WO2003072797A1 - Novel trans-viral vectors comprising multicistronic expression constructs and methods of use - Google Patents

Abstract

The present invention provides novel methods and compositions for the expression of a polypeptide of interest. In particular, the methods and compositions of the invention combine a trans-viral vector system (Figure 1) with a gene transfer vector having at least one DNA expression cassette comprising multiple open reading frames separated by internal ribosomal entry sites (IRES) or an active fragment or variant thereof.

Description

NOVEL TRANS-VIRAL VECTORS COMPRISING MULTICISTRONIC EXPRESSION CONSTRUCTS AND METHODS OF USE

FIELD OF THE INVENTION The present invention is drawn to methods and compositions for the expression of a polypeptide of interest.

BACKGROUND OF THE INVENTION

Recently, progress has been made in the development of virus-based vectors that are capable of efficient gene transfer and include important safety design features. For example, a new class of vectors have been introduced to split the Gag-Pro-Pol component of the packaging construct into two separate parts: one expresses Gag/Gag-Pro and the other expresses Pol (Reverse Transcriptase and Integrase) fused with Vpr (Vpr-RT-IN) (Liu et. al. (1997) J. Virol. 77:7701-7710; Wu et al. (1997) EMBOJ. 16:5113-5122; and Wu et al. (1995) J. Virol. 69:3389-3398), (Wu et al. (2000) Mol. Therapy 2,47-55). This "trαπs-viral" vector design reduces the risk of generating replication competent retrovirus through genetic recombination, and enables in vitro monitoring of trans -viral stocks for the existence of recombinants containing functional Gag-Pol as a means to quality assure safety against generating a replication competent virus.

Various systems and methods have been developed for the introduction of nucleotide sequences into target cells for the production of recombinant polypeptides at high levels of purity and quantity. Frequently, however, the gene product of interest and the cell lines expressing the polypeptides result in the expression levels of certain polypeptides to be lower than desirable. In many instances, poor recovery of clones containing the gene of interest due to deletion or inactivation or alternatively, the isolation of high levels of false positive clones carrying only the marker and not the gene of interest result. Accordingly, methods are needed for the improved expression of recombinant proteins. SUMMARY OF THE INVENTION Methods and compositions for the expression of a polypeptide of interest are provided. Compositions of the invention comprises a trans-viral vector system comprising a gene transfer vector having a DNA expression cassette comprising at least a first nucleotide sequence encoding a first polypeptide and a second nucleotide sequence encoding a second polypeptide, wherein said first and said second nucleotide sequence are operably linked by an internal ribosomal entry site (IRES) or an active variant or fragment thereof. In specific compositions of the invention, the first or the second nucleotide sequence of the gene transfer vector encodes a polypeptide selected from the group consisting of an integral membrane protein (i.e., a GPCR or a multidrug resistance protein) an immunomodulatory polypeptide, and a growth factor.

In other compositions of the invention, at least one of the first or the second nucleotide sequences of the gene transfer vector encodes a selectable marker. In more specific embodiments, the selectable marker confers resistance to a cytotoxic agent including, for example, puromycin and neomycine.

In other compositions of the invention, the traλ.s-viral vector is from a retrovirus or a lentivirus. In yet other embodiments, the lentivirus is a human immunodeficiency virus or a simian immunodeficiency virus; and, in other embodiments, the human immunodeficiency virus is HIV-1 or HIV-2.

Also provided are methods for expressing at least two polypeptides of interest. Particularly, the methods of the invention comprise providing a target cell and transducing the target cell with a trans-viral vector particle comprising a gene transfer vector comprising a DNA expression cassette, wherein the DNA expression cassette comprises a first nucleotide sequence encoding a first polypeptide and a second nucleotide sequence encoding a second polypeptide wherein, the first nucleotide sequence is operably linked to a promoter active in the target cell; and, the first and the second nucleotide sequence are operably linked by an internal ribosomal entry site (IRES) or an active fragment or variant thereof; and, culturing said target cell under conditions that allow for expression of said first and said second nucleotide sequence of interest. In other methods, the first or the second nucleotide sequence of the DNA expression cassette encodes a selectable marker. In yet other methods, the second nucleotide sequence encodes a selectable marker. When a selectable marker is used in the gene transfer vector, the methods may further comprise selecting the target cells which express the selectable marker and further culturing said target cells which express the selectable marker under cell growth conditions.

The methods of the invention find use the production of various polypeptides include, for example, integral membrane proteins, immunomodulatory polypeptides, and growth factors.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 schematically illustrates a non-limiting example of a viral vector that can be used in the methods of the present invention. Specifically, Figure 1 provides an illustration of the "trans- viral" vector design comprising four (4) different DNA constructs used to generate a trans-viral particle. Inactive Reverse Transcriptase and Integrase are unboxed in the diagram.

Figure 2 provides a non-limited illustration of the trans-lenti viral vector design.

Figure 3 provides a non-limited illustration of the trans -retro viral vector design.

Figure 4 illustrates a non-limiting example of a gene transfer vector that can be used in the methods and compositions of the present invention. The construct illustrated comprises a bicistronic DNA expression cassette having the following operably linked components: 5' LTR sequences: a packaging signal; an RRE; a central polypurine tract (cts/ppt); a CMV promoter; a cDNA of interest: an IRES; a sequence conferring puromycin resistance; a WPRE; and a 3' -LTR.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides novel methods and compositions for the expression of a polypeptide of interest. In particular, the methods and compositions of the invention combine a trans-viral vector system with a gene transfer vector having at least one DNA expression cassette comprising multiple open reading frames separated by internal ribosomal entry sites (IRES) or an active fragment or variant thereof.

More specifically, the methods of the invention comprise transducing a target cell with a trans-viral vector particle comprising a gene transfer vector having at least one DNA expression cassette. The DNA expression cassette employed in the trans- viral vector system comprises a first nucleotide sequence encoding a first polypeptide which is operably linked to a second nucleotide sequence encoding a second polypeptide by an internal ribosomal entry site or an active variant or fragment thereof. The transduced cells are cultured under conditions that allow for the expression of the first and the second nucleotide sequence contained within the expression cassette. As the expression of the first and the second nucleotide sequences are under the control of the same promoter, the two sequences are transcribed as a multicistronic mRNA. The presence of the IRES element in the transcript permits the translation of the second open reading frame from the multicistronic message.

In one embodiment of the present invention, a nucleotide sequence of the DNA expression cassette encodes a selectable marker, while the other nucleotide sequence encodes a polypeptide of interest. In this particular embodiment, the transduced target cells are cultured under conditions that allow for the transcription of the multicistronic transcript and the expression of both the selectable marker and the polypeptide of interest. Based on the expression of the selectable marker, target cells expressing the polypeptide of interest are then selected away from cells that are either not expressing the polypeptide of interest or are expressing the polypeptide at low levels. The method thereby reduces the problem of false positive clones that express high levels of the selectable marker, while not expressing the polypeptide of interest. More details regarding various embodiments of the invention are provided below.

I. The Trans-Viral Vector The methods and compositions of the invention combine a trans-viral vector system with a gene transfer vector having at least one DNA expression cassette comprising multiple open reading frames separated by an internal ribosomal entry site (IRES) or an active variant or fragment thereof. As used herein, a viral vector having a "trans-viral vector" design is characterized as separating, at least in part, nucleotide sequence encoding the Gag and the Pol polyproteins. By "polyprotein" is intended a single precursor polypeptide which is processed into individual proteins. For example, the HIV Pol polyprotein comprises Reverse Transcriptase and Integrase. The HIV Gag polyprotein comprises, for example, MA, CA, NC, and p6.

In the trans-viral vector design, the nucleotide sequence encoding the Gag- Pro-Pol polyprotein is split into at least two separate parts: a) at least a first nucleic acid segment comprising a nucleotide sequence encoding at least a functional portion of a Gag polypeptide, and the first nucleic acid segment does not encode at least one of a functional Reverse Transcriptase polypeptide and a functional Integrase polypeptide; and, b) at least a second nucleic acid segment comprising at least one nucleotide sequence encoding a polypeptide selected from the group consisting of a functional portion of a Reverse Transcriptase polypeptide; and, a functional portion of an Integrase polypeptide; wherein said second nucleic acid segment does not encode a functional Gag polypeptide.

In other words, a trans-viral system is distinguishable from other viral vector systems in that the polypeptides encoding Reverse Transcriptase and Integrase are supplied in trans from at least one other DNA segment than the DNA segment encoding a functional Gag polypeptide. Consequently, the trans-viral vector system allows for a safer viral vector, in part, by diminishing the likelihood of generating replication competent retrovirus through genetic recombination. As used herein, a "trans-viral vector system" comprises a composition comprising at least the two nucleic acid segments described above. In one embodiment, the trans vector design encompasses a "trans-lenti viral vector." The "trans-lenti" viral vector design is characterized by expressing the Gag- Pro-Pol polyprotein in at least two parts: a first DNA segment that expresses Gag or Gag-Pro and at least a second DNA segment that expresses Reverse Transcriptase and/or Integrase. 'Trans-lenti" viral vector design is further characterized by the use of a Vpr and/or Vpx polypeptide or a functional equivalent thereof to target the Reverse Transcriptase and Integrase to the viral particle. In this design, the Vpr and/or Vpx polypeptides are used as vehicles to deliver functional Reverse Transcriptase and Integrase into the viral particle. Specifically, the trans-lenti viral vector design comprises: a) at least a first nucleic acid segment comprising a nucleotide sequence encoding at least a functional portion of a Gag polypeptide, and said first nucleic acid segment does not encode at least one of a functional Reverse Transcriptase polypeptide and a functional Integrase polypeptide; and, b) at least a second nucleic acid segment comprising at least one nucleotide sequence encoding a fusion protein selected from the group consisting of: i) a functional portion of a Vpr or a Vpx polypeptide and a functional portion of a Reverse Transcriptase polypeptide; and, ii) a functional portion of a Vpr or Vpx polypeptide and a functional portion of an Integrase polypeptide; wherein the functional portion of the Vpr or the Vpx polypeptide is capable of providing for the incorporation of the fusion protein into a viral particle and the second nucleic acid segment does not encode a functional Gag polypeptide; and, wherein the viral vector system produces an infectious viral particle. Further details regarding the design of the Vpr/Npx fusion proteins used in the trans-lenti viral vector design are outlined below.

In yet another embodiment, the trans-viral vector design encompasses a "trans-retroviral vector" design. As explained in further detail below, the "trans- retroviral" vector is characterized by expressing the Gag-Pro-Pol polyprotein in at least two parts: a first DΝA segment that expresses Gag or Gag-Pro and at least a second DΝA segment that expresses Reverse Transcriptase and/or Integrase. The "trans-retroviral" vector design is further characterized by the use of at least a fragment of the Gag polypeptide that is capable of being targeted to the viral particle, or a functional equivalent thereof, to target the Reverse Transcriptase and Integrase to the viral particle. Further details regarding the design of the trans-retro viral vector are outlined below.

