WO2007133674A2 - Compositions à base de vecteurs lentiviraux, procédés et applications - Google Patents

Compositions à base de vecteurs lentiviraux, procédés et applications Download PDF

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WO2007133674A2
WO2007133674A2 PCT/US2007/011385 US2007011385W WO2007133674A2 WO 2007133674 A2 WO2007133674 A2 WO 2007133674A2 US 2007011385 W US2007011385 W US 2007011385W WO 2007133674 A2 WO2007133674 A2 WO 2007133674A2
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cell
vector
lentiviral
repressor
groi
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WO2007133674A3 (fr
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Boro Dropulic
Yung Nien Chang
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Lentigen Corporation
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
    • C12N2830/003—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor tet inducible

Definitions

  • the present invention relates to lentiviral constructs and their use in transient production of recombinant retrovirus in mammalian cells and to methods of using such constructs to deliver and express heterologous gene products in mammalian cells with high efficiency.
  • Retroviral vectors have become the primary tool for gene delivery in human gene therapy applications.
  • the ability of retrovirus vectors to deliver a single copy gene into a broad range of rodent, primate, and human somatic cells in primary culture makes them well suited for this purpose.
  • Retroviruses are typically classified as belonging to one of three subfamilies, namely oncoviruses, spumaviruses and lentiviruses. These viruses typically contain a single stranded, plus-strand RNA genome of approximately 8,000 to 10,000 nucleotides encompassing gag, pol, and env genes, as well as long terminal repeat (LTR) sequences.
  • LTR long terminal repeat
  • lentiviruses In addition to the gag, pol, and env genes, lentiviruses possesses a number of additional genes with regulatory functions. The lentiviral group can be split further into “primate” and “non-primate.”
  • primate lentiviruses include the human immunodeficiency virus (HIV), the causative agent of human auto-immunodeficiency syndrome (AIDS), and the simian immunodeficiency virus (SIV).
  • the non-primate lentiviral group includes the prototype "slow virus” visna/maedi virus (VMV), as well as the related caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIR) and bovine immunodeficiency virus (BIV).
  • VMV visna/maedi virus
  • CAEV caprine arthritis-encephalitis virus
  • EIAV equine infectious anemia virus
  • FIR feline immunodeficiency virus
  • BIV bovine immunodeficiency virus
  • lentivirus family and other types of retroviruses are that lentiviruses have the capability to infect both dividing and non-dividing cells.
  • retroviruses such as MLV
  • are unable to infect non-dividing cells such as those that make up, for example, muscle, brain, lung, and liver tissue.
  • GROI RNAs of interest
  • Current methodologies used to design retroviral vectors that express one or more genes or RNAs of interest have relied on three general strategies: i) the expression of different proteins from alternatively spliced mRNAs transcribed from one promoter; ii) the use of the promoter in the 5' LTR and internal promoters to drive transcription of different cDNAs and (iii) the use of internal ribosomal entry site (IRES) elements to allow translation of multiple coding regions from either a single mRNA or from fusion proteins that can then be expressed from an open reading frame.
  • IRS internal ribosomal entry site
  • genes or RNAs that contain splice sites are problematic for expression from lentiviral vectors because splicing during production can result in a truncated genomic lentiviral vector RNA.
  • the present invention relates to vectors which allow efficient expression of genes or RNAs of interest (GROI) which possess internal splicing sites. Further, the invention relates to the construction of lentiviral vector where expression of such GROIs do not result a truncated genomic lentiviral vector RNA. Moreover, the lentivira! vector constructs are designed such that expression from the vector can be modulated via inducible promoters. These lentiviral vectors, and in particular HIV vectors, are useful in functional genomics, drug discovery, target validation, protein production, vaccine production, clinical gene therapy, and other applications.
  • a lentiviral transfer vector including, in operable linkage, a 5' LTR, a 3' LTR comprising a polyadenylation signal, a minimal packaging signal; an inducible promoter (iP), a nucleotide sequence encoding a cognate repressor for the inducible promoter, and a nucleic acid sequence encoding a gene or RNA of interest (GROI) operatively linked to the iP.
  • the operably linked iP-GROI is configured to be in an opposite transcriptional orientation relative to the 5' LTR and nucleic acid encoding the repressor.
  • the iP is regulated by a repressor-transactivator fusion protein where the repressor is constitutively expressed.
  • the iP is regulated by an antibiotic-responsive gene regulon.
  • the antibiotic is a small molecule includes, but is not limited to, tetracycline, streptogrami ⁇ , macrolide, or cumate.
  • the vector includes a nucleotide sequence encoding a product which inhibits cell growth.
  • an infectious lentiviral particle produced by a cell transduced by a lentiviral vector includes, in operable linkage, a 5' LTR, a 3' LTR comprising a polyadenylation signal, a minimal packaging signal, an inducible promoter (iP), a nucleotide sequence encoding a cognate repressor for the inducible promoter, and a nucleic acid sequence encoding a gene or RNA of interest (GROI) operatively linked to the iP.
  • the operably linked iP-GROI is configured to be in an opposite transcriptional orientation relative to the 5' LTR and nucleic acid encoding the repressor.
  • the cell is a producer cell.
  • a method of making lentiviral particles including providing a lentiviral vector producer cell, introducing a lentiviral vector into a producer cell in the presence of a small molecule which modulates a repressor- transactivator fusion protein, and collecting lentiviral particles from the producer cells.
  • the lentiviral vector includes, in operable linkage, a 5' LTR, a 3' LTR comprising a polyadenylation signal, a minimal packaging signal, an inducible promoter (iP), a nucleotide sequence encoding a cognate repressor for the inducible promoter, and a nucleic acid sequence encoding a gene or RNA of interest (GROI) operatively linked to the iP.
  • the operably linked iP-GROI is configured to be in an opposite transcriptional orientation relative to the 5' LTR and nucleic acid encoding the repressor.
  • the particle comprises a heterologous env protein.
  • a method of modulating expressing a gene or RNA of interest (GROI) in a host cell including, contacting a cell with a lentiviral vector and contacting the cell with a small molecule which regulates the repressor-transactivator fusion protein, where if the small molecule is present, expression from the iP is inhibited, and if the small molecule is removed, expression from the iP is disinhibited.
  • GROI RNA of interest
  • the lentiviral vector includes, in operable linkage, a 5' LTR, a 3' LTR comprising a polyadenylation signal, a minimal packaging signal, an inducible promoter (iP) comprising a cognate repressor binding site.
  • iP inducible promoter
  • the iP is regulated by a repressor-transactivator fusion protein, and a nucleic acid sequence encoding gene or RNA of interest (GROI) operatively linked to the iP, where the iP-GROI is configured to be in an opposite transcriptional orientation relative to the 5' LTR and the nucleic acid encoding the repressor.
  • GROI nucleic acid sequence encoding gene or RNA of interest
  • Figure 1 is a illustration of the restriction map of a Tet-regulated lentiviral vector for delivering heterologous RNA.
  • Lentiviruses are characterized by long incubation periods between infection of the host and the manifestation of clinical disease. Lentiviruses infect a wide variety of mammals, including humans, monkeys, sheep, goats, and horses, and includes for example retroviruses, such as immunodeficiency viruses, such as HIV-I , HIV-2, FIV, and SIV.
  • the term "packaging cell” means a cell that provides packaging functions in trans for a transgene introduced into a cell with a transfer vector, but which does not encapsidate its own viral RNA.
  • packaging defective means a packaging vector which lacks the nucleic acids necessary for packaging of an RNA corresponding to the packaging vector nucleic acid into a retroviral (e.g. HIV, SIV) capsid. That is, packaging vector nucleic acids are not themselves encapsidated in the HIV or SIV particles which they encode, i.e. they are not infective.
  • retroviral e.g. HIV, SIV
  • packaging signal means nucleic acid sequences upstream and downstream from the splice donor (SD) site which are necessary for the efficient packaging of the vector RNA genome.
  • packaging vector means a vector that lacks the nucleic acids necessary for packaging of an RNA corresponding to the packaging vector nucleic acid into a retroviral (e.g. HIV, SIV) capsid. That is, packaging vector nucleic acids are not themselves encapsidated in the HIV or SIV particles which they encode, i.e. they are not infective.
  • the packaging vector optionally includes all of the components necessary for production of HIV or SIV particles, or optionally includes a subset of the components necessary for HIV or SIV packaging. For instance, a packaging cell may be transformed with more than one packaging vector, each of which has a complementary role in the production of an HIV or SIV particle.
  • Two (or more) HIV- or SIV-based packaging vectors are "complementary" when they together encode all of the functions necessary for HIV or SIV packaging, and when each individually does not encode all of the functions necessary for packaging. For example, when two vectors transduce a single cell and together they encode the information for production of HIV or SIV packaging particles, the two vectors are "complementary.”
