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  • 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
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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
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
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  • C12N2740/00—Reverse transcribing RNA viruses
  • C12N2740/00011—Details
  • C12N2740/10011—Retroviridae
  • C12N2740/16011—Human Immunodeficiency Virus, HIV
  • C12N2740/16051—Methods of production or purification of viral material
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  • C12N2830/00—Vector systems having a special element relevant for transcription
  • C12N2830/48—Vector systems having a special element relevant for transcription regulating transport or export of RNA, e.g. RRE, PRE, WPRE, CTE
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  • C12N2830/00—Vector systems having a special element relevant for transcription
  • C12N2830/50—Vector systems having a special element relevant for transcription regulating RNA stability, not being an intron, e.g. poly A signal
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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
  • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES

Definitions

• retroviruses derived the surface and transmembrane components of the viral envelope glycoprotein.
• An additional smaller coding domain present in all retroviruses is pro, which encodes a protease enzyme that is recruited to the inside of the virion to effect maturation of fusion polyprotein products therein.
• the lentivirus subfamily of retroviruses further encodes pro, which is a part of pol (PR, RT, IN).
• Retroviruses may generally be divided into two classes: simple and complex, depending on the organization and representation of the genome. Complex retroviruses, in contrast to simple retroviruses, encode additional accessory and regulatory proteins that may be derived from differentially spliced messages coming from the genome. Complex retroviruses may further be divided into additional subgroups, among which are the lenti viruses.
• lentiviruses are known to cause disease, principally by killing or inducing loss of function of specific cells and tissues.
• HIV the causative agent of Acquired Immuno Deficiency Syndrome (AIDS) in humans.
• AIDS Acquired Immuno Deficiency Syndrome
• Man-made retroviral vectors including HIV-based lentiviral vectors exist and offer significant utility in being able to transduce a variety of nondividing tissue cells and sustain expression of transgenes in vivo.
• Akkina et al. (1996) J. Virol. 70: 2581-2585; Naldini et al. (1996) Science 272:263-267.
• Such tissues include but are not limited to brain, liver, muscle, and hematopoietic stem cells.
• Lentiviral vectors in particular hold great promise for gene therapy, and clinical trials to evaluate their safety and efficacy for treating certain human disease are being considered. However, despite the advances and the promise these vectors hold, safety concerns still exist.
• Replication incompetent retroviral vectors can potentially recombine to form replication competent retroviruses (RCRs). This possibility, especially in the instance of pathogenic lentiviruses, should be minimized to maximize safety.
• lentiviral vector systems have partially addressed this issue by expressing the essential viral genes on separate genetic elements (so-called "split-function") vectors. Naldini et al., supra; Kafri et al. (1999) J. Virol. 73:576-84; Wu et al. (1997) EMBO, vol. 16, no. 16, pp. 5113- 5122.
• RCRs can form from these split function systems, and current in vitro cell culture and animal model systems are limited in their ability to monitor and evaluate this.
• a retroviral assay system that can sensitively monitor genetic recombination events is therefore needed.
• the invention features methods, systems, and indicator cells that provide for a sensitive determination of retroviral recombination and/or the potential for production of RCR or the emergence in vivo of potentially pathogenic viral forms.
• the recombinants can contain one or more retroviral genetic determinants, e.g., gag, pol, env, tat, and /or rev, which can originate from one or more packaging constructs such as is generally used in split function retroviral packaging systems.
• Env is usually supplied in its own construct.
• Nucleic acids that encode the packaging component of the vector particles are generally not efficiently packaged into retroviral virion.
• the principal function of the packaging construct(s) is to package/encapsidate and help transfer a gene- transfer construct.
• These packaging constructs supply in trans the necessary structural and enzymatic retroviral proteins.
• "SIN" vectors are self-inactivating vectors that have, for example, deleted U3 sequence in the 3' LTR. Upon one round of replication, these changes are copied into both the 5' and 3' LTRs, thereby producing a less transcriptionally active pro virus.
• the invention can measure recombination events that unite one or more of the necessary genetic determinants contained within a wide variety of packaging and gene- transfer constructs. The result is detected by the establishment of a new, "recombinant" structure that contains genetic elements or portions thereof that were formerly found separately in the packaging and gene transfer construct components of the system. It is possible, although less likely so in the instance of SIN vector use, that these recombinant nucleic acids can then be packaged and mobilized to other cells because the recombinant nucleic acids contain, and thereby may encode, at least some of the necessary components for replication and mobilization.
• the functional retroviral gag-pol genetic structure is required for any type of RCR or viral DNA mobilization
• monitoring for its presence in the form of a retroviral genetic recombinant allows a means to assess the risk of a retroviral/lentiviral vector creating an RCR event.
• the invention provides a basis to measure the safety of vector stocks for use in gene transfer by using gag and pol, and more specifically gag-pol, as a surrogate marker for RCR.
• the invention features a method of detecting a retroviral genetic recombinant, preferably a recombinant having gag and pol functions which can be complemented by helper function(s) supplied by or to the cell, and which helper function(s) facilitate propagation of the recombinant to permit the recombinant' s detection.
• This detection can take place using any one of a variety of biochemical, diagnostic, and/or functional assays that can identify the recombinant.
• the assay makes used of the gag and pol functions contained within the recombinant, and a marker gene to facilitate the detection.
• gag and pol functions is meant to either a gag-pol fusion arrangement or to the supply of the individual gag and pol elements separated from one another.
• retroviruses e.g., lentiviruses are characterized by the former, and others, e.g., spumaviruses, by the latter.
• gag function is meant a viral packaging function sufficient to produce a virion or virus-like-particle that is capable of encapsidating a retroviral genome. Such gag function does not necessitate expression of the entire gag sequence (Accola and Gottlinger (2000) J. Virol. 74:5395).
• poly function is meant any one of the activities (enzymatic or non-enzymatic) or combinations of activities provided by protease (PR), reverse transcriptase (RT), and integrase (IN). These functions may be provided by the expression of the entire pol gene (PR, RT, IN), or by separate expression of the individual components thereof.
• retroviral genetic recombinant is meant a species of retrovirus that has undergone a nucleic acid recombination event, e.g., between one or more nucleic acid strands or constructs, e.g., packaging and vector constructs of split function retroviral systems.
• the retroviral genetic recombinants of the invention may, in addition to, and as a consequence of being derived from the events described above, also contain man-made or artificial sequences, including heterologous sequences such as reporter/marker gene sequences and mutations introduced into coding and non-coding regions.
• Propagating refers to the ability of the recombinant to integrate and duplicate in the host cell, e.g., as part of a mitosis event, and/or to support viral replication and mobilization, e.g., using various helper functions that are supplied by or to the host cell.
• Helper functions are used to help facilitate propagation of the recombinant when the recombinant is one that is incapable or inefficient at replicating, packaging, and/or infecting by itself Examples of helper functions include but are not limited to the retroviral env gene product and pseudotypes thereof, and/or the retroviral tat and rev gene products. Depending on the embodiment, various helper functions can be provided individually or in combination.
• helper functions will originate from a different nucleic acid strand in the cell relative to the recombinant strand, although each may ultimately share the feature of being integrated into the host genome. This is usually a consequence of the "split function" aspect of most retroviral systems that is directed to “disarming" viruses and promoting biological containment and safety. "Providing a cell suspected of having said recombinant” embraces any manner in which the introduction or establishment of such recombinant can occur, including but not limited to infection, transfection, transduction, and transformation, as those techniques are commonly understood.
• the recombinant may be formed in the cell itself, e.g., from different or related genetic elements present on different nucleic strands, at a step upstream to this, e.g., in another cell from which the intact recombinant is then transferred to the present cell, e.g., via mobilization, or formed in a virion particle.
• a step upstream to this e.g., in another cell from which the intact recombinant is then transferred to the present cell, e.g., via mobilization, or formed in a virion particle.
• the recombinant is formed in the cell itself, e.g., from different or related genetic elements present on different nucleic strands, at a step upstream to this, e.g., in another cell from which the intact recombinant is then transferred to the present cell, e.g., via mobilization, or formed in a virion particle.
• exactly how the recombinant is formed is not intended to be limiting
• the term "mobilization" is broad. It can relate to the packaging and/or export of a nucleic acid sequence, usually to another cell.
• the nucleic acid sequence is preferably either a retroviral genetic recombinant nucleic acid sequence or a marker gene flanked by sequences that facilitate its mobilization and expression, e.g., packaging into a retroviral- like particle, reverse transcription, integration, and promoter-specific expression.