One of skill will recognize that the trans- viral vector design can be used in viral vectors derived from any retroviral source. Accordingly, in specific embodiments of the present invention, a trans- viral vector is derived from any retrovirus. Such as, but not limited to, Moloney Leukemia Virus (MLV), Abelson murine leukemia virus, AKR (endogenous) murine leukemia virus, Avian carcinoma, Mill Hill virus 2, Avian Leukosis virus - RSA, Avian myeloblastosis virus, Avian myelocytomatosis virus 29, Bovine syncytial virus, Caprine arthritis encephalitis virus, Chick syncytial virus, Equine infectious anemia virus, Feline leukemia virus, Feline syncytial virus, Finkel-Biskis-Jinkins murine sarcoma virus, Friend murine leukemia virus, Fujinami sarcoma virus, Gardner- Arnstein feline sarcoma virus, Gibbon ape leukemia virus, Guinea pig type C oncovirus, Hardy-Zuckerman feline sarcoma virus, Harvey murine sarcoma virus, Human foamy virus, Human spumavirus, Human T-lymphotropic virus 1, Human T-lymphototropic virus 2, Jaagsiekte virus, Kirsten murine sarcoma virus, Langur virus, Mason-Pfizer monkey virus, Moloney murine sarcoma virus, Mouse mammary tumor virus, Ovine pulmonary adenocarcinoma virus, Porcine type C oncovirus, Reticuloendotheliosis virus, Rous sarcoma virus, Simian foamy virus, Simian sarcoma virus, Simian T- lymphotropic virus, Simian type D virus 1, Snyder-Theilen feline sarcoma virus, Squirrel monkey retrovirus, Trager duck spleen necrosis virus, UR2 sarcoma virus, Viper retrovirus, Visna/maedi virus, Woolly monkey sarcoma virus, and Y73 sarcoma virus human-, simian-, feline-, and bovine immunodeficiency viruses (HIV, SIV, FIV, BIV). See also, U.S. Patent Application No. 09/578,548.

In other embodiments of the present invention, a trans-viral vector (i.e., the trans-retroviral vector or the trans-lenti viral vector) is derived from a retrovirus, particularly a lentivirus. Lentiviral vectors are derived from viruses of the family Retroviridae and the subfamily lentivirinae. The Lentiviruses are associated with slow, progressive disease affecting the immune system (Coffin et al. (1997) Retroviruses, Cold Spring Harbor Laboratory Press, herein incorporated by reference) and are characterized by the ability to integrate into the genome of non-dividing cells. The lentiviruses include a variety of primate (e.g. human immunodeficiency viruses [HIV-1 and 2], and simian immunodeficiency viruses [SIV]) and non-primate viruses (e.g. maedi-visna virus [MW], feline immunodeficiency virus [FIN], equine infectious anemia virus [ELAN], caprine arthritis encephalitis virus [CAEV] and bovine immunodeficiency virus [BIN] viruses. For a review, see for example, Romano et al. (2000) Stem Cells 75:19-39. In addition, one of skill in the art will recognize that the trans-viral vector system used in the method of the present invention is "replication defective". By "replication defective" is intended the viral vector viral particle is unable to reconstitute a complete viral particle in the target cell and consequently, is unable to multiply and spread to other cells.

The trans-viral vector used in the methods of the invention is "infectious". By "infectious" is intended the viral particle is able to gain entry into the target cell. In other embodiments the trans-viral vector used in the methods of the invention is capable of "transducing" the target cell. By "transducing" is intended the viral vector gains entry into the target cell and integrates the gene transfer vector into the genome of the target cell.

A complete description of the "trans" viral vector design, the "trans-lenti" viral vector design, and the "trans-retroviral" vector design and the viral vectors systems used to produce these viral particles has been described in detail in U.S. Patent Application Nos. 09/089,900; 09/709,751; 09/460,548; U.S. Patent No. 6,001,985; PCT Patent Application No. PCT/USOO/18597, in Wu et al. (1997) EMBO 7(5:5113-5122; all of which are herein incorporated by reference. A non-limiting illustration of the trans- viral vector system used in the methods of the present invention is provided in Figures 1-3. In these examples, the trans- vector system comprises the following components: an env construct, a packaging construct, a trans-enzyme construct, and a retroviral gene transfer vector. The "packaging construct" of the trans-viral system comprises a nucleotide sequence encoding Gag/Pro (represented as boxed structures in Figures 1-3, while the nucleotide sequences encoding Reverse Transcriptase (RT) and/or Integrase (IN) have been either deleted completely from the construct or disrupted in some manner that prevents the expression of a functional polypeptide. The nucleotide sequences encoding the Reverse Transcriptase and Integrase polypeptides are provided in trans to the packaging construct on a stretch of DNA referred to herein as the "trans- enzyme construct". The viral expression system thereby disarms the Gag-Pro-Pol structure by splitting Gag-Pro from the nucleotide sequences encoding Reverse Transcriptase and Integrase.

The trans-viral vectors produced by the trans-viral system can be distinguished physically from viral vectors that use a three-vector system where the Gag/Pol is expressed as a polyprotein. See, for example, Wu et al. (1997) EMBO J. 76:5113-5122 and Wu et al. (2000) Mol. Therapy 7:47-55, which provide assays to identify uncleaved Vpr/Vpx fusion proteins in the trans-virus particles and assays that measure a reduced level of genetic recombination in the trans-viral vector when compared to the three vector lenti viral vectors.

As used herein "nucleic acid sequences" will sometimes be used as a generic term encompassing both DNA and RNA fragments. As the materials of the invention include modified retroviral genomes and their proviral counterparts, particular functional sequences referred to will occur both in RNA and DNA form. The corresponding loci will be referred to interchangeably for their occurrences in both DNA and RNA. For example, the xp packaging signal functions in the retroviral RNA genome as a packaging signal; however, the corresponding sequences occur in the proviral DNA. Similarly, promoter, enhancer, and terminator sequences occur, though in slightly different forms, in both the genomic RNA and proviral DNA forms. The interchangeability of these functionalities in the various phases of the viral life cycle is understood by those in the art, and accordingly, rather loose terminology in regard to DNA or RNA status is often used in referring to them. Specifically, sequences specified by a progression of bases should be understood to include these specific sequences and their complements, both in DNA and RNA forms.

While a description of the various elements of the trans-viral vector system, including, for example, the components used on the gene transfer vector, the packaging construct, the envelope construct, and the trans-enzyme construct of the trans -viral vector system are provided below, it is recognized that one of skill in the art can readily generate "functionally equivalent" constructs. By "functionally equivalent" construct is intended each DNA construct (i.e., the packaging construct, the gene transfer vector, the envelope construct, and the trans-enzyme construct) have substantially the same function as the specific vector constructions illustrated herein. It is further recognized that the genetic elements in the various vectors of the trans- viral vector system may be from any source (i.e., viral, cellular, or synthetic) including, for example, a retrovirus, and more particularly a lentiviral source.

Examples and assays for the functional equivalence of the various components of the trans-vector system are described more fully below. While, Table 1 provides a reference for various genetic elements of the HIN-1 genome and is based on ΝCBI Genbank Accession Number AF033819, it is recognized that sequences from other retroviruses and/or lentiviruses are known in the art and can be used to construct functionally equivalent vectors and vector systems directed to a given host species of animal. A more detailed explanation of the components outlined in Table 1 and their function may be found in Coffin et al. (1997) Retroviruses, Cold Spring Harbor Laboratory Press, New York, herein incorporated by reference. Moreover, those of skill will appreciate that allelic variations in the various genetic elements exist between different isolates of the viruses and such variants may be used in the constructs of the present invention. For instance, such viral isolates are described in Li et al. (1992) J. Virol. (56:6587; Ghosh et al. (1993) Virology 794:858; and, U.S. Patent No. 5,869,313; all of which are herein incorporated by reference.

TABLE 1

Genetic Elements and Coordinates of a Human HIV-1 Isolate

Genetic Element Coordinates

R: (1-96)

U5: (97-181)

PBS: (182-199) gag: (336-1836) pro: (1637-2099) pol: (2102-4640) vif: (4587-5163) vpr: (5105-5339) tat: (5377-5591, 7925-7968) rev: (5516-5591, 7925-8197) vpu: (5608-5854) env: (5771-8339) nef. (8343-8710)

PPT: (8615-8630)

U3: (8631-9085)

R: (9086-9181)

Functionally equivalent sequences of the present invention also encompass various fragments of a retroviral genome or any other organism that retain substantially the same function as the respective native sequence. Such fragments will comprise at least about 10, 15 contiguous nucleotides, at least about 20 contiguous nucleotides, at least about 24, 50, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 340, 360, 380, or up to the entire contiguous nucleotides of the specific genetic element of interest. Such fragments may be obtained by use of restriction enzymes to cleave the native viral genome; by synthesizing a nucleotide sequence from the native nucleotide sequence of the virus genome; or may be obtained through the use of PCR technology. See particularly Mullis et al. (1987) Methods Enzymol. 755:335-350, and Erlich, ed. (1989) PCR Technology (Stockton Press, New York). Again, variants of the various vector components, such as those resulting from site-directed mutagenesis, are encompassed by the methods of the present invention. As described in more detail below, methods are available in the art for determining functional equivalence.

By "variant" is intended substantially similar sequences. Thus, for nucleotide sequences or amino acid sequences, variants include sequences that are functionally equivalent to the various components of the trans-viral vector system (i.e., the IRES element of the DNA expression cassette). Variant nucleotide sequences also include synthetically derived nucleotide sequences that have been generated, for example, by site directed mutagenesis but which still retain the function of the native sequence. Generally, nucleotide sequence variants or amino acid sequence variants of the invention will have at least 70%, generally 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%o, 91%, 98%), or 99% sequence identity to its respective native nucleotide sequence.

Variants of the nucleotide sequences can encode amino acid sequences that differ conservatively because of the degeneracy of the genetic code. These naturally occurring allelic variants can be identified with the use of well-known molecular biology techniques, such as polymerase chain reaction (PCR) and hybridization techniques. Variant nucleotide sequences also include synthetically derived nucleotide sequences that have been generated, for example, by using site-directed mutagenesis, but which still remain functionally equivalent. With respect to the amino acid sequences for the various full length or mature polypeptides used in the viral vector system used in the present invention, variants include those polypeptides that are derived from the native polypeptides by deletion (so-called truncation) or addition of one or more amino acids to the N-terminal and/or C-terminal end of the native polypeptide; deletion or addition of one or more amino acids at one or more sites in the native polypeptide; or substitution of one or more amino acids at one or more sites in the native polypeptide. Such variants may result from, for example, genetic polymorphism or from human manipulation. Methods for such manipulations are generally known in the art.

For example, amino acid sequence variants of a polypeptide can be prepared by mutations in the cloned DNA sequence encoding the specific vector element of interest. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York); Kunkel (1985) Proc. Natl. Acad. Sci. USA 52:488-492; Kunkel et al. (1987) Methods Enzymol. 154:361-382; Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York); U.S. Patent No. 4,873,192; and the references cited therein; herein incorporated by reference. Guidance as to appropriate amino acid substitutions that may not affect biological activity of the various vector polypeptide may be found in the model of Dayhoff et al. (1978) Atlas of Polypeptide Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.), herein incorporated by reference. Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be preferred. Examples of conservative substitutions include, but are not limited to, Gly<=> Ala, Nal«Ile<= Leu, Asp»Glu, Lys<= Arg, Asn<s»Gln, and Phe »Trp«>Tyr.