  • the use of complementary vectors increases the safety of any packaging cell made by transformation with a packaging vector by reducing the possibility that a recombination event will produce an infective virus.
  • 3' LTR refers to a 3' retroviral or lentiviral long terminal repeat, which may or may not be modified from its corresponding native (i.e., that existing in the wild-type retrovirus) 3' LTR by deleting and/or mutating endogenous sequences and/or adding heterologous sequences.
  • the 3' LTR may be natural or synthetic.
  • 5' LTR refers to a 5' retroviral or lentiviral long terminal repeat, which may or may not be modified from its corresponding native 5' LTR by deleting and/or mutating endogenous sequences and/or adding heterologous sequences.
  • the 5' LTR may be natural or synthetic.
  • 3' LTR polyadenylation signal refers to the polyadenylation signal present in the 3' LTR of retroviruses or lentiviruses.
  • the polyadenylation signal may be natural or synthetic.
  • Upstream enhancer and "UE” are used interchangeably, and refer to a control element present in the 3' untranslated region of various eukaryotic and viral genes that enhances transcriptional termination by a polyadenylation signal located downstream of the enhancer.
  • UEs are found in the SV40 late polyadenylation signal (USE), the HIV-I LTR (UHE) and the ground squirrel hepatitis virus (UGE).
  • USE SV40 late polyadenylation signal
  • UHE HIV-I LTR
  • UGE ground squirrel hepatitis virus
  • the UEs may be natural or synthetic.
  • Upstream enhancer sequence and "UE sequence” are used interchangeably, and refer to the sequence of a UE or an active segment thereof. Like a UE, an active segment of a UE increases the transcriptional termination activity of a polyadenylation signal when it is placed 5' upstream of that signal.
  • a UE may comprise many active segments that may or may not be overlapping in sequence.
  • a "heterologous" UE sequence is a UE sequence from a UE not identical to the one present in the native 3' LTR of the virus from which the viral vector of the invention is derived.
  • an "endogenous" UE sequence is a UE sequence from a UE present, such as UHE of HIV-I, in the native 3' LTR of the virus from which the viral vector of the invention is derived.
  • 3' transcription termination structures of a lentiviral vector refer to structures within and proximal to the 3' LTR that effect termination of transcriptions initiated upstream of the structures.
  • Such structures comprise the 3' LTR polyadenylation signal and may additionally comprise endogenous UE sequences and heterologous UE sequences operatively associated with that signal.
  • the structures may be natural or synthetic.
  • Retrovirus denotes a class of viruses which use RNA-directed DNA polymerase, or "reverse transcriptase” to copy a viral RNA genome into a double-stranded DNA intermediate which integrates into the chromosomal DNA of a host cell.
  • Retroviruses include lentiviruses. Examples of retroviruses include, but are not limited to, Moloney murine leukemia virus, spleen necrosis virus, Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus.
  • viral vector genome refers to a polynucleotide comprising sequences from a viral genome that are sufficient to allow an RNA version of that polynucleotide to be packaged into a viral particle, and for that packaged RNA polynucleotide to be reverse transcribed and integrated into a host cell chromosome by the action of the viral enzymes, such as reverse transcriptase and integrase, contained in the viral particle.
  • a "heterologous" gene or coding sequence is a gene or coding sequence that is not identical to any gene or coding sequence found in the virus from which the viral vector of the invention is derived.
  • Packaging construct refers to a nucleic acid containing (and in a producer cell expressing) at least lentiviral or retroviral gag, preferably expressing both lentiviral or retroviral gag and pol, and optionally also containing (and in a producer cell expressing) additional lentiviral genes, including but not limited to Tat, Rev, Vif, Vpu, Vpr, Nef and Env.
  • Producer cell refers to (i) cells that stably and constitutively express the proteins required for packaging of vector particles, (ii) cells that stably and inducibly express the proteins required for packaging of vector particles, and (iii) transiently transfected cells that express the proteins required for packaging of the vector particles for a limited period of time.
  • Minimal packaging signal refers to a packaging signal in a transfer vector construct that has been reduced in size (e.g., to include not more than 40, 60, or 80 continuous nucleic acids of the gag gene) and/or a packaging signal in a transfer vector construct that contains one or more substitution mutations therein to reduce or minimize homology to a corresponding portions of the gag ORF in separate nucleic acid construct(s) in the producer cell, in like manner as described with respect to mutated gag sequences for packaging constructs below.
  • An antibiotic-responsive gene regulon means a gene expression control system comprising transcriptional signals and protein repressors/activators which are modulated in concert by specific antibiotics.
  • the most widely used antibiotic-responsive gene regulation systems are derived from prokaryotic regulons consisting of a repressor (i.e., TetR; PlP) that dissociates from cognate promoters/operators P P [ R //?ZV) in response to tetracyclines/streptogramins.
  • the vectors of the invention comprise a viral vector genome having a 5' LTR, a 3' LTR comprising a polyadenylation signal, and a packaging signal.
  • the viral vector genome may be from a lentivirus including, but not limited to, human immunodeficiency virus, simian immunodeficiency virus, equine infectious anemia virus, feline immunodeficiency virus, visna virus.
  • the vectors comprise a viral vector genome that is replication defective. Typically, such a defect is due to a mutation and/or deletion of one or more viral structural and replication functions (e.g., gag, pol, env). Accordingly, vectors of the invention may be derived from replication defective viral vectors in the art as noted below.
  • Vectors of the invention may be derived from replication defective lentiviral vectors including, but not limited to, those based on: HlV-I, such as pHR'CMVlacZ, pHR'CMVIacZ SIN18, and pRRLPGK-GFP (Zufferey et al., J. Virol. 72:9873-9880 (1998)), LL-CG, CL-CG, LS-CG, CS-CG and CL-G (Miyoshi et al., J. Virol. 72.
  • HIV-2 (Arya et al., Hum Gene Ther.9: 1371-80 (1998)); simian immunodeficiency virus, such as pVG (Schnell et al., Hum Gene Ther 1 1 :439-47 (2000)); feline immunodeficiency virus, such as F ⁇ V-gal (Wang et al., J Clin Invest.104.-R55-62 (1999)), PTFIV (Johnston et al., J Virol 73:4991-5000 (1999)); equine infectious anemia virus, such as pONY2.101acZ, pONY4.0Z (Mitrophanous et al., Gene Ther. 6:1808-18 (1999)).
  • pVG Schonell et al., Hum Gene Ther 1 1 :439-47 (2000)
  • feline immunodeficiency virus such as F ⁇ V-gal (Wang et al., J Clin Invest.104.-R55-62 (1999)),
  • the vectors of the invention may be derived from non-mobilizing or self-inactivating (SIN) lentiviral vectors.
  • the 5' LTR of the vectors may be an unmodified viral 5' LTR. That is, a native 5' LTR as it exists in a lentivirus or retrovirus.
  • the endogenous U3 promoter of the 5' LTR has been inactivated by mutation and/or deletion and replaced with a heterologous promoter.
  • the activity of the heterologous promoter may be constitutive, inducible or target cell-specific (i.e., expression is preferential or limited to one or several specific cell types and not or less so in other cell types).
  • Useful heterologous promoters include, but are not limited to, CMV (Miyoshi et al., J. Virol. 72.
  • Rous sarcoma virus promoter Dull et al., J. Virol. 72:8463-71 (1998)
  • tetracycline-inducible promoter Hwang et al., J. Virol. 71 :7128-31 (1997)).
  • the 3' LTR of the vectors may be an unmodified lentiviral or retroviral 3' LTR.
  • the endogenous U3 promoter of the 3' LTR has been inactivated by mutation or deletion.
  • such inactivation is specific to the U3 promoter. That is, the inactivation does not adversely affect other structures, such as the att sequence needed for integration and any endogenous UE, of the 3' LTR.
  • the promoter is inactivated by mutating or deleting sequences in the region that corresponds to about residues 91 13 to 9506 of the pNL4-3 strain of HIV- I .
  • the vectors of the invention have an enhanced 3 ' transcription termination structure, which may comprise one or several UE sequences operably associated with the polyadenylation signal in the 3' LTR.
  • the UE sequence may be a heterologous UE sequence or an additional copy of any endogenous UE sequence which may be present in the 3' LTR.
  • the 3' transcription termination structure comprises one or several heterologous UE sequences.
  • the 3' transcription termination structure comprises one or several additional copies of an endogenous UE sequence.
  • the 3' transcription termination structure comprises both heterologous and an additional copy of endogenous UE sequences.
