EP0511311A1 - Novel retroviral vectors - Google Patents

Abstract

A new retroviral vector includes at least four different enzyme restriction sites, at least two of which have an average frequency of appearance in eukaryotic genes less than once in ten thousand base pairs. Such a vector can be used in combination with a cloning shuttle vector having complementary cloning sites to accomplish gene and / or promoter transfers between the cloning shuttle vector and the retroviral vector. Such a system ensures the efficient transfer of genes and / or promoters to a retroviral vector without the need to carry out a reconstruction of all the retroviral vectors. The invention also relates to a retroviral vector having a 3 'LTR where at least the promoter sequence of 3' LTR is mutated so that this promoter sequence becomes non-functional.

Description

NOVEL RETROVIRAL VECTORS

This invention relates to retroviral vectors. More particularly, this invention relates to

retroviral vectors having multiple cloning, or restriction enzyme recognition sites, and to systems for the exchange of gene sequences between vectors having compatible or complementary multiple cloning sites.

Retroviral vectors are useful as agents to mediate retroviral-mediated gene transfer into eukaryotic cells. Such vectors are generally

constructed such that the majority of sequences coding for the structural genes of the virus are deleted and replaced by the gehe(s) of interest.

These new genes have been incorporated into the proviral backbone in several general ways. The most straightforward constructions are ones in which the structural genes of the retrovirus are replaced by a single gene which then is transcribed under the control of the viral regulatory sequences within the long terminal repeat (LTR). Retroviral vectors have also been constructed which can introduce more than one gene into target cells. Usually, in such vectors one gene is under the regulatory control of the viral LTR, while the second gene is expressed either off a spliced message or is under the regulation of its own, internal promoter.

Efforts have been directed at minimizing the viral component of the viral backbone, largely in an effort to reduce the chance for recombination between the vector and the packaging-defective helper virus within packaging cells. A packaging-defective helper virus is necessary to provide the structural genes of a retrovirus, which have been deleted from the vector itself.

Bender et al., J. Virol. 61:1639-1649 (1987) have described a series of vectors, based on the N2 vector (Armentano, et al., J. Virol., 61:1647-1650) containing a series of deletions and substitutions to reduce to an absolute minimum the homology between the vector and packaging systems. These changes have also reduced the likelihood that viral proteins would be expressed. In the first of these vectors,

LNL-XHC, there was altered, by site-directed

mutagenesis, the natural ATG start codon of gag to TAG, thereby eliminating unintended protein synthesis from that point. In Moloney murine leukemia virus (MoMuLV), 5' to the authentic gag start, an open reading frame exists which permits expression of another glycosylated protein (pPr80^gag). Moloney murine sarcoma virus (MoMuSV) has alterations in this 5' region, including a frameshift and loss of

glycosylation sites, which obviate potential

expression of the amino terminus of pPr80^gag.

Therefore, the vector LML6 was made, which

incorporated both the altered ATG of LNL-XHC and the 5' portion of MoMuSV. The 5' structure of the LN vector series thus eliminates the possibility of expression of retroviral reading frames, with the subsequent production of viral antigens in genetically transduced target cells. In a final alteration to reduce overlap with packaging-defective helper virus, Miller has eliminated extra env

sequences immediately preceding the 3' LTR in the LN vector (Miller et al., Biotechniques, 7:980-990, 1989).

The paramount need that must be satisfied by any gene transfer system for its application to gene therapy is safety. Safety is derived from the combination of vector genome structure together with the packaging system that is utilized for production of the infectious vector. Miller, et al. have developed the combination of the pPAM3 plasmid (the packaging-defective helper genome) for expression of retroviral structural proteins together with the LN vector series to make a vector packaging system where the generation of recombinant wild-type retrovirus is reduced to a minimum through the elimination of nearly all sites of recombination between the vector genome and the packaging-defective helper genome (i.e. LN with pPAM3).

Although the LN series of vectors has generated a vector backbone which incorporates several safety features, the LN vector contains a very limited number of potential cloning sites for the insertion of additional genes into the vector.

Gene therapy or drug, delivery via gene transfer entails the creation of specialized vectors each vector being applicable only to a particular disease. Thus, it is desirable that a vector cloning system be available which consistently maintains the necessary safety features yet permits maximal flexibility in vector design. Subtle changes in gene position, or in the specific combination of regulatory sequence(s) with the gene of interest, can lead to profound differences in vector titer or in the way that transferred genes function in target cells. Current vector designs require that for each combination of genes and promoters, the entire vector be

reconstructed, and even then comparisons between different vectors are difficult because of

inconsistencies in the detail of their construction. These inconsistencies in vector structure can also lead to questions of vector safety which need answering on a case by case basis.