A variant of a native nucleotide sequence or native polypeptide has substantial identity to the native sequence or native polypeptide. A variant may differ by as few as 1 to 10 amino acid residues, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue. A variant of a nucleotide sequence may differ by as low as 1 to 30 nucleotides, such as 6 to 20, as low as 5, as few as 4, 3, 2, or even 1 nucleotide residue.

By "sequence identity" is intended the same nucleotides or amino acid residues are found within the variant sequence and a reference sequence when a specified, contiguous segment of the nucleotide sequence or amino acid sequence of the variant is aligned and compared to the nucleotide sequence or amino acid sequence of the reference sequence. Methods for sequence alignment and for determining identity between sequences are well known in the art. With respect to optimal alignment of two nucleotide sequences, the contiguous segment of the variant nucleotide sequence may have additional nucleotides or deleted nucleotides with respect to the reference nucleotide sequence. Likewise, for purposes of optimal alignment of two amino acid sequences, the contiguous segment of the variant amino acid sequence may have additional amino acid residues or deleted amino acid residues with respect to the reference amino acid sequence. The contiguous segment used for comparison to the reference nucleotide sequence or reference amino acid sequence will comprise at least 20 contiguous nucleotides, or amino acid residues, and may be 30, 40, 50, 100, or more nucleotides or amino acid residues. Corrections for increased sequence identity associated with inclusion of gaps in the variant's nucleotide sequence or amino acid sequence can be made by assigning gap penalties. Methods of sequence alignment are well known in the art.

The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, percent identity of an amino acid sequence can be determined using the Smith- Waterman homo logy search algorithm using an affine 6 gap search with a gap open penalty of 12 and a gap extension penalty of 2, BLOSUM matrix 62. Alternatively, percent identity of a nucleotide sequence is determined using the Smith- Waterman homology search algorithm using a gap open penalty of 25 and a gap extension penalty of 5. Such a determination of sequence identity can be performed using, for example, the

DeCypher Hardware Accelerator from TimeLogic Version G. The Smith- Waterman homology search algorithm is taught in Smith and Waterman (1981) Adv. Appl. Math 2:482-489, herein incorporated by reference. Alternatively, the alignment program GCG Gap (Wisconsin Genetic Computing Group, Suite Version 10.1) using the default parameters may be used. The GCG Gap program applies the Needleman and Wunch algorithm and for the alignment of nucleotide sequences with an open gap penalty of 3 and an extend gap penalty of 1 may be used. Another preferred, non- limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA

90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990) J. Mol. Biol. 275:403. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12, to obtain nucleotide sequences having sufficient sequence identity. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3, to obtain amino acid sequences having sufficient sequence identity. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov. Another preferred, non- limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller (1988) CABIOS 4:11- 17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

Within preferred embodiments of the invention, the trans-enzyme construct, the env construct, the gene transfer vector, and the packaging construct described below are contained on one or more vectors or plasmids. The plasmid may contain a bacterial origin of replication, one or more selectable markers, a signal that allows the plasmid construct to exist single stranded (i.e., a Ml 3 origin of replication), a multiple cloning site, and a "mammalian" origin of replication (i.e., a SV40 or adeno virus origin of replication). Such vectors are known in the art.

A. Gene Transfer Vector As noted above, the present invention combines a trans-viral vector system with a gene transfer vector having at least one DNA expression cassette comprising multiple open reading frames separated by an IRES or an active fragment or variant thereof.

As used herein the "gene transfer vector" refers to a nucleotide sequence that has the necessary "cis acting" components that allow for the transcription of the gene transfer vector; encapsudation of the gene transfer vector mRNA (i.e., modified proviral genome) into the viral particle; reverse transcription of the gene transfer vector mRNA; and integration of the gene transfer vector into the genome of the target cell.

Cis acting nucleic acids sequences that carry out the functions described above are well know in the art. For instance, a gene transfer vector can comprise the following components: a 5' LTR; a packaging signal; a Rev Responsive Element (RRE); and a 3' LTR or any functional variant or derivative of each of these elements. In the methods and compositions of the invention, the gene transfer vector further comprises at least one DNA expression cassette comprising multiple open reading frames separated by an IRES or a functional variant or fragment thereof. Each of these components is described in more detail below.

The 5' and 3' LTR sequences flank the other elements of the gene transfer vector. The LTR sequences contain multiple elements including, for example, promoter/enhancer elements along with other cts-acting sequence elements important for integration and integration of the proviral genome into the genome of the target cell. Various c/s-acting elements of the LTR include, for example, the U5 region (nt 97-181 of GenBank Accession No. AF033819) and the U3 region (nt 8631-9085 of GenBank Accession No. AF033819) which comprises viral promoter and enhancer sequences that direct the expression of the retroviral gene transfer vector into a single precursor mRNA. Other LTR components include the R region which comprises sequences required for RNA transcription initiation (i.e., the transactivating region (TAR)), the polyadenylation signals (nt 1-96 of GenBank Accession No. AF033819). A more complete description of LTR sequences and functional variants for the HIV-1 virus can be found in Pereira et al. (2000) Nucleic Acid Research 25:663-668, herein incorporated by reference. One of skill in the art will recognized that the various components of the gene transfer vector can be arranged in any order as long as they are internal to the 5' and 3' LTR elements. The transfer vector can further comprise tRNA primer binding site sequences (nt 182-199 of GenBank Accession No. AF033819 which functions in the initiation of reverse transcription. Such alterations are known to one of skill in the art. It is further recognized that modifications to the LTRs can be made, such as those of the self-inactivating (SIN) vectors. Such alterations are known to one of skill in the art.

As used herein the "packaging signal" or "ψ signal" refers to a nucleic acid sequence that is required in cis for the encapsudation of the viral RNA into the viral particle. The packaging signal used in the methods of the present invention may be a minimal packaging signal required for encapsudation of the gene transfer vector into the viral particle. This minimal packaging sequence for the preferred retroviral gene transfer vector of the present invention will be sufficient to direct the incorporation of the modified proviral genome (i.e., gene transfer vector) into the viral particle.

It is recognized that variants or fragments of known packaging signals may be used in the methods of the present invention so long as the variants direct the encapsudation of the retroviral gene transfer vector into the viral particle. For instance, extended packaging signals which encompass sequences surrounding the minimal packaging sequence may increase the efficiency of encapsudation of the gene transfer vector in the viral particle. The HTV packaging signal has been further characterized in Mcbirde et al. (1997) J. Virol. 77:4544-4554, which is herein incorporated by reference.

The gene transfer vector may also contain a Rev Responsive Element (RRE). The presence of this element allows Rev to direct the nuclear export of the RRE- containing mRNAs. The sequence of the RRE and functional variants thereof are known in the art. See, for example, Berchtold et al. (1995) Virology 277:285-289; Dillon et al. (1990) J. Virol. 64:4428-4431; Le et al. (1990) Nucleic Acid Research 75: 1613-1623; all of which are herein incorporated by reference. It is further recognized that Rev/RRE can be substituted with other elements, including for example, the c/s-acting 219-nucleotide constitutive transport element (CTE) from the Mason-Pfizer monkey virus (MPMV) that has been shown to allow Rev-independent HIV-1 replication. See, for example, Bray et al. (1994) Proc. Natl. Acad Sci USA 97:1256-1260. The gene transfer vector may further contain at least one DNA construct comprising a selectable marker operably linked to a promoter. Details regarding the use of selectable markers are describe more fully below. One of skill will appreciate that numerous possibilities exist. It is further recognized that the promoter selected for the expression of selectable marker will vary depending on if the marker is being used to monitor incorporation of the gene transfer vector into the packaging cell line or into the genome of the target cell. 7. The DNA Expression Cassette

The gene transfer vector further comprises a DNA expression cassette. The DNA expression cassette comprises a promoter that is active in the target cell operably linked to a first nucleotide sequence encoding a first a polypeptide operably linked to an IRES or an active fragment or variant thereof, operably linked to a second nucleotide sequence encoding a second polypeptide. Thus, the DNA expression cassette contained in the gene transfer vector, when introduced into the target cell via the viral particle, is expressed as a multicistronic message. By "operably linked" is intended the individual nucleotide sequences are joined such that expression of the nucleotide sequences contained within the expression cassette are under the regulatory control of the 5' and 3' regulatory sequences of the DNA expression cassette. When one of the nucleotide sequences encodes a polypeptide, "operably linked" further encompasses the joining of the nucleotide sequences such that expression of the coding sequences occurs in the proper reading frame. Similarly, an "operably linked" IRES element permits the translation of the downstream open reading frame. It is recognized that "operably linked" elements need not be contiguous with one another so long as the elements are able to carryout their desired function. For example, a promoter need not be contiguous with a coding sequence so long as it functions to direct expression of the sequences.

As used herein, an "internal ribosomal entry site" or "IRES" is a cis acting nucleic acid element that mediates the internal entry of ribosomes on an RNA molecule and thereby regulates translation in eukaryotic systems. In the methods and compositions of the present invention, the IRES element or an active variant or fragment thereof is contained in a DNA expression cassette and permits the translation of two or more open reading frames from a single messenger RNA. Such constructs having two open reading frames joined in this fashion are referred to in the art as "dicistronic" or "bicistronic." See, for example, Kaufman et α/.(1991) Nuc. Acids Res. 79:4485-4490; Gurtu et al. (1996) Biochem. Biophys. Res. Comm.

229:295-298; Rees et al. (1996) BioTechniques 20:102-110; Kobayashi et al. (1996) BioTechniques 27:399-402; and Mosser et al. (1997) BioTechniques 22:150-161; all of which are herein incorporated by reference. Many IRES elements have been identified in both viral and eukaryotic genomes. In addition, synthetic IRES elements have also been developed. For example, IRES elements have been found in a variety of viruses including members of the genus Enterovirus (e.g. human poliovirus 1 (Ishii et al. (1998) J Virol. 72:2398- 405 and Shiroki et al. (1997) J. Virol. 77:1-8), human Coxsackievirus B); Rhinovirus (e.g., human rhinovirus); Hepatovirus (Hepatitis A virus); Cardiovirus (Encephalomyocarditis virus ECMV (nucleotides 2137-2752 of GenBank Accession No. AB041927 and Kim et al. (1992) Mol Cell Biology 72:3636-43) and Etheirler's encephalomyelitis virus); Aphtovirus (Foot- and mouth disease virus (nucleotides 600-1058 of GenBank Accession No. AF308157; Belsham et al. (1990) EMBO 77:1105-10; Poyry et al. (2001) RNA 7:647-60; and Stoneley et al. (2000) Nucleic Acid Research 25:687-94), equine rhinitis A virus, Ewuine rhinitis B); Pestivirus (e.g., Bovine viral diarrhea virus (Poole et al. (1995) Virology 206:150-154) and Classical swine fever virus (Rijnbrand et al. (1997) J. Virol 77:451-7); Hepacivirus (e.g., Hepatitis C virus (Tsukiyama-Kohara et al. (1992) J. Virol. 66:1476-1483, Lemon et al. (1997) Semin. Virol. 5:274-288, and nucleotide 1201-1812 of GenBank Accession No. AJ242654.) and GB virus B). Each of these references is herein incorporated herein by reference.