  • the vectors of the invention may additionally comprise a microbial origin of replication and a microbial screenable or selectable marker for use in amplifying vector sequences in microbial cells, such as bacteria and yeast.
  • the transcriptional termination structures used in the construct may comprise the 3' untranslated region of an eukaryotic or viral gene.
  • the 3' untranslated region comprises an endogenous polyadenylation signal.
  • the transcription termination structure comprises a viral 3' LTR.
  • a transcription termination structure may comprise a modified 3' LTR, where the U3 promoter is inactivated by deletion or other means.
  • the transcription termination structure comprises a 3' LTR that is to be incorporated into a vector of the invention.
  • the vectors of the present invention may include one or more posttranscriptional regulatory element (PRE).
  • PREs such as the WPRE are' known and described in, for example, U.S. Pat. Nos. 6,312,912 and 6,287,814, which disclose posttranscriptional regulatory elements useful for efficient RNA export of RNA is provided. WPRE was originally derived from woodchuck hepatitis virus.
  • Other PREs sometimes also termed "RNA export elements," are described in U.S. Pat. No. 6,677,500, and include, but are not limited, to Mertz sequences (also described in U.S. Pat. Nos. 5,914,267 and 5,686, 120).
  • PREs and particularly WPREs are further described in U.S. Pat. Nos. 6,555,342; 6,312,912; 6,287,814; 6,808,905; 6,800,281; and 6,712,612.
  • the vectors include one or more central polypurine tract element, or "cPPT".
  • cPPT elements are known and described in, for example, U.S. Pat. Nos. 6,800,281 ; 6,649,159; 6,627,442; and 6,682,907.
  • U.S. Pat. No. 6,682,907 the identification of cPPT sequences is facilitated by the fact that a polypurine sequence located at the upstream edge (5') of the 3' LTR in all retroviruses is repeated in the center of the genome in lentiviruses.
  • This cPPT sequence can be an exact repeat as in the HIV-I virus, or slightly modified in other lentivirus.
  • the sequence of nucleotides comprising cPPT can be point mutated or mutated by deleting or inserting nucleotides.
  • point mutations have been produced in the cPPT sequence of HIV-I and have shown that it retained residual infectivity in the cells (Chameau et al, J. Virol. 1992, 66, p 2814-2820).
  • the vectors of the invention may be beneficially used to express desired gene products in mammalian cells and organisms. Accordingly, the vectors may additionally comprise one or more heterologous coding sequences, wherein such sequences are derived from sources other than the lentiviral genome from which the vectors are derived.
  • the heterologous coding sequences are inserted into the viral backbone, for example, between the 5' and 3' LTRs, such that they are operably associated with the 5' LTR promoter. Where such insertions lead to production of polycistronic mRNA comprising the heterologous coding sequences, it may be advantageous to also operatively associate each heterologous coding sequence with an IRES in order to achieve efficient translation of each sequence.
  • the heterologous coding sequences are each operably associated with an individual promoter to form expression constructs, and such constructs are inserted into the viral backbones, for example, between the 5' and 3' LTRs.
  • the expression constructs may comprise promoters that are constitutive, inducible, tissue-specific, or cell-cycle specific. Examples of useful promoters include, but are not limited to, the SV40 promoter, CMV promoter, adenovirus promoters, B 19 parvovirus promoters, histone promoter, pol III promoter, and beta-actin promoters.
  • a lentiviral transfer vector including, in operable linkage, a 5' LTR, a 3' LTR comprising a polyadenylation signal, a minimal packaging signal; an inducible promoter (iP), a nucleotide sequence encoding a cognate repressor for the inducible promoter, and a nucleic acid sequence encoding a gene or RNA of interest (GROI) operatively linked to the iP.
  • the operably linked iP-GROI is configured to be in an opposite transcriptional orientation relative to the 5' LTR and nucleic acid encoding the repressor.
  • the iP is regulated by a repressor-transactivator fusion protein where the repressor is constitutively expressed.
  • vectors of the invention include polypeptides, proteins and RNAs such as structural RNAs, anti-sense RNAs, short interfering RNAs, and ribozymes.
  • the vectors of the invention comprise and express one or more heterologous sequences encoding therapeutic polypeptides.
  • Example therapeutic polypeptides include cytokines, growth factors, hormones, kinases, receptors, receptor ligands, enzymes, antibody polypeptides, transcription factors, blood factors, and artificial derivatives of any of the foregoing.
  • the preintegration complex of lenti viruses have been shown to possess nuclear targeting signals which allow these viruses to infect non-dividing cells including macrophages.
  • the capacity of HIV-I (P. Lewis et al., EMBO J., 1 1 :3053-3058 (1992); M. Burinsky et al., Proc. Natl. Acad. Sci. USA, 89:6580-6584 (1992)) vectors to stably transduce non-dividing cells has been demonstrated in vitro (J. Reiser et al., Proc. Natl. Acad. Sci. USA, 93:15266-15271 (1996)) and also in vivo (L. Naldini et al., Science, 272:263-267 (1996)).
  • these vectors are capable of long-term expression.
  • a second feature of HIV-I based vectors is the ability to manipulate the target cell range by substituting the HIV-I envelope glycoprotein, gpl60, with envelope proteins from other viruses which confer an extended host range that can be specifically targeted.
  • VSV vesicular stomatitis virus
  • a second feature of HIV-I based vectors is the ability to manipulate the target cell range by substituting the HIV-I envelope glycoprotein, gpl60, with envelope proteins from other viruses which confer an extended host range that can be specifically targeted.
  • VSV vesicular stomatitis virus
  • Lentiviral vectors such as HIV-I vectors have therefore been developed to a point of clinical utility and offer considerable potential as an in vivo tool for the manipulation of both dividing and non-dividing cells.
  • the lentiviral virion is expressed by a vector system encoding the necessary viral proteins to produce a virion (viral particle).
  • these include at least one vector containing a nucleic acid sequence encoding the lentiviral pol proteins necessary for reverse transcription and integration, operably linked to a promoter.
  • the pol proteins may be expressed by multiple vectors.
  • Such vector may contain a nucleic acid sequence encoding the lentiviral gag proteins necessary for forming a viral capsid operably linked to a promoter.
  • this gag nucleic acid sequence is on a separate vector than at least some of the pol nucleic acid sequence, and may be on a separate vector from all the pol nucleic acid sequences that encode pol proteins.
  • gag, pol and env vector(s) do not contain nucleotides from the lentiviral genome that package lentiviral RNA, referred to as the lentiviral packaging sequence.
  • this region corresponds to the region between the 5' major splice donor and the gag gene initiation codon (nucleotides 301 -319).
  • the lentivirus is a primate lentivirus (U.S. Pat. No. 5,665,577) or a feline immunodeficiency virus (FIV) (Poeschla, E. M., et al., Nat. Medicine 4:354-357 (1998))
  • FMV feline immunodeficiency virus
  • the pol/gag nucleic acid segment(s) and the env nucleic acid segment will when expressed produce an empty lentiviral particle.
  • modifications such as deleting the tat coding region, the MA coding region, or the U3 region of the LTR, the possibility of a reversion to a wild type virus has been reduced to virtually nil.
  • a desired family of heterologous nucleic acid segments (sometimes referred to as the target molecules) can be inserted into the empty lentiviral particles by use of a plurality of vectors each containing a nucleic acid segment of interest and a lentiviral packaging sequence necessary to package lentiviral RNA into the lentiviral particles (the packaging vector).
  • the packaging vector contains a 5' and 3' lentiviral LTR with the desired nucleic acid segment inserted between them.
  • the nucleic acid segment can be an antisense molecule or encodes a protein such as an antibody.
  • the packaging vector may contain a selectable marker.
  • genes that change the sensitivity of a cell to a stimulus such as a nutrient, an antibiotic, ant the like.
  • Genes include those for neo, puro, tk, multiple drug resistance (MDR), and the like.
  • Other genes express proteins that can readily be screened for such as green fluorescent protein (GFP), blue fluorescent protein (BFP), luciferase, LacZ, nerve growth factor receptor (NGFR) 3 and the like.
  • GFP green fluorescent protein
  • BFP blue fluorescent protein
  • NGFR nerve growth factor receptor
  • transduction the introduction of DNA into a host cell is referred to as transduction, sometimes also known as transfection or infection.
  • An automatic sorter that screens and selects cells displaying the marker, e.g. GFP, can be used in the present method.
  • an inducible promoter When an inducible promoter is used with the target molecule, minimal selection pressure is exerted on the transformed cells for those cells where the target molecule is "silenced.” Thus, identification of cells displaying the marker also identifies cells that can express the target molecule. If an inducible promoter is not used, it is preferable to use a "forced- expression" system where the target molecule is linked to the selectable marker by use of an internal ribosome entry site (IRES) (see Marasco et al., PCT/US96/ 16531). In this manner, virtually all cells selected on the basis of the marker also contain and can express the target molecule.
