfection of the pMGP/REV/RRE gag-pol expression plasmid, the pHGVSV-G (SEQ ED NO: 74) env expression plasmid, and the pCAH/SINdl/hlacZ (SEQ ED NO: 79) transfer vector plasmid into human 293T cells.
• the pCMV ⁇ R8.2 gag- pol expression plasmid, the pHGVSV-G (SEQ ID NO: 74) env expression plasmid, and the pHR/lacZ transfer vector were co-transfected into the 293T cells to produce the HIV-I vector particles.
• the CAEV-based transfer vector of the present invention was integrated at a level comparable to that of the HIV-I- based lentiviral transfer vector.
• 293T cells were treated with the DNA synthesis inhibitor, aphidicolin, plated on a 6-well culture plate, and then transduced with the CAEV vector particles encoding a lacZ marker gene.
• cells were infected side-by-side with a lacZ expressing MuLV retroviral vector and HIV-I lentiviral vector.
• expression of the transduced lacZ gene was counted by X-gal staining.
• the MuLV- derived vector efficiently infected cells not treated with the DNA synthesis inhibitor.
• the transduction efficiency was dropped markedly.
• CAH/SINdl/hlacZ (SEQ ID NO: 79) CAEV vector is used to transduce muscle cells in vivo.
• the hind-legs of mice (Beige strain) are intramuscularly injected with 100 ⁇ l of the CAEV vectors in the presence of 4 ⁇ g/ml of polybrene.
• the mice are sacrificed two days later and the injected tissue is prepared for frozen section and for ⁇ -galactosidase analysis.
• the expected result is that CAH/SINdllacZ (SEQ ID NO: 79) CAEV vector transduces muscle cells efficiently in vivo.
• Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells. Proc Natl Acad Sd USA 90(17), 8033-7. Carswell, S., and Alwine, J. C. (1989). Efficiency of utilization of the simian virus 40 late polyadenylation site: effects of upstream sequences. MoI Cell Biol 9(10), 4248-58. Cech, T. R. (1988). Ribozymes and their medical implications. Jama 260(20), 3030-
• the vif gene is essential for efficient replication of caprine arthritis encephalitis virus in goat synovial membrane cells and affects the late steps of the virus replication cycle. J Virol 69(6), 3247-57. Harmache, A., Russo, P., Guiguen, F., Vitu, C, Vignoni, M., Bouyac, M., Hieblot, C, Pepin, M., Vigne, R., and Suzan, M. (1996). Requirement of caprine arthritis encephalitis virus vif gene for in vivo replication. Virology 224(1), 246-55.
• Dystrophin the protein product of the Duchenne muscular dystrophy locus. Cell 51(6), 919-28. Horwich, A. L., Fenton, W. A., Williams, K. R., Kalousek, F., Kraus, J. P., Doolittle, R. F., Konigsberg, W., and Rosenberg, L. E. (1984). Structure and expression of a complementary DNA for the nuclear coded precursor of human mitochondrial ornithine transcarbamylase. Science 224(4653), 1068-74. Karlin, S., and Altschul, S. F. (1990). Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes.
• DNA triple-helix formation an approach to artificial repressors? Antisense ites Dev 1(3), 277-81. Marcus-Sekura, C. J. (1988). Techniques for using antisense oligodeoxyribonucleotides to study gene expression. Anal Biochem 172(2), 289-95.
• NC_001463 TCCTGTACAA AAAAAAGGAG GGATCTCGGT CAGGACCAGG ACCCCTGGGA AF322109 TCTTGTT...