• the packaged nucleic acid may be contained within a retroviral virion, virus like particle, or capsid structure, which may also be isolated and assayed, e.g., extracellularly as a viral supernatant. Usually, although not always, the packaging of the nucleic acid is facilitated by a viral packaging signal.
• the nucleic acid sequence will also preferably contain cis-acting signals that facilitate reverse transcription and integration.
• the flanking sequences will also preferably contain a promoter for the expression of the marker gene.
• the promoter may be an inducible promoter or a constitutive promoter.
• a preferred embodiment for the latter is the human phosphoglycerate kinase gene promoter, "pGK". See Michelson et al. (1985) PNAS 82:20, pp. 6965-6969 and GenBank accession Ml 1958.
• determining the presence of said retroviral genetic recombinant can mean any assay that can detect mobilization or a recombinant, including but not limited to e.g., hybridization assays, i.e., fluorescent in situ hybridization (FISH), antigen-detection (e.g., ELISA), polymerase chain reaction (PCR), genetic marker response/mobilization, and/or recombinant mobilization.
• hybridization assays i.e., fluorescent in situ hybridization (FISH), antigen-detection (e.g., ELISA), polymerase chain reaction (PCR), genetic marker response/mobilization, and/or recombinant mobilization.
• FISH fluorescent in situ hybridization
• ELISA antigen-detection
• PCR polymerase chain reaction
• both the marker gene and one or more other ways of determining the presence of the recombinant are used, e.g., PCR and antigen-detection.
• the marker is used and must be present in the recited cell, e.g., to denote the presence of and/or enrich for recombinants from among a greater population of non-recombinants, e.g., in a viral stock.
• the retroviral genetic recombinant is integrated into a cellular chromosome and propagated or capable of propagation along with the cell, e.g., as a provirus.
• the marker gene if it is present in the cell, is preferably also integrated.
• retroviral sequences e.g., coding and/or cis-acting sequences
• other sequences including heterologous sequences may also be included.
• retroviral coding sequences include gag, pro, rt, in, pol, gag-pol, gag-pro, tat, rev, env, vpr, vpu, and vif.
• "Cis-acting" sequences include those that possess functions in addition to or separate from coding sequences, e.g., promoters, enhancers, frame-shift signals, polyadenylation signals, primer binding recognition sites (PBRs), LTRs, portions thereof, etc.
• Heterologous sequences may include reporter genes and inducer or repressor molecules, e.g., that can activate or repress a marker gene.
• the recombinant contains at least a gag sequence and at least some pol sequence, or derivative sequences thereof.
• Derivative sequences may include mutations consisting of silent mutations, stop codons, deletions, and/or insertions.
• the recombinant may also consist, at least in part, of a synthetic sequence, e.g., one having silent mutations in one or more codons, or with stronger or weaker codon usage in the target host than the natural retroviral coding sequence.
• gag and pol functions is not necessarily meant "functional gag-pol".
• a mobilization may be of the recombinant itself and/or of a marker gene.
• the marker gene preferably has a retroviral packaging sequence, a promter, and flanking LTR elements, like the recombinant.
• the promoter may be an inducible promoter or a constitutive promoter. Mobilization may be fostered by the in trans supply of an env gene or pseudotype thereof.
• the env gene may have previously been introduced to the cell, but preferably is introduced immediately prior to a desired mobilization event, e.g., in the preferred form of a pseudotyping agent such as a VS V-G protein, however introduced.
• a pseudotyping agent such as a VS V-G protein
• "Pseudotyping" is a commonly understood term of art used to denote the combining of heterologous envelope species and/or other retroviral components, which may allow for modified function over the wild-type retrovirus, e.g, the ability to infect or replicate in a broader or narrower host cell range.
• the marker gene is a selectable marker gene, preferably one that encodes antibiotic resistance, preferably resistance to an antibiotic, and most preferably resistance to the antibiotic puromycin.
• the marker gene can be synonymous with a reporter gene, e.g., luciferase, green fluorescent protein (GFP), or B-galactosidase.
• luciferase e.g., green fluorescent protein (GFP), or B-galactosidase.
• GFP green fluorescent protein
• B-galactosidase e.g., luciferase, green fluorescent protein (GFP), or B-galactosidase.
• the marker gene is affixed to a promoter, inducible or constitutive, that is used to drive expression of the gene within a host cell.
• the invention features a method for detecting a retroviral genetic recombinant having gag and pol functions.
• the method includes providing the recombinant in a cell as described for the first aspect, and additionally includes a marker gene in the cell that can be mobilized in the presence of the recombinant and one or more helper functions as described previously to thereby facilitate detection of the recombinant.
• the marker gene and recombinant are integrated into the host genome, with each capable of expressing a gene.
• the gene expressed by either, including the recombinant may be detected using an assay.
• the assay for detecting the recombinant' s expressed gene can include, but is not limited, to FISH, PCR, antigen detection, Tat transfer, Gag transfer, and nucleic acid or gene mobilization.
• the recombinant includes one or more retroviral coding and cis-acting sequences, with the coding sequences preferably encoding one or more members selected from the group: gag, pro, pol, gag-pol, gag-pro, and derivatives thereof. Derivatives may include mutations such as insertions, deletions, silent mutations, and stop codon introduction.
• the recombinant has genetic elements that are used to mobilize itself and or another nucleic acid sequence, e.g., a marker gene provided in the host cell.
• helper functions as described above are used to help facilitate propagation of the recombinant.
• helper functions can include, for example, the retroviral env gene product and pseudotypes thereof, and/or the retroviral tat and rev genes.
• various helper functions can also be provided in combination, e.g., by providing each of env, tat, and rev where appropriate.
• the marker is, once again, integrated into the host cell genome, e.g, into one or more of its chromosomes.
• the host cell is assumed to be a eukaryotic host cell, preferably a mammalian cell, and preferably one capable of being infected or otherwise supporting replication of the subject recombinant. This may require helper functions.
• the marker gene is a selective marker gene, more preferably an antibiotic resistance gene, and most preferably a gene encoding resistance to the antibiotic puromycin.
• the gene is capable of expression from a constitutive or inducible promoter, which is preferably provided and operatively attached.
• the marker gene is further preferably flanked by cis-acting sequences that provide for its encapsidation, reverse transcription, integration, and/or expression.
• the invention features a method for detecting a retroviral genetic recombinant in a cell that also includes a marker gene that is responsive to the presence of the recombinant within that cell. The method includes measuring the response of the marker gene to thereby detect the recombinant.
• the marker gene response is the result of a specific gene product encoded by the recombinant.
• This gene product may be a retroviral gene, e.g., tat or rev, or else one that encodes a heterologous inducer or repressor protein.
• the gene product influences marker gene expression, preferably through the presence of a corresponding cis-acting promoter and/or other response elements.
• the marker gene is also a mobilizable marker gene, meaning that it can be mobilized.
• the marker marker gene can be a reporter gene or a selective marker gene.
• it is a selective marker gene, more preferably an antibiotic resistance gene, and most preferably a puromycin resistance gene.
• the marker preferably has an expression that is controlled by a promoter that is present, whether inducible or constitutive.
• the invention features a retroviral assay system for detecting a retroviral genetic recombinant having gag and pol functions.
• the system features a cell suspected of containing the recombinant, propagation of which is facilitated in the presence of one or more helper functions.
• the system further features a means for detecting the recombinant.
• the means includes, but is not limited to expressing a_marker gene that is also present in the cell.
• the marker gene is responsive to the presence of the recombinant, preferably to a gene product encoded therein.
• the means for detecting the recombinant can include mobilizing the marker gene, mobilizing the recombinant; and/or assaying for a product encoded or otherwise produced by the recombinant or said marker gene.
• the recombinant derives from a retroviral vector, and more preferably from a lentiviral vector, which is a preferred embodiment for all the aspects.
• the lentiviral-based vector may include, but is not limited to, HIV- 1 , SIV, HIV-2, FTV, EIA V, BIV, CAEV, and OVINE.
• the one or more helper functions is further included to allow or help propagate the recombinant.
• the helper functions that can be used include, but are not limited to, env and pseudotypes thereof.
• the means for detecting the retroviral genetic recombinant is the expression of a marker gene that is responsive to the presence of the retroviral genetic recombinant.
• the means may further include detecting one or more genes expressed by the recombinant.
• the marker gene encodes a product that can be selected for under one or more environmental conditions.