  • IRS internal ribosome entry site
  • IRES sequences are known in the art and include those from encephalomycarditis virus (EMCV) (Ghattas, I. R. et al., MoI. Cell. Biol., 11 :5848-5849 (1991)); BiP protein (Macejak and Sarnow, Nature, 353:91 (1991)); the Antennapedia gene of Drosophila (exons d and e) (Oh et al., Genes & Development, 6:1643-1653 (1992)); those in polio virus (Pelletier and Sonenberg, Nature, 334:320-325 (1988); see also Mountford and Smith, TIG, 11: 179-184 (1985)).
  • EMCV encephalomycarditis virus
  • BiP protein Macejak and Sarnow, Nature, 353:91 (1991)
  • Antennapedia gene of Drosophila exons d and e
  • polio virus Pelletier and Sonenberg, Nature, 334:320-3
  • the target molecule may be operably linked to an inducible promoter.
  • inducible promoter Such systems allow the careful regulation of gene expression. See Miller, N. and Whelan, J., Human Gene Therapy, 8:803-815 (1997).
  • Such systems include those using the lac repressor from E. coli as a transcription modulator to regulate transcription from lac operator-bearing mammalian cell promoters (Brown, M. et al., Cell, 49:603-612 (1987), and those using the tetracycline repressor (tetR) (Gossen, M., and Bujard H., Proc. Natl. Acad. Sci. USA 89:5-547- 5551 (1992); Yao, F.
  • the lac repressor from Escherichia coli can function as a transcriptional modulator to regulate transcription from lac operator-bearing mammalian cell promoters (M. Brown et al., Cell, 49:603-612 (1987)). M. Gossen et al. (Proc. Natl. Acad. Sci.
  • this tetracycline inducible switch does not require the use of a tetracycline repressor- mammalian cell transactivator or repressor fusion protein, which in some instances can be toxic, to cells (M. Gossen et al., Proc. Natl. Acad. Sci. USA 89:5547-5551 (1992); P. Shockett et al., Proc. Natl. Acad. Sci. USA 92:6522-6526 (1995)), to achieve its regulatable effects.
  • the repressor is constitutively expressed.
  • the inducible system is a tetR system.
  • a general design concept may include an antibiotic-responsive repressor-DNA interaction which is converted into a mammalian expression configuration by placing (tandem) operator modules (tet7; pirS) adjacent to a 3' minimal eukaryotic promoter (P h C M Vmi n ; Phs P 70mi n )- Such a hybrid promoter is then activated upon binding of a transact! vator (tTA; PIT) 3 assembled by fusing repressor proteins to eukaryotic transactivating domains in an antibiotic adjustable manner.
  • tTA transact! vator
  • E.REX/E-ON E. c ⁇ // Tf481 A repressor; ET4[E-KRAB] transactivator/transrepressor; P ETR ON8 P SV40 -ETR8 promoter; and macrolide antibiotic small molecule regulator
  • Q-mateTM CymR Pseudomonas putida Fl repressor protein
  • CymR transactivator/transrepressor P C MV S - (Cu O) P CM V 5 -CUO promoter
  • cumate small molecule regulator E.REX/E-OFF
  • E.REX/E-OFF E. coli Tf481A repressor; ET [E-VP 16], transactivator/transrepressor; P ⁇ .2 ETR-P n cMVmm; and macrolide small molecule regulator
  • the inducible promoter is turned on and one screens for cells and/or animals displaying a particular phenotype. For example, enhanced expression or lack of expression of a particular receptor, selective killing of abnormal cells, etc.
  • the cells displaying the desired phenotype are selected for and depending upon the phenotype, the selection can be by a high throughput automated screening. For example, beads to select cells displaying a particular receptor. FACS analysis can be used to identify the change in expression of particular receptors. Other systems can readily be identified.
  • the tag contained on the molecule e.g., antibody
  • one can obtain the molecules, e.g., antibody that resulted in the desired phenotype.
  • the antibody can then be used to identify the antigen it bound to, if that is desired.
  • This method permits one to use a multitude of molecules to identify a specific molecule providing the desired function from a large group of molecules without first needing to know the specific identity of any member.
  • a lentiviral transfer vector including, in operable linkage, a 5' LTR, a 3' LTR comprising a polyadenylation signal, a minimal packaging signal; an inducible promoter (iP), a nucleotide sequence encoding a cognate repressor for the inducible promoter, and a nucleic acid sequence encoding a gene or RNA of interest (GROI) operatively linked to the iP.
  • the operably linked iP-GROI is configured to be in an opposite transcriptional orientation relative to the 5' LTR and nucleic acid encoding the repressor.
  • the iP is regulated by a repressor-transactivator fusion protein where the repressor is constitutively expressed.
  • the iP is regulated by an antibiotic-responsive gene regulon.
  • the antibiotic is a small molecule including, but is not limited to, tetracycline, streptogramin, macrolide, or cumate.
  • a method of modulating expressing a gene or RNA of interest (GROI) in a host cell including contacting a cell with a lentiviral vector and contacting the cell with a small molecule which regulates the repressor-transactivator fusion protein, where if the small molecule is present, expression from the iP is inhibited, and if the small molecule is removed, expression from the iP is disinhibited.
  • GROI RNA of interest
  • the lentiviral vector includes, in operable linkage, a 5' LTR, a 3' LTR comprising a polyadenylation signal, a minimal packaging signal, an inducible promoter (iP) comprising a cognate repressor binding site.
  • iP inducible promoter
  • the iP is regulated by a repressor-transactivator fusion protein, and a nucleic acid sequence encoding gene or RNA of interest (GROI) operatively linked to the iP, where the iP-GROI is configured to be in an opposite transcriptional orientation relative to the 5' LTR and the nucleic acid encoding the repressor.
  • GROI nucleic acid sequence encoding gene or RNA of interest
  • the vector is a tat deleted vector. This can be accomplished by inactivating at least the first exon of tat by known techniques such as deleting it. Alternatively, one can extend the U3 LTR deletion into the R region to remove the TAR element. The tat deleted vectors result in high titer of virus.
  • Variations can be made where the lentiviral vector has multiple modifications as compared to a wildtype lentivirus. For example, with HIV being nef-, rev-, vif- and vpr-. In addition one can have MA- gag, 3' and 5' U3 deleted LTR and variations thereof.
  • the envelope protein is not from the lentivirus, but from a different virus.
  • the resultant particle is referred to as a pseudotyped particle.
  • envelopes By appropriate selection of envelopes one can "infect" virtually any cell.
  • an env gene that encodes an envelope protein that targets an endocytic compartment such as that of the influenza virus, VSV-G, alpha viruses (Semliki forest virus, Sindbis virus), arenaviruses (lymphocytic choriomeningitis virus), flaviviruses (tick-borne encephalitis virus, Dengue virus), rhabdoviruses (vesicular stomatitis virus, rabies virus), and orthomyxoviruses (influenza virus).
  • envelopes that can preferably be used include those from Moloney Leukemia Virus such as MLV-E, MLV-A and GALV. These latter envelopes are particularly preferred where the host cell is a primary cell.
  • Other envelope proteins can be selected depending upon the desired host cell. For example, targeting specific receptors such as dopamine receptor for brain delivery. Another target can be vascular endothelium. These cells can be targeted using a filovirus envelope. For example, the GP of Ebola, which by post-transcriptional modification become the GPi and GP 2 glycoproteins.
  • a SHIV pseudotyped vector can readily be used in animal models such as monkeys.
  • the packaging sequence can be excluded from the vector(s) by any of a variety of techniques well known to the person of ordinary skill in the art. For example, one can simply delete the entire sequence. Alternatively, one can delete a sufficient portion of a sequence to render it incapable of packaging. An alternative strategy is to insert nucleotides into such a site to render it non-functional. Most preferably, one will delete the site entirely to prevent homologous recombination.
  • the lentiviral vectors can express the desired viral proteins, but because the packaging site has been removed, and the lentiviral LTRs are not operational their mRNA will not be effectively packaged into the lentiviral particles, and the recombinant virus will not be able to replicate and infect other cells.
  • the lentiviral vectors can also contain sequences encoding desired lentiviral regulatory proteins such as Tat, Rev, and the like. However, in a number of embodiments it is preferable not to contain such regulatory genes. If RRE and CAR sequences are included in the gene, the inclusion of sequence encoding REV is necessary, unless the virus is expressed in the cytoplasm. These regulatory sequences can be on the other lentiviral vectors (e.g., gag vector, pol vector, gag-pol vector, or env, vector), or on their own lentiviral vector. Alternatively, one can use constitutive transport elements (CTE) in place of RRE, to make the vector REV independent. Also, there is less sequence homology.