It is therefore an object of the present invention to provide a rapid system for the

construction of retroviral vectors which also permits consistent vector construction, whereby genes, promoters, or combinations of genes and promoters may be rapidly exchanged and the vectors evaluated to achieve optimal results in tissues of interest.

In accordance with an aspect of the present invention, there is provided a retroviral vector which includes at least four cloning, or restriction enzyme recognition sites, wherein at least two of the sites have an average frequency of appearance in eukaryotic genes of less than once in 10,000 base pairs, i.e., the restriction product has an average DMA size of at least 10,000 base pairs. Preferred cloning sites are selected from the group consisting of Notl, SnaBI, Sall, and Xhol.

Such vectors may be engineered from existing retroviral vectors through genetic engineering techniques known in the art such that the resulting retroviral vector includes at least four cloning sites wherein at least two of the cloning sites are selected from the group consisting of the NotI, SnaBI, Sall, and Xhol cloning sites. In a preferred embodiment, the retroviral vector includes each of the Notl, SnaBI, Sall, and Xhol cloning sites.

Examples of retroviral vectors which may be transformed to include the above-mentioned cloning sites include Moloney Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus and Harvey Sarcoma Virus. Specific vectors which may be constructed in

accordance with the present invention are described in the examples hereinbelow.

Such a retroviral vector may serve as part of a cloning system for the transfer of genes to such retroviral vector. Thus, in accordance with another aspect of the present invention, there is provided a cloning system for the manipulation of genes in a retroviral vector which includes a retroviral vector including multiple cloning sites of the type

hereinabove described, and a shuttle cloning vector which includes at least two cloning sites which are compatible with at least two cloning sites selected from the group consisting of Notl, SnaBI, Sall, and Xhol located on the retroviral vector. The shuttle cloning vector also includes at least one desired gene which is capable of being transferred from said shuttle cloning vector to said retroviral vector.

The shuttle cloning vector may be constructed from a basic "backbone" vector or fragment to which are ligated one or more linkers which include cloning or restriction enzyme recognition sites . Included in the cloning sites are the compatible, or

complementary cloning sites hereinabove described. Genes and/or promoters having ends corresponding to the restriction sites of the shuttle vector may be ligated into the shuttle vector through techniques known in the art. The shuttle cloning vector can be employed to amplify DNA sequences in prokaryotic systems. The shuttle cloning vector may be prepared from plasmids generally used in prokaryotic systems and in

particular in bacteria. Thus, for example, the shuttle cloning vector may be derived from plasmids such as pBr322; pUC 18; etc.

Although the scope of the present invention is not to be limited to any theoretical reasoning, the present invention provides for retroviral vectors having a larger choice of cloning sites, and shuttle vectors with complementary cloning sites, which provides for the rapid exchange of genes and/or promoters from the shuttle vector to the retroviral vector. The increased number of cloning sites also provides for greater flexibility in vector

construction.

In addition, the Notl, SnaBI, Sall, and Xhol cloning sites are sites which are of extreme rarity in eukaryotic genes. The use of such "rare" sites enables one to extract a first gene from the

retroviral vector, and replace the first gene with a second gene without altering the retroviral vector backbone structure. Thus, reconstruction of the entire retroviral vector is not necessary. To aid in the effective transfer of genes and/or promoters batwaan the retroviral vector and the shuttle vector, it is preferred that the order of the cloning sites in the retroviral and shuttle vectors be

complementary. Through such exchange of genes and promoters, vectors are constructed which may be efficiently evaluated to achieve optimal results in tissues of interest.