• the marker gene is an
• antibiotic resistance gene and more preferably one that encodes resistance against puromycin.
• Preferred embodiments for the assay system include components that perform or assist in performing PCR, FISH, antigen-detection, Tat transfer, Gag transfer, and gene mobilization, or any other biochemical assay.
• the invention features an indicator cell for indicating the presence of a retroviral recombinant, an RCR event, and or a wild-type retrovirus.
• the cell has an integrated marker gene, preferably a selectable marker gene.
• the marker gene is responsive to the presence of one or more genetic elements encoded by the retrovirus, e.g., tat, or a heterologous inducer or repressor protein.
• the marker gene may also be capable of mobilization as described above, and to that end may include cis-acting elements suitable for accomplishing or facilitating such.
• the indicator cell may also include helper functions, e.g., env or a pseudotype thereof, when the retrovirus is one incapable of replicating, packaging, and/or infecting by itself.
• helper functions may be heterologously engineered into the cell.
• tat is built into the cell to assist retroviral expression, replication, and/or packaging.
• the indicator cell is preferably an immortalized mammalian cell, more preferably one that is capable of propagating an animal retrovirus, preferably a lentivirus, more preferably a human lentivirus, and most preferably a human HIV lentivirus.
• the indicator cell can be HeLa, 293T, a hematopoietic stem cell, or a derivative thereof. Most preferably, the cell is a 293T cell.
• the selectable marker gene of the indicator cell is an antibiotic resistance gene, preferably one that encodes a product that is resistant to puromycin.
• the invention features a method, retroviral assay system, or indicator cell of any of the preceding aspects and embodiments that is further used to evaluate the risk of producing a replication-competent retrovirus from a retroviral vector.
• the starting product may be a retroviral recombinant, e.g., one lacking one or more functional members selected from gag, pro, rt, int, env, tat, rev, and/or other gene(s).
• a retroviral recombinant e.g., one lacking one or more functional members selected from gag, pro, rt, int, env, tat, rev, and/or other gene(s).
• the invention may further feature a method, system, or cell used for determining the risk of producing a replication-competent retrovirus (RCR) from a test vector particle.
• RCR replication-competent retrovirus
• This aspect may include any one or more of the embodiments described previously.
• the method, system, or cell includes providing a cell with a selectable marker gene having an expression that is activated or enhanced in response to a protein encoded by a recombinant.
• the selectable marker gene encodes a product which can be selected for in a growth media for said cell under a defined environmental condition.
• the method, system, or cell may also include features that facilitate contacting the cell with a test vector particle that is potentially capable of producing the recombinant in the cell and then selecting for the expression of the selectable marker gene in the environmental condition to thereby determine the risk of the test vector particle producing a replication competent retrovirus.
• a "retrovirus” is any ribonucleic acid sequence that may be reverse transcribed into double stranded DNA and subsequently integrated into a host cell genome.
• preferred retroviruses and vector systems for use with the invention include the lentiviruses, most preferably those selected from the group consisting of HIV- 1, SIV, HIV-2, FIV, EIAV, BIV, CAEV, and OVINE.
• the genomes for these lentiviruses, including the locations of the cis and trans elements contained therein, are all well known and available in the art, e.g., as found in GenBank.
• HIV-1 see Table 1, below, for genetics
• other systems may be similarly designed, and with similar effect.
• heterologous coding and cis elements combining elements from different retroviral species
• RCR replication-competent retrovirus
• LTR long terminal repeat
• ⁇ retroviral packaging signal
• ⁇ retroviral packaging signal
• ⁇ polyadenylation signal
• promoter or promoters for expressing the genes usually within the 5' LTR sequence
• PBS transfer RNA binding site
• PPT polypurine tract
• Retrovirus particles can be pseudotyped with non-env protein that mediates infection.
• endres et al. “Targeting of HIV- and SlV-infected cells by CD4-chemokine receptor pseudotypes", Science. 278:1462-1464; Mebatsion et al. (1997) "A CXCR4/CD4 pseudotype rhabdovirus that selectively infects HIV-1 envelope protein-expressing cells", Cell. 90:841-847; Schnell et al.
• test vector particle When integrated into a host cell, this structure constitutes one form of a "provirus.”
• test vector particle is preferably meant a retroviral virion, virus-like particle, or retroviral vector particle bearing a nucleic acid construct that is suspected of possessing, or having the potential to generate, a recombinant in an infected host cell.
• the test vector particle may be present as part of a larger heterogenous population, and may have a minority representation in that population.
• the test vector particle is preferably capable of infecting a host cell. In preferred embodiments, that cell harbors a selective marker gene.
• test vector particle is able to form a recombinant nucleic acid species, that species is the product of genetic recombination involving one or more constructs, e.g., packaging and gene-transfer vector constructs.
• the test vector particle reflects a potential recombination event between such constructs, and it is a purpose of the assay to test for this. It is not necessary that the packaging and vector constructs from which the
• test vector particle derives have homologous sequences, e.g., gag coding sequences.
• one or more mutations is present in the retroviral sequence of a recombinant, including but not limited to internal stop codons, silent mutations and/or deletions within the gag and gag-pol sequences of either of these constructs.
• a "gene-transfer vector” or “gene-transfer construct” as used herein does not require gag, pol, and env, and need only contain the "cis" signals noted above, or equivalents thereof, for vector packaging, reverse transcription, and integration.
• a transfer vector can be illustrated symbolically as LTR- ⁇ -seq-LTR.
• recombinant provirus is meant one that is produced by at least one genetic recombination event between one or more nucleic acid strands or constructs, e.g., packaging and vector constructs of split function retroviral systems. Although preferable, such a recombinant may not be integrated into a chromosome of a cell.
• a "risk of producing a replication-competent retrovirus” occurs by any recombination event that produces a recombinant provirus.
• the preferred purpose of the assay is to measure the possibility for creation of LTR- ⁇ -gag-pol-env-LTR (an RCR), and not necessarily its actual occurrence.
• the assay is meant to serve, at least in part, as a means to assess the safety of administering a given vector (e.g., in gene therapy applications) to an animal, especially in the long term.
• a “selectable marker gene” may be a reporter gene but is preferably a gene having an expressed product capable of affecting the growth characteristics of a cell under one or more environmental conditions.
• the expressed product can encode, for example, a product that endows resistance to an antibiotic. Examples include dhfr, which confers resistance to methotrexate (Miller et al. (1985) Mol. Cell. Biol. 5:431-437); neomycin/hygromycin phosphotransferase, which confers resistance to G418 and hygromycin (Palmer et al. (1987) Proc. Natl. Acad. Sci: 84:1055-1059; Yang et al. (1987) Mol Cell. Biol.
• 14 expressed product may overcome a metabolic deficiency of the host.
• Examples include hprt (Miller et al (1983) Proc. Natl. Acad. Sci. 80:4709-4713) and thymidine kinase (tk) (Shimotohno and Temin (1981) Cell 26:67-77; Wei et al (1981) J. Virol. 39:935-944), which allow host cells that are otherwise deficient for the functional genes to grow in minimal media supplemented with, e.g., hypoxanthine and thymidine.
• the selectable marker gene is preferably integrated into the host cell genome, and is preferably flanked by one or more nucleic acid sequences not natural to the host cell it is in.
• the gene is flanked on its 5' end by a retroviral LTR sequence that may promote expression of the gene, and a retroviral packaging signal, e.g., ⁇ .
• the gene is flanked on both sides by an LTR or SIN sequence that, under appropriate conditions, can be packaged, transferred to other host cells, and selected for under one or more environmental conditions.
• the selectable marker gene is under control of an LTR promoter that is responsive to the retroviral Tat protein.
• Tat refers to a retroviral protein encoded by the retroviral tat gene. Tat activates transcription from the retroviral LTR promoter by binding to a proximal sequence, "TAR", that is located within the 5' LTR (the "R" region).
• the terms “mobilized” and “mobilizing” as used herein denote the process, method, or ability of replicating a recombinant and/or other provirus, e.g., one containing a marker gene, and transmitting it to another cell. This may require helper virus or transfected genes that encode and express these products to supply helper functions, e.g., packaging, virion production, and receptor binding capabilities (e.g., env).
• helper virus or transfected genes that encode and express these products to supply helper functions, e.g., packaging, virion production, and receptor binding capabilities (e.g., env).
• the recombinant provirus includes a gag sequence that encodes a Gag protein.