  • CTE constitutive transport elements
  • the vector system can be used to package a wide range of desired nucleotide segments into an empty lentiviral particle because of the large genomes of lentiviruses.
  • the use of promoters and enhancers can also significantly add to the length of an insert.
  • the system can be used with groups containing multiple molecules displaying diversity such as genetic diversity. Accordingly, the system of the present invention provides a significant advantage over currently available vectors by allowing for inserts that can contain a number of promoters and genes and that can be used to transfect resting cells as well as dividing cells.
  • the packaging vector contains (a) a promoter sequence operably linked to at least one heterologous nucleic acid sequence and (b) at least one sequence sufficient to permit transcription and processing of mRNA, the translation of which results in an expressed protein.
  • the processing sequence may be a polyadenylation sequence, including that the promoter is part of an inducible system.
  • the heterologous nucleotide sequence can encode a wide variety of proteins such as a therapeutic protein, i.e., one that compensates for an inherited or acquired deficiency.
  • therapeutic proteins include neurotransmitter biosynthetic enzymes, e.g., tyrosine hydroxylase for the treatment of Parkinson's disease; neurotrophic factors including neutrophins, e.g., nerve growth factor for the treatment of Alzheimer's disease, one can also use nerve growth factor receptor and the trk receptor; hypoxanthine-guanine porphoribosyl transferase (HGPRT) for the treatment of Lesch Nyhan disease; ⁇ -hexosaminadase ⁇ chain for the treatment of Tay Sachs disease; insulin for the treatment of diabetes.
  • HGPRT hypoxanthine-guanine porphoribosyl transferase
  • Receptors can also be prepared, e.g. the nerve growth factor receptor, the trk receptor, and the like. Because the insert can be large, it is possible to encode a series of different proteins. For example, one can encode a series of proteins that form a receptor-ligand complex.
  • proteins include, for example, signal transduction enzymes, e.g., protein kinase c; transcription factors, e.g., c-fos, NF-PB; oncogenes, e.g., erbB, erbB-2/neu, ras; neurotransmitter receptors, e.g., glutamate receptor, dopamine receptor, and the like.
  • signal transduction enzymes e.g., protein kinase c
  • transcription factors e.g., c-fos, NF-PB
  • oncogenes e.g., erbB, erbB-2/neu, ras
  • neurotransmitter receptors e.g., glutamate receptor, dopamine receptor, and the like.
  • One preferred group of proteins are antibodies. Included are single chain antibodies and Fabs. Libraries of antibodies are known and can be used in the present invention. For example, using a phage display library both generalized and specialized libraries can be used. A specialized library would be one where the member antibodies are generated to a specific group of antigens, e.g., a specific tumor. The diversity of the members of a specialized library is less than that of a generalized library.
  • the heterologous nucleotide sequence can also encode antisense molecules (DNA or RNA). These molecules can be used to regulate gene expression associated with a particular disease.
  • the antisense molecules are obtained from a nucleotide sequence by reversing the orientation of the coding region with regard to the promoter.
  • the antisense RNA is complementary to the corresponding mRNA.
  • the antisense sequence can contain modified sugar phosphate backbones to increase stability and make them less sensitive to RNA sensitivity. Examples of the modifications are described by Rossi, et al., Pharmacol. Ther. 50(2):245-354 (1991).
  • Another class of molecule includes ribozymes. Ribozymes and antisense molecules that engage in, as well as those that do not show transplicing can be used.
  • heterologous nucleotide sequence is preferably operably linked to an inducible promoter sequence which is constructed such that the orientation of transcription is opposite of the 5' LTR.
  • Cells can be transfected by the vectors to prepare the viral particle.
  • a method of making lenti viral particles including providing a lentiviral vector producer cell, introducing a lentiviral vector into a producer cell in the presence of a small molecule which modulates a repressor-transactivator fusion protein, and collecting lentiviral particles from the producer cells.
  • the lentiviral vector includes in operable linkage, a 5' LTR, a 3' LTR comprising a polyadenylation signal, a minimal packaging signal, an inducible promoter (iP), a nucleotide sequence encoding a cognate repressor for the inducible promoter, and a nucleic acid sequence encoding a gene or RNA of interest (GROI) operatively linked to the iP.
  • the operably linked iP-GROI is configured to be in an opposite transcriptional orientation relative to the 5' LTR and nucleic acid encoding the repressor.
  • the particle comprises a heterologous env protein.
  • the vectors may be prepared in vitro, where one may then harvest the particles, purify them and inject them by means well known in the art. In another embodiment, one may purify the particles, and then use those purified particles to infect desired cells.
  • producer cell lines expressing virions may be generated and used to transform cells with a packaging vector.
  • the producer cell lines or any cell can be transformed by standard techniques.
  • One method is to use an inactivated adenovirus vector linked to the packaging vector by a condensing polycation such as polylysine or polyethylanimine (PEI) (see Baker, A. et al., Nucleic Acids Res., 25(10): 1950-1956 (1997); Baker, A. et al., Gene Ther., 4(8):773-782 (1997); Scaria, A. et al., Gene Ther., 2:295-298 (1995)).
  • PEI polylysine or polyethylanimine
  • the vectors express proteins and mRNA which assemble into particles and hence can be used to express large amounts of viral particles. This requires transfecting a cell with the particle vector system described herein, the packaging vector, and culturing the cell line under conditions and time sufficient to express the viral proteins, which then form the particles. Thereafter, the particles can be purified by known techniques with care taken to insure that the struclure of the particle is not destroyed.
  • the particles can be used in a variety of areas. For example, they can be used to generate a desired immune reaction, to transform a cell with a heterologous nucleic acid sequence and/or to deliver a nucleic acid sequence to a desired host cell.
  • transient or stable cell lines may be generated that express the lentiviral particles by standard techniques. Thereafter, if stable cell lines are desired, cells may be screened for stably transfection by standard technique.
  • Such stable producer cell lines are a preferred source for obtaining packaged particles.
  • the particles of the present invention can be used to deliver heterologous DNA to a target cell.
  • the target cell may be in vivo, in vitro or ex vivo.
  • the target cell can be a dividing or preferably a quiescent cell.
  • Quiescent cells include nonmitotic or postmitotic cells, including, but not limited to macrophages.
  • the target cells also include cells of the nervous system, e.g., neural or neuronal cells.
  • quiescent or slowly dividing target cells include glia cells, myocytes, hepatocytes, pneumocytes, retinal cells, and hematopoietic stem cells.
  • Pancreatic islet cell may also a be targeted by the particles of the present invention.
  • the viral particle carrying the heterologous gene to be delivered to a target cell may be effected by any method known to those of skill in the art.
  • techniques include the use of catheters, injection, scarification, and the like.
  • stereotaxic injection can be used to direct the viral particles to a desired location in the brain. Stereotaxic surgery is performed using standard neurosurgical procedures (Pellegrino and Clapp, Physiol. Behav. 7: 863-8 (1971)).
  • the particles can be delivered by intracerebroventricular ("icv") infusion using a minipump infusion system, such as a SynchroMed Infusion System,
  • icv intracerebroventricular
  • a minipump infusion system such as a SynchroMed Infusion System
  • convection A recent method based on bulk flow, termed convection, has also proven effective at delivering large molecules to extended areas of the brain and may be useful in delivering the viral particle to the target cell (R. Bobo et al., Proc. Natl. Acad. Sci. USA 91 : 2076-80 (1994); P. Morrison et al., Am. J. Physiol. 266: R292-305 (1994)).
  • Other methods can be used including oral or other known routes of administration.
  • these vectors are used to transform host cells in vivo to look for the effects of specific genes in a living system.
  • the vectors may be injected in a sufficient amount as separate vectors or packaged viral particles to obtain a serum concentration in the tissue containing the target cell of the therapeutic protein ranging between about 1 pg/ml to 20 ⁇ g/ml.
  • this may be carried out by expressing a specific protein or, alternatively stopping the function of a protein. such as by expressing an antibody to a specific sequence intracellularly.
  • the amount may be between about 0.1 ⁇ g/ml to 10 ⁇ g/ml. In another embodiment, the amount may be between about 0.5 ⁇ g/ml to 10 ⁇ g/ml.