IRES elements have also been found in viruses from the family Retroviridae, including members of the Lentivirus family (e.g., Simian immunodeficiency virus (Ohlmann et al. (2000) Journal of Biological Chemistry 275:11899-906) and human immunodeficiency virus 1 (Buck et s/. (2001) J Virol. 75:181-91); the BLV-HTLV retroviruses (e.g., Human T-lymphotrophic virus type 1 (Attal et al. (1996) EEES Letters 392:220-4); and the Mammalian type C retoviral family (e.g., Moloney murine leukemia virus (Vagner et al. (1995) J. Biol. Chem 270:20316-83), Friend murine leukemia virus, Harvey murine sarcoma virus, Avian retriculoendotheliosis virus (Lopez-Lastra et al. (1997) Hum. Gene Ther 5:1855-65), Murine leukemia virus (env RNA) (Deffaud et al. (2000) J. Virol. 74:846-50), Rous sarcoma virus (Deffaud et al. (2000) J. Virol. 74:11581-8). Each of these references is herein incorporated by reference.

Eukaryotic mRNAs also contain IRES elements including, for example, BiP (Macejak et al. (1991) Nature 355:91); Antennapedia of Drosophilia (exons d and e) (Oh et al. (1992) Genes and Development 6:1643-1653; c-myc; and, the X-linked inhibitor of apoptosis (XIAP) gene (U.S. Patent No. 6,171,821).

Various synthetic IRES elements have been generated. See, for example, De Gregorio et al. (1999) EMBO J. 75:4865-74; Owens et al. (2001) PNAS 4:1471-6; and Venkatesan et al. (2001) Molecular and Cellular Biology 21 :2826-37. For additional IRES elements known in the art, see, for example, www.rangueil.inserm.fr/IRESdatabase.

In a specific embodiment, the IRES sequence is derived from the encephalomyocarditis virus (ECMV). It is further recognized that an IRES used in the methods and compositions of the invention may be a variant or a fragment of a naturally occurring sequence or even a synthetic sequence. By an "active variant or fragment of an IRES" is intended a cis- acting element that retains the activity of an IRES (i.e., mediates the internal entry of ribosomes on an RNA molecule and thereby regulates translation in eukaryotic systems). Assays for measuring such activity are known in the art. For example, a bicistronic DNA construct can be constructed wherein a segment of the IRES variant to be tested is inserted between two open reading frames. The construct is then transcribed and translated, either in vivo or in vitro. If a functional IRES element is present, translation of the downstream open reading frame will occur independent of the cap-mediated translation of the first open reading frame. See, for example, Sachs et al. (2000) Cell 101:243-245 and Kozak et al. (2001) Mol. Cell. Biol. 27:1899-1907. Additional assays for IRES activity include using a bicistronic plasmids encoding the enhanced blue and green fluorescent proteins (EBFP and EGFP) separated by a potential IRES element. The construct is delivered into mammalian cells. Cells that received a functional IRES element can be isolated using the EBFP and EGFP reporters and fluorescence-activated cell sorting. See, for example, Venkatesan et al. (2001) Molecular and Cellular Biology 27:2826-37.

In one embodiment of the present invention, one of the nucleotide sequences of the DNA expression cassette encodes a selectable marker, while the other nucleotide sequence encodes a polypeptide of interest. The nucleotide sequence encoding the selectable marker can be either the first or the second nucleotide sequence in the DNA expression cassette. One of skill in the art will recognize that nucleotide sequences expressed in the DNA expression cassette and their order in the DNA expression cassette will vary depending on the desired outcome. Illustrative examples of the variations on the DNA expression cassette that can be used in the methods and compositions of the present invention are provided below in the section related to methods of expressing a sequence of interest. As used herein a "selectable marker" is a nucleotide sequence that confers a phenotype on a cell expressing the marker, such that the cell can be identified under appropriate conditions. Generally, a selectable marker allows for the selection of transduced cells based on their ability to thrive in the presence or absence of a chemical or other agent that inhibits an essential cell function. Suitable markers, therefore, include genes coding for proteins which confer drug resistance or sensitivity thereto, impart color to, or change the antigenic characteristics of those cells transfected with a nucleic acid element containing the selectable marker when the cells are grown in an appropriate selective medium.

For example, selectable markers useful in the methods and compositions of the present invention include, cytotoxic markers and drug resistance markers, whereby cells are selected by their ability to grow on media containing one or more of the cytotoxins or drugs; auxotrophic markers by which cells are selected by their ability to grow on defined media with or without particular nutrients or supplements, such as thymidine and hypoxanthine; metabolic markers by which cells are selected for, e.g., their ability to grow on defined media containing the appropriate sugar as the sole carbon source, or markers which confer the ability of cells to form colored colonies on chromogenic substrates or cause cells to fluoresce.

Selectable markers which impart resistance to a cytotoxic agent to the transformed target cell are useful in the methods and compositions of the present invention. A selectable marker used in the methods of present invention when expressed in a cell confers resistance to a cytotoxic agent on a transduced cell. As used herein, by "confers resistance to a cytotoxic agent" is intended expression of the selectable marker results in the attenuated resistance to the appropriate agent as compared to cells that are either not expressing the marker or are expressing the marker at lower levels and thus have a weaker resistance conferred by the selectable marker.

The selectable markers that can be used in the methods and compositions of the invention include, but are not limited to, the nucleotide sequence encoding Adenosine deaminase (confers resistance to Adenosine, alaosine, and 2'- deoxycoformycine); Adenylate deaminase (confers resistance to Adenine, azaserine, and coformycine); Asparagine synthetase (confers resistance to -aspartyl hydroxamate or albizzin); Aspartate transcarbamolyase (confer resistance to PALA); Dihydrofolate reductase (confers resistance to Methotrexate); Glutamine synthetase (confers resistance to Methionine sulfoximine); Metallothionein (confers resistance to Cadmium). Additional selectable markers are known in the art. See, for example, Ausubel et al. (1998) Current Protocols in Molecular Biology John Wiley & Sons, Inc. which is herein incorporated by reference. In another embodiment, the selectable marker confers resistance to neomycin and neomycin analogues such as geneticin, hygromycin, and the like. For example, the gene encoding aminoglycoside-phosphotranferase (APH) allows selection in mammalian cells by conferring resistance to neomycin. Several mutations to this gene have been described which impart resistance to the neomycin analogue geneticin (G418) (available from Sigma, St. Louis, Mo.). For example, an APH with aspartic acid at position 261 replaced by an asparagine, confers reduced resistance to gentamicin. See, e.g., Blazquez et al. (1991) Molec. Micro. 5:1511-1518. Alternatively, the selectable marker can confer resistance to puryomycin. For example, expression of the Pac gene which encodes a puromycin N-acetyl-tranferase (PAC) is capable of conferring resistance to puromycin (De La Luna et al. (1992)

Methods In Enzymology 276:376-385; Vara et al. (1985) Biochemistry 24:8074-8081; and Lacalle et al. (1989) Gene 79:375-380.

Additional selectable markers include the Sh ble gene which confers resistance to antibiotics in the bleomycin family, and the bsd gene from Aspergillus terreus, which confers resistance to the nucleoside antibiotic blasticidin S HC1.

Other selectable markers useful in the methods and compositions of the invention include cell surface markers such as alkaline phosphatase, nerve growth factor receptor, or any other suitable membrane-associated moiety. Representative examples of such markers and associated prodrug molecules include alkaline phosphatase and various toxic phosphorylated compounds such as phenolmustard phosphate, doxorubicin phosphate, mitomycin phosphate and etoposide phosphate; β- galactosidase and N-[4-( β -D-galactopyranosyl) benyloxycarbonyl]-daunorubicin; azoreductase and azobenzene mustards; β -glucosidase and amygdalin; β - glucuronidase and phenolmustard-glucuronide and epirubicin-glucuronide; carboxypeptidase A and methotrexate-alanine; cytochrome P450 and cyclophosphamide or ifosfamide; DT diaphorase and 5-(aziridine-l-yl)- 2,4,dinitrobenzamide (CB1954) (Cobb et al. (1969) Biochem. Pharmacol 75:1519, Knox et al. (1993) Cancer Metastasis Rev. 72:195); β -glutamyl transferase and β- glutamyl p-phenylenediamine mustard; nitroreductase and CB1954 or derivatives of 4-nitrobenzyloxycarbonyl; glucose oxidase and glucose; xanthine oxidase and hypoxanthine; and plasmin and peptidyl-p-phenylenediamine-mustard. Nonimmunogenic markers may also be made by expressing an enzyme in a compartment of the cell where it is not normally expressed.

Still other suitable selectable markers are genes which impart color to those cells transfected with a nucleic acid element expressing the selectable marker such that detection can be achieved by virtue of a color change (either visible or fluorescent). For example, the gene encoding Green Fluorescent Protein (GFP) may be used as the selectable marker, as can derivatives thereof such as Enhanced Green Fluorescent Protein (EGFP), and like molecules. See, for example, Venkatesan et al. (2001) Molecular and Cellular Biology 27:2826-37.

One of skill in the art will be apprised of additional selectable markers that can be used in the methods and compositions of the invention. See, for example, Ausubel et al. (1998) Current Protocols in Molecular Biology John Wiley & Sons, Inc. which is herein incorporated by reference.

As described above, the DNA expression cassette can include in the 5 '-3' direction of transcription, a transcriptional and translational initiation region, a first nucleotide sequence, an IRES, a second nucleotide sequence encoding a polypeptide and a transcriptional and translational termination region functional in the targeted host cell. The transcriptional initiation region, the promoter, may be native or foreign to the target cell. Additionally, the promoter may be the natural sequence or, alternatively, a synthetic sequence. By "foreign" is intended that the transcriptional initiation region is not found in the target cell into which the trans-viral vector is introduced. While it may be preferable to express the sequences using heterologous promoters, the native promoter sequence may be used. The termination region may be native with the transcriptional initiation region, may be native with the operably linked DNA sequence of interest, or may be derived from another source. Any promoter may be operably linked to the first nucleotide sequence of the DNA expression cassette so long as the promoter is active in the target cell. Such promoters may be constitutive promoters (i.e., Beta actin promoter (Balling et al. (1989) Cell 55:337-347 and Beddington et al. (1989) Development 105:133-131, the metallothionein promoter (Palmiter et al. (1983) Science 222:809-814 and Iwamoto et al. (1991) EMBO J. 10:3161-3115, the HMGCR promoter (Mehtali et al. (1990) Gene 97:179-184 and Tarn et al. (1992) Development 775:703-715, the histone H4 promoter (Choi et al (1991) Mol. Cell. Biol. 77:3070-3074); the SV40 early promoter, a CMV promoter, such as the CMV immediate early promoter, the mouse mammary tumor virus promoter, the adenovirus major late promoter, the herpes simplex virus promoter, and the proximal promoter for the human elongation factor 1 alpha (EFlα) gene. Additional promoter or enhancer elements can be found, for example, in the eukaryotic promoter database and in U.S. Patent No. 6,271,436, herein incorporated by reference. Alternatively, the promoter may be conditionally active. By a "conditional" promoter is intended the promoter is silent (or shows a reduce expression) until specifically activated. The promoter may be activated by an experimental manipulation, such as the administration of a drug or other activating agent, or alternatively, the promoter may be activated at a specific developmental stage or in a specific tissue. Conditional promoters include, but are not limited to, promoters that are regulated by heavy-metal ions, heat shock, growth factors, steroid hormones, or various synthetic promoters and inducible activators that often contain cis and trans elements derived from bacteria or yeast. Examples of conditional promoters include, for example, the tetracycline-responsive promoter. See, for example, Ausubel et al. (1998) Current Protocols in Molecular Biology, John Wiley & Sons, Inc.