• a "Gag” protein is a retrovirus structural protein that is processed into several smaller proteins that form the virion matrix, the capsid, and the nucleoprotein structures of a retrovirus.
• one can detect Gag using one or more of several techniques common in the art, e.g., as an additional measure of determining the risk of producing a replication-competent retrovirus (RCR) from said test vector particle. These techniques include the use of the polymerase
• PCR 15 chain reaction
• antibody detection e.g., as in a p24 assay
• biochemical methods e.g., as in a p24 assay
• the recombinant provirus includes a gag-pol sequence that can transcribe and translate into Gag, Pro, RT, and INT proteins.
• gag-pol refers to a continuous nucleic acid sequence that includes at least a portion of the gag, pro, rt, and int retroviral genetic loci, preferably in that order.
• functional gag-pol is meant a nucleic acid sequence that encodes functional Gag, Pro, and Pol protein products which together can promote the packaging and mobilization of a retroviral and/or recombinant proviral nucleic acid sequence.
• a step is also incorporated in which the recombinant provirus' presence is determined using an inhibitor of reverse transcripase, e.g., Nevirapine.
• an inhibitor of reverse transcripase e.g., Nevirapine.
• RCR replication-competent retrovirus
• the method, system, or cell detects env minus recombinant provirus.
• Env or “env” is meant, respectively, the retroviral envelope glycoprotein and its corresponding genetic locus. Retroviral surface and transmembrane components derive from the envelope protein and are necessary for the production of infectious retroviral virion particles.
• An "env minus” provirus is one that lacks one or more of these capabilities.
• the recombinant provirus protein that promotes expression of the selectable marker gene is a Rev protein that functions by virtue of its ability to shuttle retroviral response element (RRE)-containing retroviral transcripts from the nucleus to the cytoplasm of a host cell for suitable expression.
• the marker gene includes an RRE element that can interact with the Rev protein.
• the test vector particle derives from a recombination event that occurs in a split function lentiviral packaging system, e.g., one that includes at least one packaging construct and a vector construct, as those constructs are known in the art.
• An "env construct” or pseudotyping equivalent may also be included. These constructs, as explained above, typically supply in trans one or more of the necessary gag, pol, and env retroviral genes whose products are necessary for gene transfer.
• test vector particle of the method, system, or cell may be present as a product of an earlier transfection event which yields a heterogenous population of virion encapsidated retroviral sequences, of which the test vector particle, if it represents or is to produce a recombinant provirus in an indicator cell of the assay method, system, or cell, may represent but a minority.
• a "test vector particle" may contain one or more nucleic acid sequences that constitute, or upon infection are capable of producing, a recombinant retrovirus.
• the envelope construct may include a pseudotype envelope sequence, such as derived from the G protein of Vesicular Stomatavirus (VSV).
• the packaging construct may further include a promoter and a retroviral nucleic acid sequence that encodes one or more products necessary for retroviral packaging.
• the promoter can promote expression of at least one of the retroviral gene products necessary for retroviral packaging, e.g., Gag, Pro, and Pol.
• the promoter can optionally be an inducible promoter, preferably selected from the group consisting of TK, RSV, CMV, and TRE promoters, which promoters are known and widely available in the art for use with the invention.
• the vector construct may further include a gene of interest, e.g., a reporter or marker gene, that can be used to monitor the relative success or absence of recombination in the assay.
• a reporter or marker gene e.g., a reporter or marker gene
• Preferred reporter genes are selected from the group consisting of green fluorescent protein (GFP), luciferase, and B-galactosidase (B-Gal).
• the reporter may express from a promoter other than an LTR promoter, and may be an "inducible” or “constitutive" promoter as such promoters are known in the art.
• Promoters for use in the vector construct may include the same promoters listed above as possibilities for the packaging construct, e.g., the TK, RSV, CMV, and TRE promoters
• the selectable marker gene encodes resistance to an antibiotic, e.g., puromycin.
• the cell in which it is provided is preferably infectable with the test vector particle used, e.g., a lentiviral based test vector particle can infect a HeLa or 293T human cell, or a derivative thereof.
• the cell line used is an immortalized mammalian cell line.
• the selection can occur within a period of time of two days less, and with a clear distinction of "positives" from "non-positives.”
• the invention features a marker rescue assay system for detecting and/or analyzing a recombinant.
• the system includes an "indicator cell" having a selectable marker gene whose expression is activated or enhanced in the presence of a recombinant provirus, and whose gene product can be specifically detected or selected for in a growth media for said cell under a defined environmental condition.
• the recombinant may be a functional gag-pol recombinant in which the Gag and Pol retroviral gene products are expressed and can be transcomplemented with Env to result in packaging and mobilization of the marker gene, which is preferably integrated in the genome of the indicator cell line.
• the selectable marker gene is also present, it too can be mobilized and selected for in the cell it is mobilized to.
• the invention features an indicator cell as noted above.
• the cell preferably has a marker gene, preferably a selectable marker gene, operably linked to a promoter.
• the marker gene exhibits altered expression in the presence of one or more proteins supplied, e.g,.
• the proteins are retroviral proteins selected from the group consisting of Tat and Rev.
• the selectable marker gene is flanked by LTR sequences and can be mobilized from said cell in the presence of functional Gag and Pol gene products. Env or pseudotypes thereof may also be used to accomplish this.
• the indicator cell line contains a stably integrated copy of the marker gene.
• the marker gene is preferably on a nucleic acid strand that contains a signal for packaging, i.e., a retroviral packaging signal.
• the indicator cell line may contain nucleic acid sequences for the expression of an env gene.
• the expression of env can pseudotype gene products of the recombinant provirus that contact the indicator cell line allowing the recombinant provirus to mobilize to another cell.
• Mobilization can be to another env containing cell or to other cell types. Mobilized recombinants can be detected using a variety of methods as described herein, including detection of gag gene product, i.e., HIV- 1 p24 core protein.
• a common theme that forms the basis for preferred embodiments of the indicator cell systems of the invention is the generation of recombinants preferably containing gag- pol.
• Such recombinant contains the minimal requirements, minus env and/or another helper function, that is necessary for replication and/or packaging. Therefore, it is principally gag-pol that the majority of assays, systems, and indicator cells described herein are intended to detect.
• Tat transfer and other marker transfer assay aspects may not require this.
• Features of the claimed indicator cells may further include any use, feature, or combination of uses of features recited for the methods and system aspects.
• the invention features a Tat transfer assay in which an indicator cell of the type described above is used.
• the indicator cell includes a selectable marker gene that is expressed in response to a retroviral Tat protein encoded by a recombinant provirus, and wherein the selectable marker gene encodes a product, preferably a product endowing puromycin resistance, which can be detected or selected for in a growth media for the cell under a defined environmental condition, preferably in two days time or less.
• the invention features a Gag transfer assay that includes an indicator cell having a selectable marker gene.
• the selectable marker gene is expressed in response to a retroviral protein encoded by a recombinant provirus.
• the recombinant provirus includes functional gag and pol genes that are capable of producing functional Gag and Pol proteins.
• the selectable marker gene encodes a product which can be selected for in a growth media for the cell under a defined environmental.
• the invention features a marker mobilization assay that includes an indicator cell.
• the indicator cell contains a selectable marker gene that is expressed in response to a protein encoded by a recombinant provirus.
• the marker gene can be mobilized from the indicator cell to another cell in the presence of functional Gag, Pol, and Env retroviral gene products.
• the marker gene encodes a product which can be selected for in a growth media for the cell under a defined environmental condition.
• Preferred embodiments for the Tat transfer, Gag transfer, and marker mobilization assay aspects may include any embodiment or combination of embodiments noted for any of the aspects that is consistent with a useful purpose. Further, the method,system, and indicator cell embodiments may combine any of the other aspect embodiments described herein as appropriate to a purpose and practice within the broad spirit of the invention.
• Advantages of the invention include the ability to detect retroviral genetic recombinants in sensitive fashion, and the use of gag-pol as a surrogate marker for identifying RCR potential.
• the invention further affords improved safety and quality control in the design, production, and implementation of existing and future retroviral vector systems, preferably in prelude to the use of such vectors in gene therapy.
• the invention is especially well-suited in assaying for recombinant provirus produced from packaging constructs in which gag-pol has been disarmed, e.g., through the inclusion of mutations and/or deletions.
• FIG. 1 shows an exemplary retroviral genome (HIV-l) having genetic components that can be used in conjunction with the methods, assay systems, and indicator cells of the invention. See Table 1 for more details.