  • the present invention utilizes standard laboratory practices for the cloning, manipulation and sequencing of nucleic acids, purification and analysis of proteins and other molecular biological and biochemical techniques, unless otherwise stipulated. Such techniques are explained in detail in standard laboratory manuals such as Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989, and Ausubel et al., in Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley-Intersciences ( 1987). Although the virus from which the transfer and packaging vectors are derived is an RNA virus (HIV-2 or SIV), the molecular cloning may be done using pro viral DNA clones, thus allowing the use of standard cloning techniques.
  • HIV-2 or SIV RNA virus
  • the deletion clone (or homologous clones) may be cut (e.g., by subcloning) into multiple expression clones with complementary functions. This decreases the chances that a recombinant event will result in an infectious particle.
  • a convenient and well-defined provirus that may be used for this purpose is the provirus molecular clone (pRODl), from HIV-2 ROD (the sequence of which is available under Genbank accession no. M 15390, and is further described in Arya et al., J. Acquir. Immune. Defic. Syndr. 6:1205-1211, 1993; Arya et al., J. Gen. Virol. 75:2253-2260, 1994; and Arya et al., Hum. Gene Ther. 9: 1371 - 1380, 1998).
  • Other retroviral provirus constructs may also be used, for instance an HIV-I or SIV provirus.
  • Genbank accession numbers for these proviruses are available and include, but are not limited to, Genbank Accession Nos. AF075702 (HIV-I isolate SE8603 from Kenya), M 17449 (HIV-I isolate MN) and AFl 31870 (SIV).
  • AF075702 HIV-I isolate SE8603 from Kenya
  • M 17449 HIV-I isolate MN
  • AFl 31870 SIV
  • Such a provirus (or combination of complementary viruses) used to produce the packaging vector, may contain a substantially complete retroviral genome including the gag, pol, and env genes, a leader sequence and the 3' and 5' LTRs, and may contain the other HIV-2 structural genes.
  • Deletions may be introduced using standard restriction vectors at appropriate sites. .Sites may be selected using a restriction map of the pROD sequence. Restriction may be done upon the provirus in situ, or for convenience, fragments of the pROD proviral vector that contain the SD and surrounding sequence, may be subcloned into a plasmid vector. The inserted nucleotides in such a subclone may be restricted or altered as desired, and then reinserted into an appropriately modified pROD clone. Clones thus constructed may then be confirmed by DNA sequencing.
  • the packaging vector thus produced will not be able to package its own genome, and is therefore not infective, but it will be able to package the genome of another virus that possesses the wild-type packaging sequence(s), for example, the lentiviral vector of the invention.
  • the packaging vector may optionally be surrounded by a capsid to comprise a viral particle.
  • the engineered proviral DNA packaging vector can be used to co-transfect cultured mammalian (e.g., human) cells in vitro or to produce a packaging cell line.
  • the packaging vector can be functionally and structurally divided into two parts.
  • One part will be as described above, that is it will contain deletions upstream and downstream of the SD.
  • it may also contain mutations or deletions which prevent the production of an envelope.
  • the second part will provide the envelope only, thereby complementing the first.
  • a transfer vector of the present invention includes a nucleotide construct that delivers a transgene (for example a non-native gene) into a target cell.
  • the transgene is then generally integrated into the genome of the target cell where it is expressed.
  • the transfer vector contains the nucleotide sequences required for efficient packaging of its RNA genome (including the transgene) and can be made from an HIV-2 proviral clone, such as, for example, HIV-2/ST (Genbank Accession no. M31 1 13, Kumar et al., J. Virol. 64:890-901, 1990, which discloses the complete sequence of HIV-2/ST; Arya et al., J. Acquir. Immune. Defic. Syndr.
  • the transfer vector of the invention can be derived from the provirus of another retrovirus such as HIV-I or SIV. Standard genetic engineering techniques can be used to manipulate the proviral genome.
  • the SD portion of the genome can be functionally deleted in many ways.
  • the SD can be functionally deleted by changing the nucleotide sequence, by physical excision of all or part of the SD sequence, by a frameshift mutation, or by introduction of a foreign gene sequence within the SD sequence, for instance, a foreign gene sequence carrying a reporter molecule.
  • Functional deletion also includes use of substitution mutants which disrupt the function of the SD, or any other mutation that disrupts the SD and enhances packaging of progeny transfer vector genomes. This effect is enhanced when the transfected cell is co- transfected with a packaging-defective packaging vector such as described herein.
  • packaging of progeny transfer genomes is enhanced (for example by a factor of at least 2, 5, 10, 20 or even 30) in comparison to a co- transfection wherein the transfer vector is not deleted for SD.
  • the transfer and packaging vectors constructed as described herein can be used to transfect mammalian cells.
  • cells that can be transfected include, but are not limited to: human epitheloid 293 or 293T cells, human lymphoid CEM cells, human SupT cells, human HeLa cells (ovarian epitheloid ATCC #CCL-2) human fresh PBMC cells (lymphocytes), human monocytic cells, such as U937 cells, human fibroblasts (such as HS27, ATCC #CRL-1634), normal human skin fibroblasts CD-27sk (ATCC #CRL-1475), fetal brain cells (such as SVG (ATCC #CRL8621), HFGC cells, and SVG-neural differentiated cells), glimoa cells (such as U281 and U373, ATCC #HTB-17) and human neuroblastoma cells (such as SKN-MC, ATCC #HTB-10 and SKN-SH).
  • human epitheloid 293 or 293T cells human lymph
  • Transfection can be performed by routine methods whereby naked nucleic acids are transferred across the cell membrane thereby entering the interior of the cell where the proviral DNA can be subject to transcription and translation using the host's cellular machinery.
  • the naked proviral DNA can be transfected into the cell using the well-known calcium phosphate transfection method.
  • the conditions for transfection can be varied widely, for instance with regard to the amount of DNA applied and the components used in the medium to make the host cell membrane permeable to the naked DNA.
  • the cells and supernatant from such a transfected cell culture can be harvested after a few days, for example 3-5 days. Such transfected cell cultures can be examined visually for syncytial formation, indicative of cytopathy.
  • the number of virus particles in the supernatant can be estimated by the standard antigen capture assay scoring for the p27 core protein.
  • Cells can also be transfected using DEAE-dextran (Arya, New Bio. 2:57-65, 1990; Arya and Sethi, AIDS Res. Hum. Retroviruses. 6:649-658, 1990), lipofectamine (as per the manufacture's instructions, GIBCO-BRL, Gaithersburg, Md.) or any other method used by those skilled in the art.
  • Transfected cells can be evaluated by a variety of techniques including ELISA, Northern blot and other standard protein assays which allow one to determine that the transgene is being expressed (for example assaying for the conversion of L- dopa to L-dopamine after transfecting cells with the AADC gene).
  • Transfected cells can be analyzed for cellular RNA by extraction of the RNA by standard methods, and by measurement ' of absorbance of light at set wavelengths.
  • Northern blot and slot-blot hybridization can be used to quantify RNA.
  • nucleic acid compositions of this invention are isolated from biological sources or synthesized in vitro.
  • the nucleic acids of the invention are present in transformed or transfected whole cells, in transformed or transfected cell lysates, or in a partially purified or substantially pure form.
  • nucleic acid constructs disclosed yield a functionally identical construct.
  • silent variations 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 sequences substituted with different amino acids with highly similar properties.
  • the vectors of the invention comprises and express one or more heterologous sequences encoding negative selectable markers.
  • the negative selectable markers may be cytotoxins that directly or indirectly inhibit or kill a host cell. Examples of "direct" cytotoxins include the active moieties of cholera and botulism toxins.
  • the vectors of the invention comprise and express one or more heterologous sequences encoding indirect cytotoxins.
  • the indirect cytotoxins by themselves are not toxic but achieve cellular inhibition by interacting with another agent.
  • An example is HSV-thymidine kinase (TK) which is non-toxic but can activate drugs like ganciclovir into a toxic nucleotides that kill mammalian cells.
  • TK HSV-thymidine kinase
  • a pharmaceutical accepted solution of proteins expressed from lentiviral vectors involved concentrating the resulting protein from the supernatant of cultures that produce the protein without diluting the sample.
  • the protein sample can be optionally formulated in its final formulation solution during the concentration process without diluting the sample.
  • Such methods are known in the art.
  • One preferred method is to use Tangential Flow Filtration (TFF) as the method to concentrate and formulate the protein sample without diluting the sample during the process.
  • TMF Tangential Flow Filtration
  • the Tangential flow filtration membrane should have the appropriate molecular weight cutoff so that the protein is retained and concentrated in the final formulation buffer (retentate) and unwanted material collected in the permeate.
  • TFF is meant as only one example of methods that can be used to concentrate and formulate desired proteins without diluting them in the process.
  • Lentiviral vectors can also be used for optimal expression of proteins in cells by knocking out endogenous genes while expressing modified genetic sequences.