• AAATAAGCCA GGATCTCGAT CAGGACCAAG ACCCCTCAGG
• NC_001463 AGAGAGTGTC TTCCCAATAG TAGTGCAAGC AGCAGGAGGG AGAAGCTGGA
• AF322109 CACATGGACA AGCAAGGATA TCTTAGAAGT ATTAGCCATG ATGCCAGGAA
• NC_001463 AGGTGGAGAA GGAATAATCC ACCACCTCCA GCAGGAGGAG GATTAACAGT
• AF322109 GGATCAGATA ATGGGGGTAG GACAAACGAA TCAGGCAGCG GCACAGGCTA
• NC_001463 CAATAGATGC AGAGCCAGTT ACACAGCCTA TAAAAGATTA TCTAAAGCTA
• AF322109 CAGTGGATGC AGAACCCGTT ACCCAACCTA TAAAAGAATA TTTAAAGGTA
• NC_001463 ACACTATCTT ATACAAATGC ATCAGCAGAT TGTCAGAAGC AAATGGATAG
• NC_001463 AACACTAGGA CAAAGAGTAC AACAAGCTAG TGTAGAAGAA AAAATGCAAG
• NC_001463 CATGTAGAGA TGTGGGATCA GAAGGGTTCA AAATGCAATT GTTAGCACAA
• AF322109 CATGCAGGGA CATCGGGGGA ACAGCATATC AGATGCAGTT GCTTGCACAA
• NC_001463 CGGACCGAAC TATAGTTAGA TGGCATGAGG GCTCGGGAAA CCCAGCCGGA
• AF322109 CAGATAAAAC GATAGTAAGA ATGCATGATG GAACAGGGAT TCCAAACGGA
• NC_001463 AGGATAAAAC TGCAAGGAAT AGGAGGAATA GTAGAAGGAG AAAAATGGAA
• NC_001463 TTAGTGGAAG AAGGAAAACT AGGAAAGGCA CCCCCACATT GGACATGTAA
• NC_001463 ATTGGACATA GGAGATGCAT ATTTTACTAT ACCCCTATAT GAACCATATC
• NC_001463 AAAAGATACT ATTGGAAAGT GCTGCCACAA GGTTGGAAAT TGAGTCCATC
• NC_001463 ATTACAGAAA TTAGTAGGAG AATTAGTATG GAGACAATCC ATAATTGGGA
• AF322109 ATTACAGAAG ATAGTAGGGG AATTAGTGTG GAGACAATCC TTGATAGGAA
• AF322109 CAAAGGAAAG GGGAACCCCT ATGGGTAAAT GTAGTACATG ACATAAAAAA
• NC_001463 CCTAAGCATC CCGCAACAGG TTATTAAAGC AGCGCAAAAA TTAACCCAAG
• NC_001463 AAAGAATAAA GTAGGAAGTC TAGGGTTCAT AGTATCAACA GGGGAAAAAT
• NC_001463 AGAATCCAAT TCAAGCAAGA ATTATGGAAA TTGCCCACAA GAAAGATAGG
• NC_001463 AATAGACAAA TATATTTCGG AAATATTTCT TGCAAAAGAA GGAGAAGGAA
• NC_001463 TTCTCCCAAA AAGAGAAGAG GATGCAGGGT ATGATTTAAT ATGCCCAGAA
• NC_001463 CAGGGACAAA TACAGGTAAT AATGTATAAT AGCAATAAAA TAGCAGTAGT
• NC_001463 CATACCCCAA GGGAGAAAAT TTGCACAATT AATATTAATG GATAAAAAGC
• NC_001463 ATGGAAAATT GGAACCCTGG GGGGAAAGCA GAAAAACAGA AAGGGGAGAA
• NC_001463 AAAGGATTTG GGTCTACAGG AATGTATTGG ATAGAAAATA TTCCTCTGGC
• NC_001463 AAAGGAGAAT CAGGGCAAGA ATTCAGAATA AAAGTGATGC ATTGGTATGC
• NC_001463 CAGAGCCCAC ACAGCTGTTA ATGCAATACC TAGGAGTAAA ACACACAACA
• NC_001463 AATCAGCCAT AGCAGCAGCC CTAGTCGCCA TAAATATAAA AAGAAAGGGT
• NC_001463 GTGAAAAGTA TTTAGTAATA CCTTACAAAG ATGCAAAATT CATCCCGCCA
• NC_001463 CAACGGGATA CACGCATGCG TGGTCTGTCC AGGAGTGCTG GTTGATGGAA
• AF322109 CAAAAGGATT CCTGGAGCCG TGGACAACGC AAGAGTGTTG GCAAATAGAG
• NC_001463 TATCTCTTAG AGGATGAGTG AAGAACTGCC TCAAAGAAGG GAGACACATC
• AF322109 TATCCCTTGG AGGATGAGTG AGGAAACCCC AGCAGGAAGA GAACCGACTG
• AF322109 CAGAGGAAAT ATTTGAGCAA GAA GCAGAAAGT. TGGAA
• NC_001463 TAAATGTAAC TAACAAGTAG CAAAAGTGTC TGTGTTAGAT GGATGCTGGG
• NC_001463 CCATGCTTTG CCTATAAAGG GATATTCCTA TGGAGGATAT CACTAACAAT
• NC_001463 CACTAATATC AGATCCCTAT GGGTTCTCAC CCATAAAAAA TGTGTCTGGG
• AF322109 ATCCAGTACC TCCCAAGGCA GAGTTATTCC CTCCAGCGTT TCAGGATTTA
• NC_001463 AGAATGGGTA AATGGGACAC CCCCGGATTG GCAAGACAGA ATTAACGGAT
• NC_001463 CCAAAGGAAT AAATGGGACG CTCTGGGGAG AGCTTAACAG TATGCATCAC
• AF322109 CCAACAGGTT CATTGGTTTG ATACCACGCC ACAATATCAT TTAGGAT...