• Fig. 2 shows an embodiment of a split function lentiviral vector assay system having a packaging construct (pTRE-gag-pol), a vector construct (pHR-CMV-GFP), and an env construct (pVSV-G; also known as pMD.G).
• pTRE-gag-pol a packaging construct
• pHR-CMV-GFP a vector construct
• pVSV-G an env construct
• Fig. 3 shows how one aspect of the invention can significantly improve on existing Tat-transfer assays by utilizing a novel indicator cell line bearing an antibiotic resistance gene (e.g., puro) that is capable of conveniently enriching for (or “capturing") the desired LTR-tat-LTR and other recombinations.
• the tat gene product is only expressed in the system when introduced in proviral form and is capable of specifically activating the LTR- driven antibiotic resistance gene, which can then be detected through routine selection means.
• Fig. 4 shows how embodiments of the inventions can be used in tat transfer, gag transfer, and marker rescue/DNA mobilization assays to enrich for and detect the occurrence of retroviral genetic recombinants.
• Fig. 5 shows a tat transfer assay embodiment that makes use of the sensitivity and enrichment provided by the specific indicator cell line HeLa-Puro. The figure shows how the desired tat-containing recombinant can be specifically selected for using an internal antibiotic resistance marker.
• Fig. 6 shows a PCR assay embodiment that may be performed to further verify the presence of a tat-containing recombinant.
• Fig. 7 shows a gag transfer assay embodiment that makes use of the sensitivity and enrichment provided by the specific indicator cell line HeLa-Puro.
• Fig. 8 shows the results of a p24 antigen assay embodiment for the invention that demonstrates gag transfer and expression.
• Fig. 9 shows a marker-gene rescue/DNA mobilization assay that makes use of the sensitivity and enrichment provided by the specific indicator cell line HeLa-Puro. Also shown is the mobilization of the puro marker gene from the HeLa-puro cell into another cell.
• Fig. 10 represents one prediction of how recombination between packaging and gene transfer constructs may occur to produce an LTR- ⁇ -gag-pol-LTR event.
• Fig. 11 shows a PCR assay embodiment that can be performed to verify the 5' recombination event that produces the LTR- ⁇ -gag-pol-LTR event.
• Fig. 12 illustrates a sequence confirmation that genetic recombination has occurred between the packaging and gene-transfer constructs.
• the sequence analysis also illustrates that the recombinants have an open gag-pol reading frame.
• Fig. 13 shows a PCR assay embodiment that may be performed to verify the 3' recombination event that produces the LTR- ⁇ -gag-pol-LTR event.
• Fig. 14 shows sequence analysis of the 5' (Panel A) and 3' (Panel B) recombinant junctions of the recombinant retroviruses obtained and their corresponding vector and packaging construct constituents as depicted in Figs. 11 and 13.
• the figure also illustrates a sequence confirmation that genetic recombination has occurred between the packaging and gene-transfer constructs.
• the sequence analysis demonstrates recombinant between the 3' end of the vector construct and the polyA tract of the packaging construct.
• Fig. 15 demonstrates validation of tat transfer via recombinant provirus using the non-nucleoside reverse transcriptase inhibitor Nevirapine.
• Fig. 16 demonstrates validation of gag transfer via recombinant provirus using the non-nucleoside reverse transcriptase inhibitor Nevirapine.
• Fig. 17 demonstrates validation of marker mobilization via recombinant provirus using the non-nucleoside reverse transcriptase inhibitor Nevirapine.
• Fig. 18 shows another split function vector (a translenti system) embodiment that is incapable of transferring functional gag-pol by virtue of abrogating the translation of RT- IN sequence from the packaging construct. This essentially "disarms" the RT and IN products downstream of pro that are part of the same mRNA transcript and normally part of the same fusion protein prior to post-translational modification and maturation of the independent proteins.
• Fig. 19 shows that the Trans-lenti viral vector cannot transfer gag in a gag-transfer assay Confirmation of this is provided when Vpr-RT-IN is supplied in trans.
• Fig. 20 shows 5' junction sequence for recombinant proviruses, demonstrating the preservation of a stop codon at the beginning of the RT coding region for the translenti system, whereas the rt (pol) gene in the lentiviral-derived recombinants has an open reading frame (no stop codon).
• Fig. 21 shows a third generation lentiviral vector system in which the tat and rev genes are supplied on a separate construct.
• Fig. 22 indicates that genetic recombination has taken place between the vector and packaging construct, since the puro marker gene is mobilized from the 293T-LTR-puro cell to the HeLa-tat cell.
• Panel A shows the assay overview using the third generation vector system.
• Panel B shows the plated/selected colonies under different experimental conditions.
• Fig. 23 illustrates a lentiviral vector packaging construction embodiment in which expression of Gag-PR-RT-IN is regulated using an Internal Ribosomal Entry Site (IRES). See Example 10.
• IRES Internal Ribosomal Entry Site
• Fig. 24 illustrates a trans-lentiviral vector split packaging system embodiment in which Gag and Gag-PR-RT-IN are expressed from separate coding sequences to mutate the highly conserved stem-loop structure in both Gag and Gag-PR-RT-LN. This does not have deleterious effects on the formation of infectious particles. See Example 10.
• Fig. 25 illustrates an MLV vector packaging construction embodiment in which expression of Gag-PR-RT-IN is regulated using an Internal Ribosomal Entry Site (IRES). See Example 10.
• IRS Internal Ribosomal Entry Site
• Fig. 26 illustrates an MLV-based vector split packaging system embodiment in which Gag and Gag-PR-RT-IN are expressed from separate coding sequences to mutate the highly conserved stem-loop structure in both Gag and Gag-PR-RT-IN. This does not have deleterious effects on the formation of infectious particles. See Example 10.
• Fig. 27a illustrates Vpr-RT-IN expression plasmids containing 1-18 kb of sniffer DNA, e.g., derived from lambda DNA ("Vpr-RT-TN/ST") for identifying which will still efficiently provide RT and IN function in trans. See Example 11.
• Fig. 27b shows how to humanize Vpr, RT and IN genes by PCR.
• Fig. 27c shows the codon usage of highly expressed human.
• Fig. 28a-d shows various embodiments for splitting the structure of Gag/Gag-PR- RT-IN into two separate coding sequences: one that express Gag and another that expresses Gag-PR-RT-IN.
• Fig. 29 illustrates an MLV-based transfer vector that contains a DNA flat sequence derived from HIV-1.
• the PPT sequence derived from MLV is inserted upstream of the cPPT sequence.
• the basic components of traditional retro viral-based gene delivery systems include packaging, vector, and envelope elements that are usually supplied on separate genetic constructs to promote safety ("split-function"; see, e.g., Figs. 2,18 and 21).
• the packaging construct traditionally encodes the structural (gag) and catalytic (pol) proteins that are necessary to generate an infectious particle.
• the vector construct typically contains the genetic material of interest that is to be transferred into the target cell, along with a packaging signal ( ⁇ ), a promoter for gene expression, and cis-acting sequences necessary for reverse transcription and integration such as the long terminal repeats (LTRs) and a primer binding site (pbs).
• the envelope (env) component also supplied on a separate construct, mediates recognition and entry of the virus into the target cell and is usually a heterologous env protein such as the G protein from vesicular stomatitis virus (VSV-G).
• VSV-G vesicular stomatitis virus
• the retrovirus gag-pol structure is so highly conserved among retroviruses that it is likely required for any type of retroviral RCR and DNA mobilization event. Therefore, sensitive assays capable of detecting the de novo establishment or absence of this entity in vector-transduced cells would be useful. Such in vitro assays could help to evaluate retroviral/lentiviral vector safety and predict the risk associated with RCR and retrovirus- induced DNA mobilization in vivo. Such assays would be especially indicated prior to use in clinical trials with humans to ensure that vector stocks are free of the potential to generate gag-pol containing recombinants.
• CBER Center for Biologies Evaluation and Research
• FDA Factorized Reduction Protocol
• MLV-based retroviral vectors include specific PCR and immuno logical assays to monitor for evidence of replication-competent MLV.
• Guidelines have not been established for the monitoring of lentiviral vectors.
• These assays include: (1) tat transfer, which monitors for the presence of the HIV t ⁇ t gene in the target cell; (2) gag transfer, based on measuring the HIV-1 capsid protein (p24Gag) in the medium of vector-transduced cells; and (3) Marker Rescue, which looks for the transfer of a marker gene from vector- transduced cells to other cells. Transfer of the tat and gag genes are used as markers to determine if recombination between the gag-pol packaging and gene transfer vector elements has occurred.