  • One method is to express modified sequence or proteins in cells while simultaneously knocking out or knocking down the expression of the native RNA sequence or protein.
  • the method involves expression of codon modified protein and simultaneously expressing a gene silencing sequence that destroys the endogenous or native protein mRNA by targeting the native coding sequence.
  • a non-limiting example is for use in the expression of T-cell receptors (TCR) for immunotherapy.
  • the vector preferably a lentiviral vector (but not limited to as such as other genetic vectors can be used in the approach) that expresses a gene silencing sequence that targets the native TCR alpha and beta subunits, preferably a conserved region of these subunits, and simultaneously expresses a desired TCR whereby the said conserved region is codon-modif ⁇ ed so that the gene silencing sequence is does not hybridize to it.
  • the desired TCR can be expressed without the concern that hybrid TCR are formed due to native alpha or beta chains complexing with the lentiviral vector expressed alpha or beta subunits.
  • silencing native alpha and beta mRNA this allows for the desired alpha and beta subunits to complex and form the desired TCR.
  • any endogenous gene can be replaced by a modified gene by introducing a lentiviral vector into a cell that expresses gene silencing sequence(s) targeted to the endogenous gene and simultaneously express a desired modified gene.
  • Lentiviral vectors can also be used for the production of genetically inactivated vaccines.
  • a non-limiting example is the influenza vaccine, for which the H5N1 variant is extremely pathogenic.
  • Influenza is a segmented negatively stranded paramyxovirus that is pathogenic in humans.
  • the method of the invention is to use a lentiviral vector to express at least one of a codon-modified influenza gene sequence from a cell. Cells are transduced with a plurality of lentiviral vectors that express one or more of the influenza virus genes.
  • codon modification single or multiple base substitutions in a single gene destroys sequences needed to package one or more of the influenza virus genomes, making the particles nonpathogenic.
  • influenza virus genes preferably a protein that is not particularly immunogenic
  • lentiviral vectors can be constructed to contain one or two modified influenza virus genes and then a mixture of lentiviral vectors comprising most or all of the modified influenza virus genes can be transduced into cells to produce the vaccine.
  • a single lentiviral vector can be constructed to express all of the influenza virus genes required to make the vaccine.
  • the advantage of the lentiviral vector is that it can transducer these genes into cells with high multiplicity and therefore all the genes can be expressed within one cell to produce whole viral vaccine.
  • lentiviral vectors expressing non-viral genes or gene silencing sequences can be added to the mixture of lentiviral vectors to further increase the safety, quality, quantity or immunogenicity of influenza vaccine particles produced.
  • a lentiviral vector expressing a cytokine or factor (non-limiting example: GM-CSF that is packaged into influenza virus genomes and expressed in influenza vaccine vector infected cells) that will further increase the immunogenicity of purified vaccine particles.
  • a cytokine or factor non-limiting example: GM-CSF that is packaged into influenza virus genomes and expressed in influenza vaccine vector infected cells
  • GM-CSF non-limiting example: GM-CSF that is packaged into influenza virus genomes and expressed in influenza vaccine vector infected cells
  • other sequences can be included or expressed from the lentiviral vector to produce a more optimized product.
  • Non-limiting examples are RMA gene inhibitory sequences to decrease the expression of a gene, resulting in a more optimized or desired vaccine, expression of proteins or factors that can increase the immunogenicity, stability or safety of the vaccine, and genetic sequences that modulate cellular function to make vaccine production more efficient.
  • Vaccines as such can be produced to multiple pathogens, including but not limited to Rabies, Rubella, RSV, Measles, Smallpox., Ebola, Huntavirus, Coronaviruses, Adenoviruses, SARS, Hepatitis viruses, HlV, and other viral infections.
  • the method is not limited to viral infections, vaccines to bacterial infection can be made by producing certain bacterial proteins that are immunogenic. These proteins could be single or produced as a complex of multiple proteins or other factors.
  • Cells e.g. VERO, 293, CHO 5 PERC6 or other mammalian cell lines
  • One mechanism for manufacture of lentiviral vectors is combining continuous flow centrifugation with either anionic exchange chromatography or tangential flow filtration.
  • continuous flow centrifugation may be performed at speeds greater than 5,000xg RCF and less than 26,000 xg RCF.
  • the continuous flow centrifugal force is about l0,500xg to 23,500 xg RCF with a spin time of between at least 1 hour, with longer centrifugal times being used with slower centrifugal force.
  • the lentiviral vector is centrifuged on a cushion of more dense material (a non limiting example is sucrose but other reagents can be used to form the cushion and these are well known in the art) so that the lentiviral vector does not form aggregates that are not filterable, as is the problem with straight centrifugation of the vector that results in a viral vector pellet.
  • a cushion can be avoided if pelleting of the material is desired.
  • Continuous flow centrifugation onto a cushion allows the vector to avoid large aggregate formation, yet allows the vector to be concentrated to high levels from large volumes of transfected material that produces the lentiviral vector.
  • the flow rate for the continuous flow centrifuge is preferably 100ml per minute, but higher flow rates can also be used (up to 100 liters per hour). The flow rate is adjusted to provide ample time for the vector to enter the core of the centrifuge without significant amounts of vector being lost due to the high flow rate. If a higher flow rate is desired, then the material flowing out of the continuous flow centrifuge can be re- circulated and passed through the centrifuge a second time.
  • the vector can be further concentrated using Tangential Flow Filtration (TFF), or the TFF system can be simply used for the buffer exchange.
  • TFF Tangential Flow Filtration
  • a non-limiting example of a TFF system is the Xampler cartridge system that is produced by GE-Healthcare. Useful cartridges are those with a MW cut-off of 500,000 MW or less. For example, a cartridge may be used with a MW cut-off of 300,000 MW. A cartridge of 100,000MW cut-off can also be used. For larger volumes, larger cartridges can be used and it will be easy for those in the art to find the right TFF system for this final buffer exchange and/or concentration step prior to final fill of the vector preparation.
  • the final fill preparation may contain factors that stabilize the vector — sugars are generally used and are known in the art.
  • Additional methods for the concentration and purification of lentiviral vectors include the use of high speed centrifugation to concentrate the lentiviral vectoi material. Also included, is the purification of lentiviral vector by high speed (but not ultra) centrifugation though a sucrose solution of less than 60% w/v or v/v.
  • This sucrose solution can be in the form of a cushion, or the entire volume of vector material can be made to contain the desired concentration of sucrose.
  • the concentration of sucrose may be between 1% and 10%.
  • Such a sample is filterable through a 0.2um or greater pore size filter for final sterilization.
  • Other solutions instead of sucrose which may be used are well known in the art.
  • the lentiviral vectors of the present invention may also be used for generation of transgenic animals.
  • the blood-brain barrier and the blood- test ⁇ s barrier is not well formed, allowing for permeability of macromolecules from the blood into the brain and testes.
  • Macromolecules also include viruses and vectors. Therefore in animals where the blood brain barrier has not formed macromolecules, viruses and vectors can pass from the blood into the brain or testes.
  • a method for generating transgenic animals involves injecting (either i.v., i.p., s.c, or any other route of injection) non-mature animals (or animals where the blood-testes barrier is made permeable by injecting the animals with a chemical, compound, protein, factor, biologic or agent, or a combination thereof to permeablize the blood-testes barrier) with at least one lentiviral vector (a plurality of lentiviral vectors can also be used) expressing a transgene sequence of interest (these are known in the art and can be a gene, two genes, a plurality of genes, a gene inhibitory sequence like a ribozyme, antisense, transdominant mutant protein, RNAi, MicroRNAi, are non-limiting examples) under a tissue specific, ubiquitous, or inducible promoter.
  • the vector is injected into the animal at concentrations that would allow for uptake in the testes and germinal cells.
  • the germinal cells then become genetically modified with the vector and accompanying transgene and after maturation will produce mature genetically modified spermatozoa.
  • Transgenic animals can then be created using the genetically modified spermatozoa by in vitro or conventional fertilization of ova.
  • the Ova are then implanted for gestation resulting in heterozygous offspring.
  • the heterozygous offspring can then be mated with other heterozygous offspring to produce homozygous offspring.
  • Lentiviral vectors can be used to generate animal models for use in research, drug discovery and target validation. Lentiviral vectors can be used to develop animals that express diseased phcnotypcs without resorting to the generation of transgenic animals.
  • a non-limiting example is the generation of animals that express tumors by directly injecting at least one lentiviral vector that express at least one oncogene (or at least one gene silencing targeted to a tumor suppressor gene) that would generate tumors in animals.
  • Another non-limiting example is the expression of at least one oncogene that is expressed from a lung specific promoter to generate tumors in the lung upon administration of the lentiviral vector to the animal, preferably directly into lung tissue.