It is further recognized that the DNA expression cassette may contain various sequences that facilitate the expression, stabilization, and/or localization of the sequences contained in the cassette and/or the resulting gene product. Such sequences include enhancers, introns, and post-transcriptional elements such as the Woodchuck Hepatitis Virus post-transcriptional region (WPRE) or functional variants and fragments thereof and the PPT-CTS or functional variants and fragments thereof. See, for example, Zufferey et al. (1999) J. Virol. 73.2886-2892 and U.S. Patent Application No. 09/709,751, filed November 10, 2000, both of which are herein incorporated by reference.

Enhancer elements may also be used in association with the promoter to increase expression levels. Such elements include, but are not limited to, the SV40 enhancer (Dijkema et al. (1985) EMBO J. 4:761), the human CMV enhancer element (Boshart et al. (1985) Cell 37:521); and, the enhancer element from the LTR of the Rous Sarcoma Virus (Gorman et al. (1982) Proc. Natl. Acad. Sci. USA 79:6111). In yet other embodiments, the DNA construct comprising the nucleotide sequence of interest further includes affinity tags for purification or labeling (e.g., with antibodies).

The DNA expression cassette can further comprise transcription termination and polyadenylation sequences. For instance, transcription termination and polyadenylation sequences can be present on the second nucleotide sequence in the DNA expression cassette. Specifically, the terminator and poly A sequence can be located 3' to the translation stop codon for the second polypeptide. Examples of transcription terminator/polyadenylation signals include, but are not limited to, those derived from SV40, as described in Sambrook et al, supra, as well as a bovine growth hormone terminator sequence.

In preparing the DNA expression cassette, the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the DNA fragments. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved. Figure 4 illustrates a non-limiting example of a gene transfer vector which can be used in the methods and compositions of the present invention. The construct illustrated comprises the following operably linked components: an LTR; a packaging signal; an RRE; a central polypurine tract (cts/ppt); a CMV promoter; a cDNA of interest: an IRES; a sequence conferring puromycine resistance; a WPRE; and an LTR. B. Trans-Enzyme Construct

The trans-enzyme construct contains the nucleotide sequences encoding Reverse Transcriptase and Integrase apart from their native configuration. In particular, the trans-enzyme construct encodes a fusion protein comprising a first polypeptide characterized by the ability to be targeted to a viral particle, operably linked to functional Reverse Transcriptase and/or Integrase polypeptide. For instance, an HIV virion-associated accessory protein (Vpr or Vpx) or a variant or fragment thereof can be used as a vehicle to deliver polypeptides having integrase activity and reverse transcriptase activity into the trans-viral vector particle. This "trans-lenti" viral vector design is illustrated in Figure 2. Specifically, the trans-enzyme construct may comprise a nucleic acid sequence encoding a fusion protein comprising a Vpr or Vpx polypeptide or functional variant or fragment thereof, fused in frame to at least one heterologous polypeptide comprising Integrase and/or Reverse Transcriptase or functional variants and fragments thereof. Such trans-enzyme constructs are know in the art. See, for example, Liu et al. (1997) J. Virol. 77:7704-7710, Wu et al. (1997) EMBO J. 76:5113-5122; U.S. Patent No. 6,001,985; U.S. Patent Application 09/089,900 filed June 3, 1998; and, U.S. Application No. 09/460,548 filed December 14, 1999; all of which are herein incorporated by reference.

As used herein, by "fusion protein" is intended a polypeptide having at least two heterologous polypeptide sequences joined for in-frame expression. That is, the nucleotide sequences encoding the heterologous polypeptides will be translated into a single translation product. In one embodiment, the fusion protein encoded by the trans-enzyme construct comprises a Vpr or a Vpx polypeptide or a functional fragment or variant thereof, while the second polypeptide comprises a polypeptide having integrase or reverse transcriptase activity. In other embodiments of the present invention, the fusion protein encoded by the trans-enzyme construct comprises nucleotide sequences encoding all three polypeptides (i.e., Vpr/Vpx, Reverse Transcriptase, and Integrase).

Sequences encoding Vpr and Vpx polypeptides are known in the art. See, for example, U.S. Patent No. 5,861,161, herein incorporated by reference. Fragments and variants of a Vpr or Vpx polypeptide can be used and will retain the ability to be incorporated into virion particles. Examples of fragments and variants of the Vpr/Vpx polypeptides that retain this activity are known. See, for example, Paxton et al. (1993) J Virol. 67:7229-7237 and U.S. Patent No. 6,043,081, both of which are herein incorporated by reference. In addition, assays to determine if a Vpr or Vpx polypeptide are incorporated into a virion are routine in the art. Briefly, a fragment or variant of a Vpr/Vpx polypeptide fused to a marker polypeptide is expressed in a packaging cell line capable of producing viral particles. The cell line is cultured and viral particles are produced. The viral particles are isolated and assayed for the presence of the marker protein. See, for example, Paxton et al. (1993) J. Virol. 67:7229-7237.

Another embodiment of the trans-vector design is illustrated in figure 3. The design in this particular non-limited example is referred to herein as the trans- reto viral vector design which uses a fragment of the Gag polypeptide which retains the ability to be targeted to the viral particle to deliver the polypeptides having integrase activity and reverse transcriptase activity into the trans-viral vector particle. Specifically, in this embodiment, the trans-enzyme construct may comprise a nucleic acid sequence encoding a fusion protein comprising a fragment of a Gag polypeptide which retains the ability to be targeted to the viral particle, fused in frame to at least one heterologous polypeptide comprising Integrase and/or Reverse Transcriptase or functional variants and fragments thereof. Such trans-enzyme constructs are known in the art. See, for example, U.S. Patent Application No. 09/578,548, filed December 14, 1999.

In one embodiment, the fusion protein encoded by the trans-enzyme construct comprises a fragment of a Gag polypeptide that retains the ability to be targeted to the viral particle, while the second polypeptide comprises a polypeptide having integrase or reverse transcriptase activity. In other embodiments of the present invention, the fusion protein encoded by the trans-enzyme construct comprises nucleotide sequences encoding all three polypeptides (i.e., a fragment of Gag that retains the ability to be targeted to the viral particle:Reverse Transcriptase: Integrase).

Sequences encoding Gag polypeptides are known in the art. Fragments and variants of the Gag polypeptide will retain the ability to be incorporated into virion particles. Examples of fragments and variants of the Gag polypeptides that retain this activity are known. See, for example, U.S. Patent No. 09/460,548.

Nucleotide sequences encoding Integrase polypeptides are also known (see Table 1). Integrase is involved in many aspects of the viral life cycle. For instance, Integrase has been shown to be involved in various steps of the virion assembly and maturation process and is involved in reverse transcription and integration of the viral genome in the host cell (Lie et al. (1999) J. Virol. 73:8831-8836 and Craigie et al. (2001) J. Bio. Chem. Manuscript R100027200). The Integrase polypeptide used in the methods of the present invention may be from any viral source, particularly a retrovirus, particularly a lentiviral source.

The Integrase polypeptide or variants or fragments thereof will have integrase activity. By integrase activity is intended the polypeptide retains sufficient activity to support the production of a viral particle (i.e., supports virus assembly, maturation, and/or integration). The regions of the Integrase polypeptide that influence virus assembly, maturation, and integration are known in the art, as are assays to determine these functions. For instance, mutations in the catalytic center of Integrase decreases infectivity of the viral particle. See, for example, Liu et al. (1997) J. Virol. 77:7704- 7710; Wu et al. (2000) Mol. Therapy 2:41-55; and Craigie et al. (2001) J. Bio. Chem. Manuscript Rl 00027200 and Liu et α/.(1999) J. Virol. 23:8831-8836; all of which are herein incorporated by reference.

Nucleotide sequences encoding Reverse Transcriptase are known in the art (see Table 1). Reverse Transcriptase is involved in the synthesis of double stranded, linear DNA from a single-stranded RNA template using cellular tRNA as a primer. The Reverse Transcriptase used in the present invention may be from any retroviral source, particularly a lentivirus including, but not limited to, HIV-1, HIV-2, and SIN. The Reverse Transcriptase polypeptide or variant or fragment thereof will have reverse transcriptase activity.

By "reverse transcriptase activity" is intended the polypeptide retains sufficient activity to support the production of a viral particle (i.e., catalyze replication of the gene transfer vector). Assays to measure reverse transcriptase activity are known in the art. For example, measurements of reverse transcriptase activity can be carried out in-vitro using an artificial template/primer construct and tritiated deoxynucleotide triphosphate as the nucleotide substrate. This assay system is based on detecting incorporation of radioactivity in RΝA/DΝA hybrids which can be precipitated with trichloroacetic acid (TCA). Other methods for determining reverse transcriptase activity can be found in, for example, U.S. Patent No. 6,132,995 herein incorporated by reference. It is well within skill in the art to generate an expression vector having at least two nucleotide sequence encoding heterologous polypeptides that will be translated into a single translation product (i.e., fused in frame). Furthermore, one of skill in the art will recognize that the linkers may be placed between the nucleotides sequences that encode the heterologous polypeptides of the trans-enzyme construct, so long as the linker sequence allows the coding regions of the peptides to remain in frame. Such linker sequences include, for example, protease cleavage sites that are recognized by the protease encoded on the packaging construct. Such sequences are known in the art and include, for example, the 33 nucleotides 5' to the Reverse Transcriptase coding sequence of the HIN-1 genome or alternatively, the 18 nucleotides located 5' to the Integrase coding sequence of GenBank Accession No. LO2317. See, for example, Wu et al. (1997) EMBO J. 76:5113-5122. One of skill in the art will recognize how to use such sequences to result in effective cleavage of the Reverse Transcriptase or Integrase from the Vpr or Npx polypeptide. To produce a replication defective trans-viral particle both Integrase and

Reverse Transcriptase are expressed in the packaging cell line in trans to the packaging construct which encodes the Gag or Gag/Pro polypeptide. In one embodiment of the present invention, fusion proteins comprising the Reverse Transcriptase and Integrase are expressed in the packaging cell lines on two separate trans-enzyme constructs. In this embodiment, the packaging cell line will contain at least two trans-enzyme constructs. Using the trans-lentiviral vector design as an example, the first fusion protein comprising Npx or Vpr and Reverse Transcriptase, and the second trans-enzyme construct encoding a fusion protein comprising Vpx or Vpr and Integrase. It is recognized that the order of the polypeptides in these constructs may vary. Moreover, it further recognized that the trans-retroviral vector design can be used in a similar manner.