• NC_001463 CTAGGATTTG CCCTTAGCCA GAACGGCAAA TGGTGTAACT ACACCGGGGA
• NC_001463 AATAAAATTA GGGCAAGAAA CATTCCAATA TCATTACAAG CCAAACTGGA
• NC_001463 CCTAACAAGG ATCCTGGGAA CAAATACAAA TTGGACAACT ATGTGGGGAA
• NC_001463 ACATATGGGG TGATAGAAAT GCCAGAAAAC TATGCAAAAA CAAGAATCAT
• AF322109 ACATACGGAG TGATTGATAT GCCAGACAAT TATG.AGACC CTACCAGGA.
• NC_001463 AAACAGGAAA AAAAGAGAAC TCAGCCACAA GAGGAAGAAG AGAGGCGTTG
• NC_001463 CCTATGCCAT GGTACAGCAT GTGGCTAAAG GCGTACGAAT CTTGGAAGCT
• NC_001463 ATTGGATTGT TGGCACTATC ATCAATACTG TATAACCTCT ACAAAAACAG
• NC_001463 CAGCAGTGGG AGAGAGGATT ACAGGGGTAT GATACAAACT TAACAATACT
• AF322109 CAAGAGTGGG AAAGGGAGAT AAGTGCGCAT GAAGGAAACA TCACTATATT
• NC_001463 GTTAAAGGAA TCAGCAGCAA TGACACAACT AGCAGAAGAG CAAGCAAGGA
• NC_001463 GCCAAATTCC TGTAAATCAC TTGGGGGGTT ATAAGAAAAG CAAGTTCACT
• NC_001463 (gag720bp) ATGGTGAGTC TAGATAGAGA CATGGCGAGG CAAGTCTCCG GGGGGAAAAG AF322109 (gag720bp) .ATGGTGAGG CAGGCCTCCG GAAGGGGAAA
• NC 001463 (gag720bp) AGATTATCCT GAGCTCGAAA AATGTATCAA GCATGCATGC AAGATAAAAG AF322109(gag720bp) GGAGTACCCC GAGCTAAAAG AATGTCTGAA AAAGGCATGC AAAATAAAAG
• NC 001463 (gag720bp) TTCGACTCAG AGGGGAGCAC TTGACAGAAG GAAATTGTTT ATGGTGCCTT AF322109(gag720bp) TAAGGGCTGG GGGGGAGCGC CTGACAGAAG GAAATTGTCT CTGGTGTATA
• NC 001463 (gag720bp) AAAACATTAG ATTACATGTT TGAGGACCAT AAAGAGGAAC CTTGGACAAA AF322109(gag720bp) AAAACACTAG AGTGTATGTA TGAGGATTGT AGGGAGGAAC CTTGGACCCC
• NC 001463 (gag720bp) AGTAAAATTT AGGACAATAT GGCAGAAGGT GAAGAATCTA ACTCCTGAGG AF322109(gag720bp) AGAAAAATGT AAACAATTAT GGAAAAAGTT GAAGCAGGTA GAGCCTGAGG
• NC_001463 (gag720bp) AGAGTAACAA AAAAGACTTT ATGTCTTTGC AGGCCACATT AGCGGGTCTA AF322109(gag720bp) AGAGTAGCAA AGCAGACTAT AACTCGTTAA AAGCAACCTT GGCGGGGATA
• NC 001463 (gag720bp) ATGTGTTGCC AAATGGGGAT GAGACCTGAG ACATTGCAAG ATGCAATGGC AF322109(gag720bp) GTCTGTGTGC AAATGGGAAT GCAGCCCGAG ACACTGCAGG ATGCGATAGC
• NC_001463 (gag720bp) TACAGTAATC ATGAAAGATG GGTTACTGGA ACAAGAGGAA AAGAAGGAAG AF322109(gag720bp) AACCTTAAAC ATGAGAGATG AAGT AAAAGGAA AGGAA..AAG