  • the promoter may be limited to a cell or tissue specific cell type, the promoter may be ubiquitous, or the promoter may be inducible, but the vector can be pseudotyped with an envelope protein that targets the vector to a specific cell or tissue type.
  • a plurality of oncogenes is used to generate the tumor in a reasonable time.
  • Other genes can also be used to generate disease specific effects.
  • a wizard software system is used to generate custom lentiviral vectors by allowing the client to choose from several options at each step of the process. Customers will choose from the gene sequence, promoters, reporter gene(s), post- transcriptional elements and envelope pseudotyping. This wizard will allow for step wise design of the vector with a illustrated diagram showing the vector design pictorially as the various options are selected. The wizard will allow the customer to save the information and edit in the future, and will allow for the purchase of the vector on-line or by phone.
  • Lentiviral vectors are useful for high efficient gene delivery.
  • the expression of genes or RNA that contain splice sites are problematic for expression from lentiviral vectors because splicing during production results in a truncated genomic lentiviral vector RNA.
  • One way to solve this problem is to express the gene or sequence in the opposite orientation. However, this leads to potentially decreased titers of vectors as an antisense is produced by placing gene sequences in the opposite orientation.
  • an inducible promoter preferably the tetracycline inducible promoter as described in Figure 1.
  • the gene sequence of interest which contains the splice site (shown as tGFP in Figure 1) is expressed in the opposite orientation relative to the genomic RNA (since splice sites work only in one direction) of the lentiviral vector, but is operably linked to a tetracycline inducible promoter, for which the repressor (tTA) is produced constitutively and binds to the inducible promoter region (Pbi) in the presence of tetracycline.
  • the vector is produced in the presence of tetracycline to repress production of the tGFP mRNA so that no antisense is produced and therefore results in useful titers of lentiviral vector.
  • the vector produced is added to the cells, they are added in the absence of tetracycline to induce tGFP expression (including splicing). If the expression needs to be turned off, then tetracycline is added back to the medium.
  • Cells are First grown and then transduced with a lentiviral vector that expresses a protein of interest (for example, the erythropoietin, growth hormone, G-CSF, viral, bacterial or protein used in vaccination, or any protein of interest) under the control of an inducible promoter (iP), where a iP/protein of interest cassette in inserted in the vector in an orientation that is transcriptionally opposite relative to the 5'LTR, and includes a second nucleotide sequence that inhibits cell growth. Further, a third nucleotide sequence encodes a repressor for the promoter, which repressor is constitutively active.
  • a protein of interest for example, the erythropoietin, growth hormone, G-CSF, viral, bacterial or protein used in vaccination, or any protein of interest
  • iP inducible promoter
  • a iP/protein of interest cassette in inserted in the vector in an orientation that is transcriptionally opposite relative to the 5'LTR, and
  • the second nucleotide sequence expresses a gene that results in a protein or a RNA sequence (such as an antisense, ribozyrne, RNAi or other gene inhibitory or gene silencing nucleotide sequence) that directly inhibits the expression or function of a target RNA.
  • the cells can be transduced with a vector, (e.g., another lentiviral vector) that optionally contains the first gene or protein of interest in the orientation as recited above, including a sequence encoding an antibiotic-responsive gene regulon, and the cells are grown in a manner which stops further cell growth, such as growing the cells at a temperature not conducive to cell growth.
  • Such cells are grown at a temperature that is less than 37°C, with an optimal temperature being between 30 0 C and 33°C.
  • Alternative methods also include chemical or drug mediated cell growth inhibition.
  • the lentiviral vector expressing the gene from the inducible promoter is transfected into cells, including human kidney cells, bone marrow cells, neural tissues, or cells of ectodermal, endodermal or mesenchymal origin. These cells can be derived from primary tissue, or can be later transformed to change or modify their properties. One such method is to transform the cells so that they are immortalized.
  • proteins are collected by standard methods known in the art, including chromatography, electrophoresis, sedimentation method, and the like. Further, protein expression can be inhibited by adding the small molecule back to the medium in which the cells are being cultured.
  • a small molecule e.g., tetracycline

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Abstract

La présente invention concerne des vecteurs permettant l'expression efficace de gènes ou d'ARN d'intérêt qui possèdent des sites d'épissage internes, comprenant la construction de vecteurs lentiviraux où l'expression de tels gènes et ARN d'intérêt ne permet pas d'obtenir un ARN de vecteur lentiviral génomique tronqué. En outre, les constructions de vecteurs lentiviraux sont conçues de sorte que l'expression provenant du vecteur puisse être modulée par des promoteurs inductibles. Ces vecteurs lentiviraux, et en particulier des vecteurs VIH, sont utiles en génomique fonctionnelle, dans la découverte de médicaments, la validation de cibles, la production de protéines, la production de vaccins, la thérapie génétique clinique, et d'autres applications.
PCT/US2007/011385 2006-05-12 2007-05-11 Compositions à base de vecteurs lentiviraux, procédés et applications WO2007133674A2 (fr)

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US10548914B2 (en) 2008-10-17 2020-02-04 American Gene Technologies International Inc. Safe lentiviral vectors for targeted delivery of multiple therapeutic molecules
US11617760B2 (en) 2008-10-17 2023-04-04 American Gene Technologies International Inc. Safe lentiviral vectors for targeted delivery of multiple therapeutic molecules
US11007209B2 (en) 2008-10-17 2021-05-18 American Gene Technologies International Inc. Safe lentiviral vectors for targeted delivery of multiple therapeutic molecules
EP3307894A4 (fr) * 2015-06-10 2019-01-16 American Gene Technologies International, Inc. Systèmes d'administration viraux non intégratifs et procédés d'utilisation associés
US11980663B2 (en) 2015-07-08 2024-05-14 American Gene Technologies International Inc. HIV pre-immunization and immunotherapy
US10472649B2 (en) 2016-01-15 2019-11-12 American Gene Technologies International Inc. Methods and compositions for the activation of gamma-delta T-cells
US10772905B2 (en) 2016-01-15 2020-09-15 American Gene Technologies International Inc. Methods and compositions for the activation of gamma-delta T-cells
US11519006B2 (en) 2016-01-15 2022-12-06 American Gene Technologies International Inc. Methods and compositions for the activation of gamma-delta T-cells
US10428350B2 (en) 2016-01-15 2019-10-01 American Gene Technologies International Inc. Methods and compositions for the activation of gamma-delta T-cells
US10420789B2 (en) 2016-01-15 2019-09-24 American Gene Technologies International Inc. Methods and compositions for the activation of gamma-delta T-cells
US10888613B2 (en) 2016-02-08 2021-01-12 American Gene Technologies International Inc. Method of producing cells resistant to HIV infection
US10767183B2 (en) 2016-03-09 2020-09-08 American Gene Technologies International Inc. Combination vectors and methods for treating cancer
US10975374B2 (en) 2016-03-09 2021-04-13 American Gene Technologies International Inc. Combination vectors and methods for treating cancer
US11976292B2 (en) 2016-06-08 2024-05-07 American Gene Technologies International Inc. Non-integrating viral delivery system and methods related thereto
US11090379B2 (en) 2016-07-08 2021-08-17 American Gene Technologies International Inc. HIV pre-immunization and immunotherapy
US11911458B2 (en) 2016-07-08 2024-02-27 American Gene Technologies International Inc. HIV pre-immunization and immunotherapy
US10494647B2 (en) 2016-07-08 2019-12-03 American Gene Technologies International Inc. HIV pre-immunization and immunotherapy
US11583562B2 (en) 2016-07-21 2023-02-21 American Gene Technologies International Inc. Viral vectors for treating Parkinson's disease
US11820999B2 (en) 2017-04-03 2023-11-21 American Gene Technologies International Inc. Compositions and methods for treating phenylketonuria
CN110520535B (zh) * 2017-04-18 2024-01-09 葛兰素史克知识产权开发有限公司 用于逆转录病毒产生的稳定细胞系
CN110520535A (zh) * 2017-04-18 2019-11-29 葛兰素史克知识产权开发有限公司 用于逆转录病毒产生的稳定细胞系
CN114181970A (zh) * 2020-09-15 2022-03-15 上海药明巨诺生物医药研发有限公司 一种慢病毒载体纯化方法
CN114181970B (zh) * 2020-09-15 2023-07-25 上海药明巨诺生物医药研发有限公司 一种慢病毒载体纯化方法
WO2022121849A1 (fr) * 2020-12-07 2022-06-16 和元生物技术（上海）股份有限公司 Vecteur de lentivirus avec expression régulable d'un gène d'intérêt, et procédé d'encapsidation associé

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