• Detecting recombinants not only provides a marker for evaluating the safety of lentiviral-based gene therapy vectors, but also provides the means to understand the nature of the recombinants at the genetic level, and additionally can help in the design of new vectors.
• gag-pol e.g., LTR- ⁇ -gag-pol-LTR
• Detecting the transfer of the tat, gag-pol and or marker gene(s) is used to determine if recombination has occurred between the gene-transfer and packaging element vectors.
• Transfer of a marker gene from one cell to another requires that a functional gag/gag-pol recombinant structure (LTR- ⁇ -gag-pol-LTR) be formed in the vector-transduced cells.
• LTR- ⁇ -marker-LTR e.g., as LTR- ⁇ -marker-LTR
• Such particles are capable of mobilizing retroviral sequences, including those having marker and/or tat, to other cells where, in the case of the marker, it can be tracked (e.g., using selective conditions).
• the pseudotyped env may be provided in trans by using a cell line capable of expressing VSV-G or other env genes capable of pseudotyping the recombinant.
• Example 1 Generation of HeLa-puro: A Tat-dependent Indicator Cell Line Capable of Capturing Lentiviral Vector Recombinants
• the puromycin N-acetyl transferase (puro) gene was integrated into a HeLa cell with its expression under control of the HIV-1 LTR. Because the HIV-1 LTR is trans-activated by the HIV-1 tat protein, puro expression within the cell is induced when the t ⁇ t gene becomes integrated into the host genome and expressed. With tat being
• the pHR-puro expression plasmid was constructed using commonly available starting materials by ligating a PCR amplified DNA fragment containing the puromycin gene (derived from pPUR; Clontech Laboratories) into the pHR- CMV-lacZ plasmid (Naldini et al.
• the vector stock encoding the puro gene was generated by cotransfection of pTRE-gag- pol, pVSV-G (Naldini et al. (1996) Science 272:263-267), and pHR-puro into 293T cells (ATCC). Culture supernatant was harvested 60 hours post-transfection and used to transduce a HeLa cell line (ATCC).
• puro gene is in part dependent on its site of integration within the HeLa cell genome, it was important to select a cell clone that not only had a high level of puro expression in the presence of tat, but also displayed a low level of background expression in the absence of tat.
• candidate indicator cell lines To select for candidate indicator cell lines, individual cell clones were expanded in culture and screened for t ⁇ t-dependent expression of the puro gene. Each clone was infected with a pseudotyped HIV-1 -derived vector encoding the tat gene.
• HeLa-LTR-puro One cell clone, henceforth termed HeLa-LTR-puro, was selected because it was able to grow and form colonies in culture media containing relatively high levels of puromycin (>30 ug/ml) following infection with the HIV vector, but displayed low level background resistance to puromycin ( ⁇ 0.5 ug/ml) in the absence of infection. Using routine methodologies, one of skill in the art can readily create equivalent, and perhaps even superior, cell lines.
• HeLa-LTR-puro line noted herein was deposited with an International Depository Authority (IDA) (American Type Culture Collection ("ATCC”; 10801 University Boulevard, Manassas, VA 20110- 2209) on 30 June 2000 under conditions satisfying the Budapest Treaty for biological deposits in support of patent applications, (accession number to be assigned).
• IDA International Depository Authority
• the component vectors are constructed as follows:
• the pCMV-gag-pol packaging plasmid was constructed by inserting an EcoRI/BamHI DNA fragment of pSG3 (coordinates 258 to 8384) into the pcDNA3.1(+) plasmid (InVitrogen) under control of the CMV promoter.
• the recombinant plasmid was then modified by introducing a 39-base pair deletion in the packaging sequence ( ⁇ ), and a 1357-base pair deletion in the envelope gene (coordinates 5827 to 7184).
• the pHR-CMV-GFP plasmid was constructed by ligating a PCR amplified DNA fragment containing the GFP gene (derived from pEGFP-Cl, Clontech Laboratories) into the BamHI/XhoI sites of the pHR-CMV-LacZ plasmid. Also, a 150 bp sequence of DNA (coordinates 4327 to 4483) containing the central polypurine tract (PPT) and central terminal site (CTS) was PCR amplified from the HIV-1 pSG3 molecular clone and ligated into the unique Clal site of pHR-CMV-GFP. The sequences in the vector construct illustrate the frameshift mutation that is used to introduce the premature stop codon in the gag gene. This was accomplished by the fill-in of a Clal restriction enzyme site that is present at the 5' end of the gag gene. The vector contains only the first 360 base pairs of the HIV-1 gag coding sequence.
• Lentiviral vector stocks were produced by transfecting 5 ug of the pCMV-gag-pol packaging plasmid, 2 ug of the pVSV-G expression plasmid (also known as pMD.G; Ory et al. (1996) PNAS 93:11400) and 5 ug of the gene transfer (vector construct) plasmid into subconfluent monolayer cultures of 293T cells by the calcium phosphate DNA precipitation method. Supernatants were harvested after 60 hrs, clarified by low speed centrifugation (lOOOg, 10 min), and filtered through 0.45-um pore-size filter.
• the vector particles were then subjected to ultracentrifugation using a Beckman SW-28 rotor (23,000 rpm, 90 min., 4°C) to concentrate vector particles.
• the pellets were resuspended in 1.0 ml of DMEM., aliquoted and frozen at -80°C.
• Example 1 that, through the expression of an inducible marker gene, can indicate when a recombinant is formed and integrated into the host cell chromosome (Figs 3-5). Because the HIV-1 LTR is trans-activated by the HJV-1 Tat protein, puro (puromycin N-acetyl transferase) expression is induced to confer resistance only if the cell becomes infected with a tat-containing recombinant that integrates and becomes expressed. Since the tat gene is present within the lentiviral packaging construct (linked to gag-pol), recombinants of the vector and packaging constructs would likely contain tat.
• puro puromycin N-acetyl transferase
• these integrated recombinant lentiviruses would express the Tat protein and transactivate the expression of the puro gene and confer resistance in puromycin-containing media.
• This novel tat transfer assay markedly improves the sensitivity for detecting genetic recombinants and importantly allows enrichment for the recombinants, making it possible for the first time to conduct detailed genetic and biologic analyses.
• 293T cells are infected with vector stocks. It is in these infected cells that recombinants are likely to be generated and stably integrated. Such recombinant genomes can be expressed and mobilize retroviral sequences, especially when helper functions are provided in trans, e.g., env.
• a Tat-inducible antibiotic resistance marker gene e.g., puro
• the HeLa-puro indicator cell line is used to capture recombinants which contain functional gag and pol genes. Utilizing puromycin selection, we are able to specifically select and enrich for cells containing recombinant retrovirus such as LTR- ⁇ -Gag-Pol-Tat/Rev-LTR.
• recombinants should also integrate into the genomes of the 293T cells, and those recombinants that contain an open gag sn ⁇ pol reading frames would express viral Gag and Gag-Pol proteins, produce progeny particles, and package the recombinant RNA genome.
• the transduced 293 T cells were transfected with the pMD.G expression plasmid in order to pseudotype any progeny vector particles that may be produced.
• a tat/rev-encoding expression plasmid was also transfected. Three days following transfection, culture supernatant was harvested and concentrated by ultracentrifugation.
• the concentrated supernatant was now used to transduce the HeLa-puro indicator cell line.
• the transduced HeLa-puro cells were placed under puromycin selection as described above followed by fixation and staining of the colonies that may have formed.
• VSV-G was supplied in trans, 390 puromycin-resistant colonies formed.
• lentiviral vector-derived recombinants could transfer functional gag and pol genes into its target cell (293T).
• This result suggested that the retroviral recombinant genome can be mobilized from the transduced 293T cells to the HeLa-puro cells if an envelope was provided in trans. Again, no colonies formed when supernatants from mock-transduced 293T cells were used to infect the HeLa-puro cells.
• Gag transfer was further confirmed by measuring the amount of HIV-1 capsid protein (p24 antigen) released into the supernatant of the puromycin-resistant HeLa-puro cells.
• Fig. 8 Method The 390 colonies which formed following selection in the puromycin-containing media were trypsinized and replated into replica cultures. One half of the cells were transfected with a plasmid expressing tat and rev while the other half was mock transfected. Culture supernatants were collected 60 hours later and a small amount was aliquoted and frozen at -80°C. Meanwhile, the remaining supernatant was concentrated by ultracentrifugation as described above and resuspended in 100 ul of DMEM.