• NC 001463 (gag720bp) GGAGGGAGAA GCTGGAAAGC AGTAGATTCT GTAATGTTCC AGCAACTGCA AF322109(gag720bp) GGAGGAAGAG CATGGAGAGC GGTAGAGCCT GCTACCTTTC AGCAGCTCCA
• NC_0014S3 (gag720bp) AACAGTAGCA ATGCAGCATG GCCTCGTGTC TGAGGACTTT GAAAGGCAGT AF322109(gag720bp) AACAGTGGCA ATGCAGCATG GACTAGTATC AGAAGAATTT GAAAGGCAGC
• NC_001463 (gag720bp) TGGCATATTA TGCTACTACC TGGACAAGTA AAGACATACT AGAAGTATTG AF322109 (gag720bp) TAGCATACTA TGCCACCACA TGGACAAGCA AGGATATCTT AGAAGTATTA
• NC 001463 (gag720bp) GCCATGATGC CTGGAAATAG AGCTCAAAAG GAGTTAATTC AAGGGAAATT AF322109(gag720bp) GCCATGATGC CAGGAAATAG AGCGCAAAAA GAACTAATAC AAGGAAAGTT
• NC_001463 (gag720bp) AAATGAAGAA GCAGAAAGGT GGAGAAGGAA TAATCCACCA CCTCCAGCAG AF322109(gag720bp) AAATGAGGAA GCAGAGAGAT GGAGAAGGCA GAATCCACAA CCTGCGG...
• NC_001463 (gag720bp) GAGGAGGATT AACAGTGGAT AF322109(gag720bp) ...GCGGGTT AACCGTGGAT CAGATAATGG GGGTAGGACA AACGAATCAG
• NC_00i463 (gag) ATGGTGAGTC TAGATAGAGA CATGGCGAGG CAAGTCTCCG GGGGGAAAAG AF322109(gag) ATGGTGAGG CAGGCCTCCG GAAGGGGAAA
• NC_001463 (gag) AGATTATCCT GAGCTCGAAA AATGTATCAA GCATGCATGC AAGATAAAAG AF322109(gag) GGAGTACCCC GAGCTAAAAG AATGTCTGAA AAAGGCATGC AAAATAAAAG
• NC_001463 (gag) TTCGACTCAG AGGGGAGCAC TTGACAGAAG GAAATTGTTT ATGGTGCCTT AF322109(gag) TAAGGGCTGG GGGGGAGCGC CTGACAGAAG GAAATTGTCT CTGGTGTATA
• NC_001463 (gag) AAAACATTAG ATTACATGTT TGAGGACCAT AAAGAGGAAC CTTGGACAAA AF322109(gag) AAAACACTAG AGTGTATGTA TGAGGATTGT AGGGAGGAAC CTTGGACCCC
• NC_001463 (gag) AGTAAAATTT AGGACAATAT GGCAGAAGGT GAAGAATCTA ACTCCTGAGG AF322109 (gag) AGAAAAATGT AAACAATTAT GGAAAAAGTT GAAGCAGGTA GAGCCTGAGG
• NC_001463 (gag) AGAGTAACAA AAAAGACTTT ATGTCTTTGC AGGCCACATT AGCGGGTCTA AF322109(gag) AGAGTAGCAA AGCAGACTAT AACTCGTTAA AAGCAACCTT GGCGGGGATA
• NC_001463 (gag) ATGTGTTGCC AAATGGGGAT GAGACCTGAG ACATTGCAAG ATGCAATGGC AF322109 (gag) GTCTGTGTGC AAATGGGAAT GCAGCCCGAG ACACTGCAGG ATGCGATAGC
• NC_001463 (gag) TACAGTAATC ATGAAAGATG GGTTACTGGA ACAAGAGGAA AAGAAGGAAG AF322109(gag) AACCTTAAAC ATGAGAGATG AA GTAAAAGGAA AGGAA..AAG