In accordance with another aspect of the present invention, there is provided a retroviral vector, said vector including a 5' LTR (long terminal repeat) and a 3' LTR. At least the promoter sequence(s) of the 3' LTR is mutated, or altered, such that the promoter sequence becomes non-functional. Such a mutation, however, does not alter the overall

structure of the LTR. In a preferred embodiment, the enhancer sequence(s) of the 3' LTR may also be mutated such that the enhancer sequence(s) also becomes non-functional. The integrator sequence, however, is maintained. Thus, when such a vector is introduced into a target cell, viral replication duplicates portions of the 3' LTR to both the 5' and 3' ends of a proviral integrant. The mutation, therefore, causes a "flipping" of the vector whereby the 5' LTR now lacks regulatory sequences, and introduced genes are completely under the regulation of their own promoters. It is also preferred that, in mutations of the 3' LTR as hereinabove described, that the approximate number of base pairs of the original 3' LTR is maintained in the mutated 3' LTR, and approximately the same proportions of types of base pairs of the original 3' LTR is maintained in the mutated 3' LTR.

Although the scope of this aspect of the present invention is not to be limited to any theoretical reasoning, the altering or mutating of only the promotar or enhancer sequences of the 3' LTR does not require a large deletion of the 3' LTR to eliminate promoter or enhancer function, and the overall structure of the 3' LTR is preserved. Because LTR structure is critical for efficient integration of the vector into the genome, one is able to maintain a high titar of retroviral vector. Such vectors, therefore, may be useful in clinical applications where the maintenance of a high titer of vector is essential or critical. Such a retroviral vector may be formed from a retroviral vector having at least four cloning sites, wherein at least two of the cloning sites are selected from the group consisting of Notl, SnaBI, Sall, and Xhol, as hereinabove described; however, the scope of the present

invention is not to be limited to such vectors, nor is the scope of this aspect of the present invention limited to vectors having multiple cloning sites.

Examples of vectors whose promoter sequence of the 3' LTR may be mutated include Moloney Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus and Harvey Sarcoma Virus. Such mutations, whereby the promoter or enhancer sequences of 3' LTR are altered, may be effected through techniques known in the art.

The invention will now be futher described with respect to the following Examples; however, the scope of the present invention is not intended to be limited thereby. EXAMPLE 1

Plasmid pG1 was constructed from pLNSX (Palmer et al., Blood, 73:438-445; 1989). The construction strategy for plasmid pG1 is shown in Figure 1. The 1.6 kb EcoRI fragment, containing the 5' Moloney Sarcoma Virus (MoMuSV) LTR, and the 3.0 kb EcoRI/Clal fragment, containing the 3' LTR, the bacterial origin of replication and the ampicillin resistance gene, were isolated separately. A linker containing seven unique cloning sites was then used to close the

EcoRI/Clal fragment on itself, thus generating the plasmid pGO. The plasmid pGO was used to generate the vector plasmid pG1 by the insertion of the 1.6 kb EcoRI fragment containing the 5' LTR into the unique EcoRI site of pGO. Thus, pG1 consists of a

retroviral vector backbone composed of a 5' portion dervied from MoMuSV, a short portion of gag in which the authentic ATG start codon has been mutated to TAG (Bender et al. 1987), a 54 base pair multiple cloning site (MCS) containing from 5' to 3' the sites EcoRI, Notl, SnaBI, SaLl, BamHI, Xhol, Hindlll, Apal, and Clal, and a 3' portion of MoMuLV from base pairs 7764 to 7813 numbered as described in (Van Beveren et al., Cold Soring Harbor, Vol. 2, pg. 567, 1985). (Figure 2). The MCS was designed to generate a maximum number of unique insertion sites, based on a screen of non-cutting restriction enzymes of the pG1

plasmid, the neo^R gene, the β -galactosidase gene, the hygromycin^R gene, and the SV40 promoter.

The pGene plasmid (Figure 3) does not exist as an independent molecular entity, but rather may be considered a construction intermediate in the process of cloning genes for subsequent insertion into pG1. The basic backone is that of pBR322 (Bolivar et al., Gene, 2:95 1977). To the 2.1 kb EcoRI/Ndel fragment containing the ampicillin resistance gene and the bacterial origin of replication two linkers were ligated. These linkers, synthesized using an

oligo-nucleotide synthesizer, contain a total of 14 unique restriction enzyme recognition sites, as well as sequences felt to enhance mRNA stability and translatability in eukaryotic cells. The restriction sites were chosen based on a screen of non-cutting restriction enzymes of the plasmid backbone, the neo^R gene, the β -galactosidase gene, the hygromycin^R gene, and the SV40 promoter. Genes can be ligated into this backbone with Ncol and Xhol ends. The resulting backbone, less the inserted gene, is roughly 2.1 kb in size and contains a 99 base pair multiple cloning site containing, from 5' to 3', the following restriction enzyme recognition sites: Sphl, Notl, SnaBI, Sall, SacII, Accl, Nrul, Bglll, Ncol, Xhol, Hindlll, Apal, and Smal (Figure 4). From the Bglll to the Ncol sites lies a 27 base pair region containing an mRNA signal based on the work of

Hagenbuchle et al. Cell, 13:551-563 (1978). It has been found that the 3' terminal sequence of 18S ribosomal RNA is highly conserved among eukaryotes, suggesting that complementary sequences between 18S RNA and mRNA may be involved in positioning the initiating start codon (AUG) on the 30S ribosome.