• Fig. 8 demonstrates that gag was transferred and expressed in the vector- transduced cell.
• the increase in p24 production with co-transfection of tat suggested that the gag gene was linked to the HIV-1 LTR, and provided additional evidence that recombination occurred between the gag-pol packaging construct and the LTR-containing gene transfer vector.
• Our results further show that the recombinants generated by the lentiviral vector system mobilized both tat and gag and pol genes from the vector transduced cells (293T) into the puro-HeLa cell when the envelope (e.g., as pseudotype VSV-G protein) was provided in trans (Fig. 9).
• the lentiviral vector can regenerate recombinants (such as LTR- ⁇ -gag-pol-tat/rev-LTR) in the vector- transduced cell, and that this envelope-deficient recombinant retrovirus can be mobilized into other cells when the envelope is provided in trans.
• recombinants such as LTR- ⁇ -gag-pol-tat/rev-LTR
• Tat expression was a product of LTR expression and not of the packaging vector
• the reverse transcription inhibitor Nevirapine (NVP) was used to identify the source of expression.
• the specific HIV-1 RT inhibitor NVP blocked the mobilization of the recombinants from the vector-transduced cell to the HeLa-puromycin cell, indicating that the retrovirus DNA mobilization is specifically mediated by HIV-1 gag/gag-pol (Fig. 16).
• DNA was extracted from the puro-resistant cells using a WizardTMgenomic DNA purification kit (Promega, Madison, WI). DNA fragments of the 5' recombinant virus region (from U3 to gag, ⁇ 2 Kb)(Figs. 11, 13,) and 3' recombinant virus region (from Tat to U3; ⁇ 2.5Kb)(Fig. 14, 15B,) were amplified by PCR, and the sequences contained therein determined.
• the 5' region of the recombinant was amplified using sense primer homologous to a sequence located in the U3 region of the 5'-LTR (sequence: 5'-ccg gaa ttc tgg eta act agg gaa ccc act gc-3'; SEQ. ID NO. 2) with an antisense primer that hybridizes to a sequence located at the 3' end of the gag gene (sequence: 5'-cgc gga tec tta ttg tga cga ggg gtc get gcc-3'; SEQ ID NO. 3).
• Amplification of a 2054 bp DNA product (the predicted size of
• the ⁇ J3-gag fragment can only occur if the vector and packaging elements have combined through genetic recombination.
• a PCR product of the predicted size was amplified from the lentiviral-derived puromycin resistant colonies. Both PCR amplified DNA fragments were cloned and sequenced. In 10 of 10 clones, sequence analysis of the 5' PCR fragment confirmed the presence of U3 R U5 (the 5' LTR), and the ⁇ packaging signal (from the gene transfer vector) linked with the gag gene from the packaging construct (Fig. 12).
• RNA could be packaged into viral particles derived from the recombinant. Such particles, if pseudotyped with VSV-G, should be capable of mobilizing the puro gene into another cell.
• gag-pol-mediated marker gene mobilization assay see Figs. 9,
• Fig. 9 shows that the puro gene was mobilized by the recombinant. This transfer was completely inhibited by NVP (Fig. 17), indicating that the marker gene mobilization was mediated by HrV-1-based retroviral recombinants (gag-pol).
• the recombinants containing the functional gag-pol structure in the vector- transduced cells can integrate into the host genome, express viral proteins (including Gag and Gag-Pol), produce retroviral particles, encapsidate viral RNA, and mobilize the viral genome to new target cells when a helper function, e.g, env, is provided in trans.
• a helper function e.g, env
• Example 7 Molecular Characterization of Recombinants Assay recombinants can be predicted to have the structure indicated in Fig 10, which shows the daughter recombinant molecule (LTR- ⁇ -gag-pol-tat rev-LTR) and parts of the parent molecules from which is formed the recombinant. To verify this, we selected for cells harboring a tat-containing recombinant genome and expanded these cells in culture to characterize the nature of the genetic element responsible for transfer of tat into the HeLa-puro cell. Chromosomal DNA from a cell pool was isolated using the WizardTM genomic DNA purification kit (Promega).
• the 5' and 3' primers used to amplify the 5' (2054bp) PCR product also encoded EcoRI and BamHI restriction sites, respectively. This allowed us to clone the PCR product into pUCl 19 vector DNA and sequence the individual clones.
• Figs. 12, 14A Our sequence analysis confirmed that the vector and packaging constructs had joined through recombination. Also, it appears that the recombination events on the 5' end occurred through homologous sequences because of the sequence overlap between the vector and packaging components. Since only recombinants with open gag reading frames are able to be transferred from 293T cells to the HeLa-puro indicator cells all recombinants sequenced (10/10) contained an open gag reading frame.
• the 3' end of the recombinant was confirmed by PCR using a sense primer specific to the 5' end of the t ⁇ t gene (sequence: 5'-ccg gaa ttc atg gag cca gta gat cct aga c- 3'; SEQ. ID. NO. 5) and with an antisense primer that hybridizes to the R region located in the 3'-LTR (sequence: 5'-cgc gga tec gca gtg ggt tec eta gtt agcc-3'; SEQ. ID. No. 6).
• Figs. 14, 15B, 24B As shown, PCR products of various molecular weights were amplified, with a predominant PCR product approximating 2500 bp in length.
• the 3' (2500 bp) PCR product was cloned into a pUC19 vector DNA and individual clones sequenced.
• Fig. 14B the junction between the 3'-LTR and the packaging construct contained a polyA sequence of various lengths. Further analysis of the sequence revealed that the polyA sequence was derived from the mRNA transcript of the pTRE-gag-pol packaging component. These results demonstrated that the polyA tract can be used to promote non-homologous recombination between the vector and packaging constructs.
• Nevirapine a non-nucleoside reverse transcriptase inhibitor specific to the HIV-1 RT.
• Nevirapine would prevent only HIV-RT mediated transfer of tat and gag into the HeLa-puro cell but not if it is transferred via plasmid DNA.
• Example 8 Assay Comparing Gag-Pro versus Gag-Pol Vector Systems
• a trans-lentivirus vector system based on a gag-pro (not gag-pol) packaging construct that promotes a "disarming" of lentiviral recombinants by eliminating production of functional reverse transcriptase (RT) and integrase (IN) from the traditional packaging vector.
• RT reverse transcriptase
• IN integrase
• RT and IN are instead expressed on their own, separate nucleic acid vector fused, e.g., to vpr, a sequence that when expressed is capable of promoting packaging of the fusion protein.
• Wu et al. (1997) EMBO, vol. 16, no. 16, pp. 5113- 5122, and United States Patent Application Serial No. 09/460,548, filed Dec 12, 1999, each of which is herein incorporated by reference.
• trans-lentiviral vector in the tat transfer assay, which does not require functional gag-pol.
• 293T cells were transfected with the four DNA components of the trans-lentiviral system (Fig. 18), and three days later the culture supernatants were collected.
• the vector particles were concentrated by ultracentrifugation, resuspended in DMEM, and titered by limiting dilution infection of the cell line, as described above. After nine day of culture in medium containing puromycin, approximately 800 resistant colonies were enumerated using bright-field microscopy (Fig. 18). This result suggests
• trans-lentiviral vector stocks of the trans-lentiviral vector were also analyzed for gag-transfer/DNA mobilization. Exactly as described above for the lentiviral vector (Fig.16), 10e7 infectious units of trans- lentiviral vector particles were used to infect 293T cells. Culture supernatants were collected and used to infect 293T cells as described above. Three days later these culture supernatants were collected and processed exactly as described above for the lentiviral vector. This analysis demonstrated that the trans-lentiviral vector is unable to produce resistant colonies, indicating added safety over the lentiviral vector.
• the resistant cells were expanded in culture and the high molecular weight DNA was extracted for genetic analysis (Fig. 20).
• This analysis demonstrated that that the RT-IN coding region was defective since 5 of 5 recombinants that were analyzed contained a translational stop codon (TAA) at the first amino acid residue of RT, as introduced into the trans-lentiviral packaging construct originally.
• TAA translational stop codon
• Third-generation lentiviral vector systems remove the tat and rev genes from the packaging construct. While tat is not needed at all, the rev gene is still required and is therefore supplied in trans from a separate genetic construct (Fig. 21). Theoretically, this reduces the possibility for an RCR event.