In another embodiment of the trans-enzyme construct, the polypeptide encoding the Reverse Transcriptase and Integrase or variants or fragments thereof are expressed as a single translation product. In this embodiment, the trans-enzyme construct encodes a fusion protein having the following polypeptides or functional variants or fragments thereof fused in frame: Vpr or Vpx, Reverse Transcriptase, and Integrase. See, for example, U.S. Patent Application No. 09/089,900 and Wu et al. (1991) EMB0 J. 76:5113-5122. The nucleotide sequence encoding the fusion protein of the trans-enzyme construct is operably linked to a promoter active in the packaging cell line. The DNA construct containing the trans-enzyme construct may further comprise transcriptional and translational termination regions that are also functional in the packaging cell line. As discussed elsewhere herein, promoters of interest include various constitutive and conditional promoters, including, for example, the chicken beta actin promoter, CMV, HIV-2 LTR, the SHVTK promoter, the RSV promoter, the adenovirus major late promoter and the SV 40 promoters.

Figure 2 illustrates one non-limiting example of a trans-enzyme construct of the trans-lentiviral vector system. The construct comprises the following operably linked components: a CMV promoter; a nucleotide sequence encoding a Vpr polypeptide; a nucleotide sequence encoding Reverse Transcriptase; a nucleotide sequence encoding Integrase; the RRE from HIV-2; and a SV40 polyadenylation signal. The trans-enzyme construct illustrated in Figure 2, preserves the N-terminal protease cleavage site of Reverse Transcriptase and the protease cleavage site between the Reverse Transcriptase and Integrase polypeptides.

C. Packaging Construct

As noted above, the trans-viral particle used in the methods of the present invention further provides a packaging construct, which in combination with the gene transfer vector, the env construct, and the trans-enzyme construct, enable the construction of a packaging cell line which precludes the formation of a replication competent virus.

The packaging construct is characterized as a nucleic acid sequence comprising at least one nucleotide sequence that encodes a truncated Gag/Pol sequence (i.e., Gag/Pro) which does not encode a functional Integrase or Reverse Transcriptase polypeptide. A nucleotide sequence that encodes a Gag/Pro polypeptide comprises a variety of structural proteins that make up the core matrix and nucleocapsid polypeptides. The sequence further encodes a functional protease. The Gag/Pro sequences may be derived from any retrovirus as described elsewhere, herein. Such Gag/Pro sequences are known in the art and include, for example, nucleotides 336-2099 of Genbank Accession No. AF033819. The packaging construct can further contain nucleotide sequences from a viral genome (particularly the retroviral genome) which are necessary for the production of a replication defective viral particle. Examples of genetic elements that may be contained in the packaging construct include, but are not limited to, nucleotide sequences encoding Vif, Tat, and Rev. The packaging construct, however, does not contain nucleotide sequences which encode a functional Envelope polypeptide, a functional Reverse Transcriptase polypeptide, and a functional Integrase polypeptide. These sequences have been totally or partially deleted or alternatively, have been altered to prevent translation of a functional polypeptide. Furthermore, the packaging construct lacks a functional packaging signal (ψ signal) thereby preventing the RNA produced from this construct from being incorporated into the viral particle.

As explained in more detail in U.S. Patent Application 09/089,900, the manner in which the Reverse Transcription and Integrase are mutated in the packaging construct may affect the infectivity of the viral particle. It is recognized that any alteration can be made to the reverse transcriptase and/or integrase sequence in the packaging construct that disrupts the function of the polypeptides and still allows for the production of an infectious, replication defective trans-viral vector when the Reverse Transcriptase and Integrase are express in trans in the trans-enzyme construct. For instance, in one embodiment, the reverse transcriptase and integrase sequences are altered to contain a stop codon 3' to the protease sequence. It is further recognized that multiple mutations may be introduced into the reverse transcriptase and integrase sequence. For example, in addition to introducing a translation stop early in the coding sequence of Reverse Transcriptase, at least one additional "fatal" mutation can be positioned within the Reverse Transcriptase and/or Integrase coding sequence. This additional mutation further decreases the likelihood of reestablishing a complete Gag-Pol coding region by genetic recombination between the packaging construct and the trans-enzyme construct.

The nucleotide sequences of the packaging construct are contained in a DNA construct which further comprises a promoter active in the packaging cell line. The DNA construct may further comprise transcriptional and translational termination regions which are also functional in the packaging cell line. As discussed elsewhere herein, promoters of interest include various constitutive and conditional promoters including, for example, CMV, HIV-2 LTR, the HCMV-IE (Naldini et al. (1996) Science 272:263-261), the SHVTK promoter, the RSV promoter, the adenovirus major late promoter and, the SV40 promoters. The packaging construct may further contain a selectable marker operably linked to an active promoter.

One of skill in the art will recognize that various functional variants of the packaging construct can be envisioned. Figure 1-3 illustrate a non-limiting example of a packaging construct useful in the methods of the present invention. The construct comprises a CMV promoter operably linked to a nucleotide sequence encoding Gag/Pro, Vif, Tat, Rev (nt 258-8384 of Genbank Accession No. L02317). The packaging construct of Figure 1 further comprises translational stop codons (TAA) at the first amino acid position of the Reverse Transcriptase and Integrase coding sequences, a deletion of the

signal, a frame shift mutation in the Vpr coding sequence, a complete deletion of the nef gene, an internal deletion that results in an inactive Vpu polypeptide, and a deletion of the nucleotides which encode the Env polypeptide. For a more detailed description of the construction of the packaging construct see U.S. Patent Application 09/089,900.

D. Env Construct

The present invention further provides an env construct that in combination with the packaging construct, gene transfer vector, and trans-enzyme construct described above, preclude formation of a replication complete trans-virus vector particle.

The env construct of the trans-viral system comprises a nucleotide sequence encoding an envelope protein or a functional variant or fragment thereof operably linked to an active promoter. A variety of envelope polypeptides are known in the art. It is recognized that the host range of cells that the viral particles of the present invention can infect can be altered depending on the envelope coding sequence used. Viral envelope proteins useful in the present invention include HTV envelope polypeptides (see Table 1), the MLV envelope glycoprotein (Page et al. (1990) J. Virol. 64:5270-5276), the vesicular stomatitis virus G-protein (VSV-G) (Yee et al. (1994) Proc. Natl. Acad. Sci. 91:9564-9568 and Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037) or the envelope polypeptides of Ebola and Makola (Kobinger et al. (2001) Nature Biotechnology 19: 225-230). In preferred embodiments of the present invention, the env coding sequence chosen will allow for the entry of the viral particle into the cells of the desired target cell. In the methods of the present invention, the G-protein of vesicular-stomatitis virus (VSV-G) or a fragment or variant thereof is used in the env construct. Pseudotyping assays to determine additional envelope polypeptides useful in the methods of the present invention are well known in the art. It is further recognized that the trans-viral vector particle may be constructed in the absence of the Env construct.

A fragment or variant of an Envelope polypeptide will retain sufficient activity to support the production of a replication defective viral particle (i.e., capable of being incorporated into the envelope of a retroviral particle and capable of binding to target cells and allowing entry of the viral particle into the target cells). Assays for determining the function of an Env polypeptide or a fragment or variant thereof are known in the art. For example, expression of a fragment or variant of an Env polypeptide of the present invention will allow vector particles produced in that packaging cell line, to transmit a selectable marker to a naive sensitive cell such that it becomes resistant to the marker drug selection.

Any promoter sequence may be used in the env construct, so long as it is active in the packaging cell line. Such promoter sequences (i.e., constitutive and conditional promoters) have been described elsewhere herein. Representative examples of suitable polyadenylation signals include the SV40 late polyadenylation signal, the bovine growth hormone termination/polyadenylation sequence, and the insulin polyadenylation signal.

The env construct may further comprise a nucleotide sequence encoding a selectable marker. Examples of such selectable markers include nucleotide sequences capable of conferring host resistance to antibiotics (e.g., puromycin, ampicillin, tetracycline, kanamycin, and the like), or conferring resistance to amino acid analogues, etc. Other selectable markers are well known in the art, including for example /3gal, GFP, and luciferase. One of skill will appreciate the numerous possibilities.

As an illustrative and non-limiting example of the env construct of the present invention, is shown in Figure 1. The construct comprises the CMV immediate early promoter operably linked to a VSV-G coding region operably linked to an SV40 polyadenylation signal. E. Packaging Cell Lines

The trans-viral particles used in the methods of the present invention are generated using techniques known in the art. See, for example, U.S. Patent No. 4,650,764, and U.S. Patent Application No. 09/089,900 herein incorporated by reference. The methods include incorporating into a packaging cell line the trans- enzyme construct, the gene transfer construct, the env construct, and the packaging construct; culturing the packaging cell line under suitable conditions that allow for the formation of viral particles; and, isolating the trans- viral particles. A wide variety of animal cells may be used to prepare the packaging cells of the present invention, including, for example, cells obtained from vertebrates, or mammals such as human, feline, goat, bovine, sheep, dog, and mice. Suitable packaging cell lines include, but are not limited to, HeLa (ATCC No. CCL2); HT1080 (ATCC No. CCL121); 293 (ATCC No. 1573); and the 293T cell line.

The various vector constructs may be introduced into the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaPO₄ precipitation. See, for example, Ausabel et al. (1994) Current Protocols in Molecular Biology; John Wileg and Sons, Inc., and U.S. Patent No. 5,739,081, both of which are herein incorporated by reference. It is further recognized that the various constructs of the trans-lentiviral system can be transiently expressed in the packaging cell line, or alternatively, any or all of the constructs can be stably incorporated into the genome of the packaging cell.

Methods for culturing the packaging cell line under conditions in which the trans-lentiviral particle is produced and the subsequent isolation of retroviral vector particles are also known in the art. For instance, the retroviral vector packaging cell lines may be cultured using standard culturing techniques, including any of a variety of monolayer culture systems. Such packaging cell lines may be cultured in T-flasks, roller bottles, or bioreactors. Any acceptable culture medium may be used, such as, AIM-V medium (Gibco BRL, Grand Island, N.Y) containing 5% fetal bovine serum, or Dulbecco's modified Eagle medium (DMEM) with high glucose (4.5 g/1) supplemented with 10% heat-inactivated fetal bovine serum

Methods of isolating retroviral vector particles are known in the art, see for example, U.S. Patent No. 5,661,022 and U.S. Patent No. 6,013,517; herein incorporated by reference. As used herein, "purified trans-virus vector particles" means a preparation of trans- viral vector particles containing at least 50%, 60%, 70%, 80% by weight, preferably at least 85% by weight, and more preferably at least 90%, 93%, 95%), 98%>, 99%) by weight, of the retroviral vector particles.