• NC_00l463 (gag) ACAAAAGAGA AAAGGAAGAG AGTGTCTTCC CAATAGTAGT GCAAGCAGCA AF322109(gag) CCATCAGAAG AAAAGAAGGG AATATATCCC ..ATATTAGT GCAGGCAGGA
• NC_001463 (gag) GGAGGGAGAA GCTGGAAAGC AGTAGATTCT GTAATGTTCC AGCAACTGCA AF322109(gag) GGAGGAAGAG CATGGAGAGC GGTAGAGCCT GCTACCTTTC AGCAGCTCCA
• NC_001463 (gag) AACAGTAGCA ATGCAGCATG GCCTCGTGTC TGAGGACTTT GAAAGGCAGT AF322109(gag) AACAGTGGCA ATGCAGCATG GACTAGTATC AGAAGAATTT GAAAGGCAGC
• NC_OO1463 (gag) TGGCATATTA TGCTACTACC TGGACAAGTA AAGACATACT AGAAGTATTG AF322109(gag) TAGCATACTA TGCCACCACA TGGACAAGCA AGGATATCTT AGAAGTATTA
• NC_001463 (gag) GCCATGATGC CTGGAAATAG AGCTCAAAAG GAGTTAATTC AAGGGAAATT AF3-22109(gag) GCCATGATGC CAGGAAATAG AGCGCAAAAA GAACTAATAC AAGGAAAGTT 651 700
• NC_001463 (gag) AAATGAAGAA GCAGAAAGGT GGAGAAGGAA TAATCCACCA ' CCTCCAGCAG
• AF322109 (gag) AAATGAGGAA GCAGAGAGAT GGAGAAGGCA GAATCCACAA CCTGCGG...
• NC_001463 GAGGAGGATT AACAGTGGAT CAAATTATGG GGGTAGGACA AACAAATCAA
• AF322109 (gag) ...GCGGGTT AACCGTGGAT CAGATAATGG GGGTAGGACA AACGAATCAG
• NC_001463 (gag) GCAGCAGCAC AAGCTAACAT GGATCAGGCA AGGCAAATAT GCCTGCAATG
• AF322109 (gag) GCAGCGGCAC AGGCTAATAT GGATCAAGCA AGACAAATAT GCCTACAATG
• NC_001463 (gag) GGTAATAAAT GCATTAAGAG CAGTAAGACA TATGGCGCAC AGGCCAGGGA
• AF322109 (gag) GGTTATAACA GCAATAAGAG GAGTTAGGCA TATGGCCCAT AGACCAGGAA
• NC_001463 ATCCAATGCT AGTAAAGCAA AAAACGAATG AGCCATATGA AGATTTTGCA
• AF322109 (gag) ATCCCATGCT GGTAAGACAA AAACCAAATG AGAACTATGA AGAGTTTGCC
• NC_001463 (gag) GCAAGACTGC TAGAAGCAAT AGATGCAGAG CCAGTTACAC AGCCTATAAA
• AF322109 (gag) GCAAGGTTGT TAGAAGCAGT GGATGCAGAA CCCGTTACCC AACCTATAAA
• NC_001463 (gag) AGATTATCTA AAGCTAACAC TATCTTATAC AAATGCATCA GCAGATTGTC
• AF322109 (gag) AGAATATTTA AAGGTAACTC TGTCTTACAC AAATGCAAAT TCGGAATGTC
• NC_001463 AGAAGCAAAT GGATAGAACA CTAGGACAAA GAGTACAACA AGCTAGTGTA
• AF322109 (gag) AAAAACATAT GGACAGAGTG TTGGGGCAAA GAGTACAGCA GGCCTCAATA

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