• tat as a part of the lentivirus trans-lentiviral vector packaging construct
• we assayed tat minus (3rd generation) vector for DNA mobilization in a tat- independent manner we assayed tat minus (3rd generation) vector for DNA mobilization in a tat- independent manner.
• Fig. 22 shows an overview of such an assay embodiment, as well as data demonstrating that the puro gene in such an assay can be transferred into HeLa Tat cells if VSV-G is supplied.
• This is the identical concept behind the marker rescue assay described above.
• the 293T-LTR puro cell line improved sensitivity.
• 293T-LTR puro is constructed in the same way as HeLa puro.
• the same puromycin resistance gene is used and positioned downstream of an HIV-1 LTR, and the genetic transcript from which it is expressed contains an HIV-1 packaging signal that allows encapsidation into an HIV particle.
• the line 293T-LTR-puro was selected for its high expression levels of the puro-containing transcript. The more puro transcript available, the better the chance of it being incorporated into a viral particle for transfer into a cell, and the more efficient the system is.
• the third generation (tat-minus) vector stock is used to infect the 293T-LTR puro cell. If one of the 293T-LTR puro cells harbors an LTR- ⁇ -gag-pol-LTR recombinant, the recombinant can express the viral Gag and Gag-Pol proteins and form a viral particle which can encapsidate the mRNA of the LTR- ⁇ - ⁇ gag-RRE-puro-LTR construct.
• Fig. 23 depicts and verifies this. Although the number of recombinants (LTR- ⁇ -gag-pol-LTR) is fewer (52/10e7), the assay is sensitive enough to detect them.
• Example 10 A Vector System Including Regulation of the Expression Level of Gag-PR-RT-IN Using an Internal Ribosomal Entry Site Using the cell-based genetic recombination assay described above, we demonstrated that a new lentivirus vector packaging construct designed by splitting gag- pol into two components (gag/gag-pro and Vpr-RT-IN) significantly improved the biosafety of the lentivirus vector by preventing the regeneration of a recombinant lentivirus containing functional gag-pol. Since the gag-pol structure is highly conserved among all retrovirus, this structure must exist in any type of replicating-competed retrovirus derived from genetic recombination.
• retrovirus-based vectors including simple retrovirus-based vectors (such as MLV) and lentivirus-based vectors such as HIV, SIV, FIV, EIAV, BIV, Visna, CAEV, and Ovine lentivirus).
• simple retrovirus-based vectors such as MLV
• lentivirus-based vectors such as HIV, SIV, FIV, EIAV, BIV, Visna, CAEV, and Ovine lentivirus.
• retrovirus-based vector packaging system which is high conserved among all retroviruses and essential for retroviruses to maintain a well regulated ratio of Gag proteins to Gag-Pol proteins in infected cells for producing infectious particles.
• This design splits the expression of Gag/Gag-PR RT-IN into two separate coding sequences: one that expresses Gag and another that expresses the Gag-PR-RT-IN.
• the production of a fixed ratio of Gag to Gag-PR-RT-IN is regulated by the internal ribosomal entry site (IRES) derived from the encephalomyocarditis virus (EMCV), rather than by the ribosomal frame-shift site.
• IRS internal ribosomal entry site
• EMCV encephalomyocarditis virus
• Vpr and Vpx can design transvector packaging systems for all retrovirus-based vectors since the approach does not require virion-associated proteins (e.g., Vpr and Vpx).
• a specific nucleotide motif, the slippery sequence, as well as a downstream mRNA secondary structure are essential for the production of the fixed ratio of Gag to Gag-PR- RT-IN (Fig. 23 for lentivirus vector; Fig. 25 for MLV vector).
• the disruption of the highly conserved stem-loop structure completely blocks the production of the fixed ratio of Gag to Gag-PR-RT-IN, which is essential to produce the infectious particles.
• Gag and Gag-PR-RT-IN are split into two separate coding regions which should prevent the regeneration of recombinant retrovirus containing the functional Gag-Pol structure; and two in that the novel packaging system completely removes the ribosomal frameshifmg site which is essential for all retrovirus to maintain the ratio of Gag and Gag-PR-RT-IN during the production of infectious particles. It is likely that without this highly conserved element, any type of recombinant retrovirus would be unable to produce the infectious particles and replicate.
• Gag-PR-RT-INT polyproteins Since we express the HIV-1 Gag and Gap-PR-RT-INT polyproteins in two separate mRNAs, and the expression of Gag-PR-RT-IN polyproteins is no longer dependent on the highly conserved stem-loop structure of mRNA, this allows us to completely humanize all of the codons in the mRNA of Gag and Gag-PR-RT-IN. Therefore, modified gag-PR-RT-LN genes would express high levels of protein to produce high titer vector.
• Example 11 Preventing Regeneration of Recombinant Lentivirus Vector Containing Functional Gag-PR-RT-IN
• trans-enzyme plasmid Vpr-RT-LN
• packaging construct gag/gag pro
• Vpr-RT-IN expression plasmid may also contain 1-18 kb of stuffer DNA, e.g., derived from lambda DNA ("Vpr- RT-IN/ST"). These plasmids transcribe greater than 4 kb of mRNAs to express the Vpr- RT-IN fusion protein. To identify which of the Vpr-RT IN/st plasmids still efficiently provide RT and IN function in trans, one can test the different constructs compared with the parent plasmid, Vpr-RT-IN. (Fig. 27a)
• (b). Eliminating incorporation of mRNA of packaging construct into vector particles One can construct the Gag-PR or Gag-PR-RT-IN expression plasmids containing 1-18 kb of stuffer DNA as described for (a), generating Gag-Pro/ST or Gag-PR-RT- IN/ST. These plasmids will transcribe greater than 7kb mRNA to express the Gag/Gag-PR or Gag/Gag-PR-RT-IN. To identify which of the Gag-Pro/ST or Gag-PR-RT-LN/ST still efficiently produce the high level of p24 antigen in the cell, one can test the different sized constructs against the parent plasmids, Gag-Pro or Gag-PR-RT-IN.
• Vpr-RT-IN gene based on optimal codon usage (humanized Vpr-RT- IN gene) will provide several advantages over the current Vpr-RT-IN gene.
• the humanized Vpr-RT-IN plasmid should dramatically reduce the homologous recombination with the RT and IN part of packaging constructs.
• the humanized synthetic gene can result in efficient expression of Vpr-RT-LN protein in a Rev- and RRE-independent fashion. Therefore, RRE sequence, which exists in both the packaging constuction and transfer vector, can be removed from the trans-enzyme plasmid.
• Fig. 27c shows the codon usage of highly expressed human genes (Haas et al (1997) Current Biology 6:315-324).
• Fig. 27b shows how to humanize Vpr, RT and IN genes by PCR.
• Lentiviruses such as HIV-1 can infect non-proliferating cells owing to the karyophilic properties of the lentiviral preintegration complex which governs recognition of the cell nuclear import machinery.
• Lenti virus-based vectors HIV, FIV and EIAV
• HIV Lenti virus-based vectors
• FIV and EIAV Lenti virus-based vectors
• MLV murine leukemia virus
• MLV-based vectors can only transduce cells that divide shortly after infection because the MLV preintegration complex cannot migrate to the nucleus in the absence of mitosis. This considerably limits the use of MLV-based vectors for gene delivery into targets such as neurons, hepatocytes, myocytes and hematopoietic stem cells.
• the modified NC containing a nuclear localization signal or other specific motif that facilitates nuclear transport will enable the PIC of an MLV-based vector to be transported into the nucleus in the absence of mitosis. Therefore, the modified MLV based vector packaging system will facilitate MLV-based vectors to transduce nondividing cells.
• central DNA flap acts as a cis-determinant of HIV-1 DNA nuclear import.
• the location of the central flap has been precisely defined in the case of HIV-1.
• Central strand displacement starts at the first nucleotide following the cPPT sequence and stops 99 nucleotides downstream at the terminator 2 site of CTS.
• HIV-1 gene transfer vectors lacking the central DNA flap exhibit a strong nuclear import defect. This defect can be rescued by reinsertion of the DNA flap sequence into the transfer vector.
• an improved MLV-based transfer vector that contains a DNA flat sequence derived from HIV-1.
• the PPT sequence derived from MLV is inserted upstream of the cPPT sequence (Fig. 29). This novel design of the MLV-based vector improves the ability to transduce non-dividing cells.

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