II. Methods of Expressing A Polypeptide of Interest

As discussed above, the present invention provides novel methods and compositions for the expression of a polypeptide of interest. In particular, the methods and compositions of the invention combine a trans- viral vector system with a gene transfer vector having at least one DNA expression cassette comprising multiple open reading frames separated by an internal ribosomal entry site (IRES) or an active fragment or variant thereof.

In one embodiment of the invention, the method comprises transducing a target cell with a trans-viral vector particle comprising a gene transfer vector having at least one DNA expression cassette. The DNA expression cassette employed in the trans-viral vector system may comprise a promoter operably linked to a first nucleotide sequence encoding a first polypeptide which is operably linked to a second nucleotide sequence encoding a second polypeptide by an internal ribosomal entry site or an active variant or fragment thereof. The transduced cells are cultured under conditions that allow for the expression of the first and the second nucleotide sequence of interest. As the expression of the first and the second nucleotide sequences are under the control of the same promoter, the two sequences are transcribed as a multicistronic mRNA. The presence of the IRES element permits the translation of the downstream open reading frame independent of the cap mediated translation event occurring in the first open reading frame. Such a system allows for the coordinated expression of the polypeptides contained on the multicistronic mRNA. This method finds use, for example, in the coordinated expression of polypeptides that form heterodimers. The method can therefore be used to produce an increased concentration of complex ed polypeptides. See, for example, U.S. Patent No. 5,655,567, which demonstrates the stoichiometric expression of a heterodiameric recombinant growth factor using a multicistronic construct.

In another embodiment of the present invention, one of the nucleotide sequences of the DNA expression cassette encodes a selectable marker, while the other nucleotide sequence encodes a polypeptide of interest. In this embodiment, the transduced target cells are cultured under conditions that allow for the expression of both the selectable marker and the polypeptide of interest. Cells expressing the marker are selected. In this manner, target cells expressing the polypeptide of interest are selected away from cells that are not expressing the polypeptide or are expressing the polypeptide at low levels. In this embodiment, the stringency of the selection system employed can be manipulated to force the selection of cell expressing high levels of the marker. Hence, cells selected based on the expression of the marker may inherently contain higher levels of the polypeptide of interest. It is further recognized that the position of the selectable marker within the

DNA expression cassette will also influence the selection of cells expressing high levels of the polypeptide of interest. For example, in specific embodiments, the selectable marker is encoded by the second nucleotide sequence located downstream of the IRES in the DNA expression cassette, while the first nucleotide sequence of the cassette encodes the polypeptide of interest. Often sequences downstream of the

IRES elements are translated at lower levels than sequences that are translated via the 5' cap mediated mechanism (i.e., the first nucleotide sequence of the DNA expression cassette). Consequently, when the sequence encoding the selectable marker is contained downstream of the IRES element, cells selected based on expression of the selectable marker will inherently be higher expressers of the polypeptide of interest.

Methods of transducing a cell with a viral particle are well known in the art.

In specific embodiments of the invention, transduction of the target cell comprises contacting the target cell with the trans-viral vector. Determining the concentration of viral titer to achieve efficient transduction of target cells is also routine in the art. In specific embodiments, the viral titer of the trans-viral vector is from about 10¹ to 10⁹ transducting units per ml.

By "target cell" is intended any cell type that the trans-viral vector is capable of infecting and transducing. Of particular interest are target cells from mammalian expression systems including, cell lines, primary cultures, stem cell cultures, tissue explants, animal organs, and whole animals. In particular embodiments, the target cells comprise slowly dividing cells or primary cells including, but not limited to, macrophage, unstimulated CD34⁺cells, hematopoietic stem cells, nerve cells, and retinal cells. One of skill in the art will be apprised of the conditions required to culture the target cells and the appropriate conditions for the expression of the first and the second nucleotide sequences contained in the DNA expression cassette. Moreover, one of skill in the art will recognize that the culturing conditions and the levels of selection pressure will vary depending on the type of target cell. For example, when puromycin is used as the selectable marker, the final concentration of puromycin can range from about 5 μg/ml to about 100 μg/ml, from about 5 μg/ml to about 75 μg/ml, or from about 5 μg/ml to about 50 μg/ml. Alternatively, the final concentration of puromycin in the cell culture can be about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 μg/ml of puromycin. In this method, cells able to grow under the highest levels of an antibiotic such as puromycin can be identified and expanded in culture and will therefore express the highest amounts of the protein of interest. Also, the toxicity of the gene of interest being expressed plays a role in determining the culturing and selection conditions. In the methods of the present invention, target cells expressing the selectable marker are selected away from the target cells that do not express, or express at a lower level, the selectable marker. One of skill in the art will recognize that the method of selection will vary depending on the selectable marker employed. For example, if the selectable marker used confers resistance to a cytotoxic agent, the cells can be contacted with the appropriate cytotoxic agent. In this embodiment, nontransfected cells, as well as nonexpressing or very low expressing cells, are negatively selected away from the transfected cells. If the selectable marker is a cell surface marker, the cells can be contacted with a binding agent specific for the particular cell surface marker, whereby the transfected cells can be positively selected away from the population.

The selection step can also entail fluorescence-activated cell sorting (FACS) techniques (i.e., the use of FACS to select cells from the population containing a particular surface marker). These cell sorting procedures are described in detail, for example, in the FACSVantage.TM. Training Manual. The selection step may also use magnetically responsive particles as retrievable supports for target cell capture and/or background removal. These and similar separation procedures are described, for example, in the Baxter Immunotherapy Isolex training manual. Once cells expressing the selectable marker are isolated, the cells can be cultured under conditions that allow for cell growth. This step allows for the amplification of cells expressing the polypeptide of interest at high levels. If desired, the polypeptide can subsequently be isolated.

7/7. Polypeptides of Interest

In the methods and compositions of the present invention, the gene transfer vector may comprise a nucleotide sequence encoding a polypeptide of interest. A nucleic acid sequence encoding the polypeptide of interest may be heterologous or homologous to the target cell. By heterologous is intended a nucleotide sequence that is not naturally found in the genome of the target cell. By homologous is intended a nucleotide sequence that is found in the target cell in nature.

One of skill will recognize that the methods and compositions of the present invention can be used to produce a variety of proteins useful for the prevention, treatment and/or diagnosis of a wide variety of diseases. For instance, the polypeptide expressed by the gene of interest may be one useful in a vaccine, therapeutic, or diagnostic and may be derived from any of several known, eukaryotes, viruses, bacteria, parasites, and fungi. Alternatively, the expressed polypeptide may be a therapeutic hormone, a transcription or translation mediator, an enzyme, an intermediate in a metabolic pathway, an immunomodulator, and the like. Thus, the methods of the present invention will find use for the expression of a wide variety of substances, including polypeptides which act as antibiotics and antiviral agents (i.e., immunogenic polypeptides for use in vaccines and diagnostics); antineoplastics; and immunomodulators. Additional polypeptides of interest that find use in the methods and compositions of the invention include integral membrane proteins, including for example ABC transporters, GPCRs, and Multidrug Resistance proteins. Other polypeptides of interest include the Beta2 Adrenergic polypeptide, Muscarinic M2 polypeptide, Angiotensin AT2 polypeptide, K-Opioid polypeptide, Dopamine, Adenosine Al, and CCR5 or active variants and fragments thereof.

Immunomodulators of interest include, but are not limited to, interleukins (i.e., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10); tumor necrosis factors (i.e., TNF-α and TNF-/3); and, interferons (i.e., IFN-α, IFN-ft IFN-γ, IFN-ω, and IFN-τ); and any biologically active variants thereof.

Other polypeptides of interest include growth factors, which include, but are not limited to, members of the neurotrophin family (i.e., nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin- 4 (NT-4, also known as NT-4/5 or NT-5); fibroblast growth factors (FGFs, i.e., basic fibroblast growth factor); epidermal growth factor family (i.e., EGF, TGFα, amphiregulin, heparin-binding EGF-like growth factor (HB-EGF), batacelluin (BTC), and the neuregulin group); platelet-derived growth factor; insulin; insulin-like growth factors (i.e., IGF-I and IGF-2); ciliary neurotrophic factor (CNTF), glia cell line-derived neurotrophic factor family (GDNF) (i.e., GDNF and neurturin (NTN), persephin (PSP), and artemin (ART)); transforming growth factor β superfamily (i.e., subfamilies include TGFβl, TGFβ2, TGFβ3, TGFβ4, TGFβ5, activin, inhibin, decapentaplegic); growth differentiation factors (GDF) (i.e., GDF1, GDF2, GDF3, GDF5, GDF6, GDF7, GDF8, GDF9, GDF9B, GDF10, GDF11, and GDF15); glia- derived nexin; activity dependent neurotrophic factor (ADNF); glial growth factor (GGF); and the like. It is further recognized that any biologically active variant of these growth factors is also useful in the methods of the present invention.

Suitable biologically active variants of the polypeptide of interest can be fragments, analogues, and derivatives of the polypeptide (i.e., growth factor polypeptides, immunomodulatory agents, and integral membrane proteins [i.e., ABC transporters, GPCRs and MRPs.]. Methods for generating active fragments and variant of polypeptides have been disclosed elsewhere herein.

EXPERIMENTAL

I. Construction of the gene transfer vector containing the DNA expression cassette Construction of the pTZV-CMV-IRES-puro gene transfer vector was carried out in two cloning steps. First, the Hpal to BamHI DNA fragment from the gene transfer vector plasmid pPCW-eGFP (described U.S. Provisional Application No. 60/344,841, filed December 21, 2001, herein incorporated by reference) was ligated into the Bglll (blunt-ended)/BamHI sites of pcDNA3.1/Hygro (Invitrogen) to generate the transition vector, ρCDNA3.1-5'-TZV. The Hpal-BamHI DNA fragment encoded the 5'-half of the gene transfer vector, which includes the 5'-LTR, psi (Ψ) packaging signal, the rev responsive element (RRE), the 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 (Ghosh et al. (1993) Virology 194: 858-864.), and the intermediate early promoter of human cytomegalovirus (CMV). The 3 '-half of the gene transfer vector was incorporated by PCR-amplification of a DNA fragment containing the internal ribosomal entry site (IRES) of the encephalomyocarditis virus (ECMV), a puromycin-N-acetyltransferase gene (puro), the post transcriptional regulator element derived from the woodchuck hepatitis virus (WPRE, Zufferey et al. (1999) J. Virol. 7:2886.), and the 3'-LTR. Xbal and Pmel restriction sites were incorporated into the 5' and 3' oligonucleotide primers for PCR, respectively, to facilitate cloning of ths DNA fragment between the Xbal/Pmel sites of the pCDNA3.1-5'-TZV transition vector.

A cDNA of interest is cloned into the pTZV-CMV-IRES-puro gene transfer vector using various restriction sites located between and including the BamHI and Xbal sites. Cloning of cDNAs between these sites puts their expression under the control of the CMV promoter and links their expression to the expression of the puro gene via the IRES sequence.