Synthesis of adenovirus 2 late proteins, particularly polypeptide IX, may also follow this rule (Lawrence and Jackson, J. Molec. Biology, 162:317-334(1982)). Following this ribosomal binding signal, a consensus signal for initiation of translation based on Kozak's rules (Kozak, Nucl. Acids Res., 12:857-872 (1984)) was also inserted. The wobble at the ATG was used which permitted use of an Ncol restriction enzyme site.

In general, genes may be inserted in between the Ncol and Xhol sites. Promoters may then be added by insertion into the Nrul site, if the restriction enzyme map of the inserted gene leaves this site as unique. However, the construction of the multiple cloning site is such that, even if some sites no longer remain unique after a gene is inserted, there is a substantial likelihood that sites 5' of the Bglll site remain available for promoter insertions. Once a promoter/gene assembly is completed, the entire combination may be removed for insertion into the pG1 backbone. Generally, this has been

accomplished by using the complementary Sall and Xhol sites of both the pGene backbone and pG1; however, enough sites are included in the multiple cloning site to generally ensure that, by simple directional cloning, promoter/gene combinations may easily be inserted into the pG1 backbone.

As an example of the utility of the pGene/pG1 system, the generation of several vectors is

described. The first is called pGlN2SvBg, a vector using the bacterial neomycin resistance (neo^R) gene as a selectable marker and also containing the bacterial β-galactosidase (β-gal) gene under regulation of the SV40 early promoter. First, a 769 base pair Eagl/HincII fragment containing all but the very 5' portion of the neo^R gene was isolated from the plasmid pMClNeo (Thomas and Capecchi, Cell

51:503-512 (1987)). To this fragment was ligated a 50 base pair Sphl/Eagl linker. This linker restored all of the 5 '-most codons of the neo^R gene, and created an Ncol site at the authentic ATG start codon. This fragment was then blunted using the Klenow polymerase, and ligated into pG1 at the unique SnaBI site, generating pGlN2. The second step in the generation of the pGlN2SvBg vector was the

construction of pBg, that is the pGene backbone with the β-gal gene inserted into the multiple cloning site. The 3.0 kb BamHI/EcoRI fragment of the lacZ gena encoding/-β-galactosidase was isolated and two linkers were added. To the 5' end an Ndel-BamHI linker, containing the 5' portion of the multiple cloning site up to the Ncol site, as well as the first 21 base pairs of the lacZ gene, was ligated. To the 3 ' end, an EcoRI/EcoRI linker completing the 3 ' sequence of the lacZ followed by sequence encoding the Xhol, HindXII, Apal, and Smal sites was ligated. The sequence of the 5' EcoRI site was mutated, maintaining amino acid coding fidelity but eliminating the internal EcoRI site to permit

directional cloning and screening of the total linkered lacZ fragment into the 2.1 kb Ndel/EcoRI of pBR322. The pBg plasmid was then used to construct an SV40 promoted β -gal gene. The 339 base pair PvuII/Hindlll SV40 early promoter fragment was then inserted into both pBg in the unique Nrul site to generate the plasmid pSvBg. Once pSvBg was obtained, it was a simple matter to obtain the Sall/Xhol fragment containing the SV 40- promoted lacZ gene and insert it into Sall/Xhol digested pG1N2, thereby generating pG1N2SvBg.