A cDNA of interest encoding a polypeptide of interest is cloned into the pTZV-CMV-IRES-puro vector between the BamHI Xbal sites to generate pTZV- CMV-NT sequence of interest-IRES-puro. To facilitate purification, a polyhistidine tag containing ten consecutive histidine residues (lOxHis) is fused in frame at the carboxy-terminus of the polypeptide encoded by the cDNA of interest. Incorporation of the lOxHis tag allows a protein to be captured by affinity chromatography via binding of the lOxHis to a metal-chelate (eg. nickel-chelate) affinity matrix.

II. Preparation of TranzVector stocks (TZV-NT of interest— IRES-puro)

The trans-lentiviral vector represents an HIV-based vector with unique safety features that have been described earlier (Wu et al. (2000) Mol. Therapy 2:47-55.). Briefly, to reduce the risk of generating a replication competent retrovirus (RCR), the vector stocks are produced using the TranzVectorTM (Tranzyme, Inc., Birmingham AL) lentiviral packaging system which separates the RT and IN from Gag-Pol and delivers it in trans as a fusion partner with the HIV-1 virion associated protein Vpr. Thus, stocks of the trans-lentiviral vector are produced by transfecting 3 μg of the pCMV-gag-pro packaging plasmid, 1.0 μg of the pCMV-vpr-RT-IN trans-enzyme plasmid, 1.5 μg of the pMD.G (VSVG) expression plasmid, and 3 μg of the gene transfer (pTZV-CMV- NT sequence of interest-IRES-puro) plasmid into subconfluent monolayer cultures of 293T cells by the calcium phosphate DNA precipitation method. Supematants are harvested after 60 h, clarified by low-speed centrifugation (lOOOg, 10 min) and filtered through 0.45-μm pore-size filters. The vector particles are concentrated by ultracentrifugation (Beckman SW28 rotor, 23,000 rpm, 2 hr). To determine vector titer, supernatant stock of 0.2, 0.04, 0.008, 0.0016, 0.00032, and 0.000064 μl are used to infect cultures of HeLa cells, and subsequently put under selection by culturing the transduced cells in media containing puromycin (2 μg/ml). Cells are cultured in selection media for an additional 9 days to allow colonies to form. To visualize the puro-resistant colonies, the cultures are fixed in methanol and stained with crystal violet and the colonies are counted. Each puro-resistant cell colony is measured as a single transduction unit (TU). Titers of the purified virus usually range between 0.1-1.0 x 10⁹ TU/ml. Aliquots of virus are stored at -80° C until use.

III. Transduction and selection of mammalian cells expressing high-levels of protein. The human embryonic kidney cell line, HEK-293, is used to express the nucleotide sequence encoding the polypeptide of interest. HEK-293 cells (1 x 10⁷ cells) are transduced with TZV-NT of interest-IRES-puro vector stock at a multiplicity of infection (MOI) of 10. Briefly, HEK-293 cells are incubated with were infected with the vector stocks in DMEM/F12 media containing 0.1 % fetal bovine serum and 8 μg/ml of Polybrene for 4 hrs at 37°C. The medium is then replaced with fresh DMEM F12 media containing 10% FBS. After allowing the cells to culture for 48 hours, the culture is evenly divided into separate flasks and cultured in media containing various concentrations of puromycin (5, 10, 20, 30, 40, and 50 μg/ml). The cells which survive the highest concentrations of puromycin (40- 50 μg/ml) are combined and expanded in culture, maintaining the puromycin selection pressure. IV. Analysis of expression of the polypeptide of interest

After culture expansion, the cells are analyzed for expression of the polypeptide of interest. Cells are lysed. If the polypeptide of interest is a membrane- bound protein, the membrane-bound polypeptides are solublized, and passed through a nickel-nitriloacetate (Ni-NTA)-agarose (Qiagen) chromatography column for purification. Briefly, cell pellet containing 5.6x10 cells is processed by the procedure of Loo et al. (1998) Methods Enzymol. 292:480-92 with further optimization. The lysates is incubated sequentially with two batches of Ni-chelate, and the two batches of the chelate are pooled, washed, and eluted with buffer 300 mM imidazole. The analysis of the pooled material can be performed using a standard coomassie-staining procedures. In addition, a Western blot of the material in the pooled fraction is electrophoresed. An antibody specific to the polypeptide of interest is used for immunodetection. This procedure has been used to obtain approximately 5 μg of a membrane-bound polypeptide in a 30 μl volume of the eluate.

The present invention has been described above with reference to the accompanying drawings, in which preferred embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Like numbers refer to like elements throughout.

Many modifications and other embodiments of the invention will come to mind to one skilled in the art to which this invention pertains having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the invention is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

EMBODIMENTS OF THE INVENTION

1. A trans-viral vector system comprising a gene transfer vector comprising a DNA expression cassette comprising at least a first nucleotide sequence encoding a first polypeptide and a second nucleotide sequence encoding a second polypeptide, wherein said first and said second nucleotide sequence are operably linked by an internal ribosomal entry site (IRES) or an active variant or fragment thereof.

2. The trans-viral vector system of embodiment 1, wherein said first nucleotide sequence and said second nucleotide sequence are operably linked by an IRES.

3. The trans-viral vector system of embodiment 1, wherein said IRES or the active fragment or variant thereof is from the encephalomyocarditis virus.

4. The trans-viral vector system of embodiment 1, wherein said first or said second nucleotide sequence encodes a polypeptide selected from the group consisting of an integral membrane protein, an immunomodulatory polypeptide, and a growth factor.

5. The trans-viral vector system of embodiment 4, wherein said integral membrane protein is selected from the group consisting of a GPCR and a multidrug resistance protein.

6. The trans-viral vector system of embodiment 1, wherein at least one of said first or said second nucleotide sequences encodes a selectable marker.

7. The trans-viral vector system of embodiment 6, wherein said selectable marker confers resistance to a cytotoxic agent.

8. The trans-viral vector system of embodiment 7, wherein said selectable marker conferring resistance to a cytotoxic agent is selected from the group consisting of puromycin and neomycine.

9. The trans-viral vector system of embodiment 6, wherein said second nucleotide sequence encodes a selectable marker.

10. The trans-viral vector system of embodiment 1, wherein said DNA expression cassette further comprises a promoter operably linked to said first nucleic acid sequence, wherein said promoter is active in a target cell.

11. The trans-viral vector system of embodiment 10, wherein said promoter is selected from the group consisting of an inducible promoter, a constitutive promoter, or a tissue-preferred promoter.

12. The trans-viral vector system of embodiment 10, wherein said promoter is a CMV promoter.

13. The trans- viral vector system of embodiment 1, wherein said trans- viral vector system is from a retrovirus.

14. The trans-viral vector system of embodiment 13, wherein said trans- viral vector system is from a lentivirus.

15. The trans-viral vector system of embodiment 14, wherein said lentivirus is a human immunodeficiency virus or a simian immunodeficiency virus.

16. The trans-viral vector system of embodiment 15, wherein said human immunodeficiency virus is HIV-1 or HIV-2.

17. The trans- viral vector system of embodiment 1, wherein the trans- viral vector system is a trans-lentiviral vector system.

18. The trans-viral vector system of embodiment 17, wherein the trans- lenti viral vector system comprises a trans-enzyme construct comprising a nucleotide sequence encoding a fusion protein comprising a functional portion of a Vpr or a Vpx polypeptide fused in frame to a functional portion of a Reverse Transcriptase polypeptide fused in frame to a functional portion of an integrase polypeptide.

19. The trans- viral vector system of embodiment 1, wherein the trans- viral vector system is a trans-retroviral vector system.

20. The trans-viral vector system of embodiment 19, wherein the trans- retro viral vector system comprises a trans-enzyme construct comprising a nucleotide sequence encoding a functional portion of a Reverse Transcriptase polypeptide fused in frame to a functional portion of an Integrase polypeptide.

21. A method of expressing at least two polypeptides of interest comprising: a) providing a target cell; b) transducing said target cell with a trans- viral vector particle comprising a gene transfer vector comprising a DNA expression cassette having a first nucleotide sequence encoding a first polypeptide and a second nucleotide sequence encoding a second polypeptide wherein, said first nucleotide sequence is operably linked to a promoter active in said target cell; and, said first and said second nucleotide sequence are operably linked by an internal ribosomal entry site (IRES) or an active fragment or variant thereof; and, c) culturing said target cell under conditions that allow for expression of said first and said second nucleotide sequence of interest.

22. The method of embodiment 21, wherein said first and said second nucleotide sequence are operably linked by an IRES.

23. The method of embodiment 21, wherein said IRES or said active fragment or variant thereof is from the encephalomyocarditis virus.

24. The method of embodiment 21, wherein said first or said second nucleotide sequence encodes a selectable marker.

25. The method of embodiment 24, wherein said second nucleotide sequence encodes a selectable marker.

26. The method of embodiment 25, wherein said selectable marker confers resistance to a cytotoxic agent.

27. The method of embodiment 26, wherein said selectable marker conferring resistance to a cytotoxic agent is selected from the group consisting of puromycin and neomycine.

28. The method of embodiment 25, wherein said first nucleotide sequence encodes a polypeptide selected from the group consisting of an integral membrane protein, an immunomodulatory polypeptide, and a growth factor.

29. The method of embodiment 24 further comprising selecting the target cells which express the selectable marker.

30. The method of embodiment 29, further comprising culturing said target cells that express the selectable marker under cell growth conditions.

31. The method of embodiment 21 , wherein said DNA expression cassette further comprises a promoter operably linked to said first nucleic acid sequence, wherein said promoter is active in the target cell.

32. The method of embodiment 31 , wherein said promoter is selected from the group consisting of an inducible promoter, a constitutive promoter, or a tissue- preferred promoter.

33. The method of embodiment 31, wherein said promoter is a CMV promoter.

34. The method of embodiment 21 , wherein said trans- viral vector particle is from a retrovirus.

35. The method of embodiment 34, wherein said trans-viral vector particle is from a lentivirus.

36. The method of embodiment 35, wherein said lentivirus is a human immunodeficiency virus or a simian immunodeficiency virus.

37. The method of embodiment 36, wherein said human immunodeficiency virus is HIV-1 or HIV-2.

38. The method of embodiment 21 , wherein the trans-viral vector particle is from a trans-lentiviral vector system.

39. The method of embodiment 38, wherein the trans-lenti viral vector system comprises a trans-enzyme construct comprising a nucleotide sequence encoding a fusion protein comprising a functional portion of a Vpr or a Vpx polypeptide fused in frame to a functional portion of a Reverse Transcriptase polypeptide fused in frame to a functional portion of an integrase polypeptide.

40. The method of embodiment 21 , wherein the trans-viral vector particle is from a trans-retroviral vector system.

41. The method of embodiment 40, wherein the trans- viral vector system comprises a trans-enzyme construct comprising a nucleotide sequence encoding a functional portion of a Reverse Transcriptase polypeptide fused in frame to a functional portion of an Integrase polypeptide.

42. A method of generating a trans- viral vector particle comprising: a) providing a packaging cell having an env construct, a packaging construct, and a trans-enzyme construct; b) introducing into said packaging cell a gene transfer vector comprising a DNA construct having a first nucleotide sequence operably linked to a second nucleotide sequence by an internal ribosomal entry site; and, c) incubating the packaging cell of step (b) under conditions wherein the trans-viral vector particle is produced.