Vector producer cell lines were prepared using established protocols. The packaging cell line PE501 (Miller and Rosman, Biotechniques 7:980-990 (1989)) was plated at a density of 5 x 10⁵ cells per 100 mm plate and the following day purified vector DNA was introduced using standard CaPO₄ precipitation (Wigler et al., Cell 14725-731 (1978)). For each plate of cells to be transfected, 20-40 μg of vector DNA was prepared with a co-precipitate consisting of 0.25M CaCl₂/l mM Hepes (pH 7.2) and 140 mM NaCl, 0.75 mM Na₂HPO₄, 25 mM Hepes (pH7.2). The DNA/precipitate was allowed to sit at room temperature for 30 min and then added (1 ml/plate) to the cells in tissue culture medium (DMEM + 10% fetal Bovine serum) for an overnight .incubation. The medium was changed to fresh DMEM + serum the following morning. The transfected cells were allowed to grow to near confluence for the next 48 hours, at which point virus supernatant was collected to infect a separate population of PA317 vector packaging cell lines at a density of 1 x 10⁵ cells per 100 mm plate seeded 24 hours prior to infection. The standard infection conditions include undiluted virus supernatant, filtered through a 0.2 uM membrane, to which 8 ug/ml polybrene is added. The transduced cells can then be analyzed directly based on β-gal expression or be selected with the neomycin analogue, G418 sulfate, to enrich for cells expressing the neo^r gene.

When G418 resistant trans-infected clones appeared, these were collected and grown up by expansion through a series of tissue culture vessels until a population great enough (over 1 x 10⁶ cells) was generated that titer could be determined.

Between 10 and 20 clones were evaluated for the number of G418-resistance conferring particles they generated (i.e. "titered"). Titers based on G418 resistance were performed using standard titering methods (Eglitis et al., Science 230:1395-1398

(1985)). Briefly, NIH 3T3 cells were plated at a density of 2 x 10⁴ cells per 35 mm dish and the following day infected for 2-4 hours with various dilutions of virus supernatant containing 8 ug/ml polybrene. The cells were allowed to grow for an additional 24-48 hours following infection and then were grown in selective medium containing G418 (800 ug/ml) for 10-12 days prior to staining with

methylene blue and counting individual G418

resistance colonies. Producer clones were identified which generated between 5 x 10⁴ and 5 x 10⁵ G418 resistant colony-forming units per ml.

The ability of the GlN2SvBg vector to transduce cells with a functional bacterial lacZ gene was established by detecting β-gal activity after reaction with the chromogenie substrate

4-Cl-5-Br-3indolyl-β-galactoside (X-gal). The X-gal staining procedure has been described in detail previously by Sanes et al. EMBO 5:3133-3142 (1986). The NIH 3T3 target cells are plated and infected with viral supernatant as described above. Infected cells are passed at a dilution of 1:20 48 hours

post-infection and allowed to grow to confluence. When confluent, plates are rinsed with PBS, brief ly fixed with 2% formaldehyde plus 0.2% glutaraldehyde in PBS and incubated overnight at 37 ° C with a reaction mixture containing 1 mg/ml X-gal. Distinct patches or "colonies" of blue cells, which arise clonally from individual infected cells, will then be visible if the vector confers the expression of active β -gal enzyme.

In other examples of the utility of the

pGene/pG1 system, the strategy for the generation of two vectors is described. The first is called pG1BgSvCb, a vector using the bacterial

β-galactosidase gene as a selectable markeer and also containing a marked truncated CD4 gene under

regulation of the SV40 early promoter. The second is called pG1BgSvCd, a vector similar to pG1BgSvCb but encoding instead the native soluble CD4. The first step in the generation of these vectors is the insertion of the 3.0 kb Ncol/Xhol fragment containing the lacZ gene obtained from pBg (see above) and inserting it by blunt ligation into the SnaBI site of pG1, thereby generating pG1Bg.

The pBg plasmid is also used to construct an SV40 promoted, truncated CD4 gene. The construction is begun with the 534 base pair Haell/Nhel fragment of the CD4 gene encoding the amino terminal 178 amino acids of the CD4 receptor. To the 5' end of this fragment is ligated an Ncol/Haell linker encoding the 23 amino acid leader sequence of CD4, including the authentic ATG start codon. To the 3' end is ligated, in one case, a 45 base pair Nhel/Xhol linker encoding for the bungarotoxin binding domain of the acetylcholine receptor (Btx). This binding domain provides a very sensitive radio-assay for the

presence of the secreted CD4 protein. The resulting 601 base pair Ncol/Xhol CD4/Btx fragment is inserted into pBg into the place of the Ncol/Xhol fragment of the lacZ gene to result in the plasmid pCb. In a second case, to the 3' end was ligated a 30 base pair Nhel/Hindlll linker coding for six natural CD4 amino acids, followed by a repetitive stop signal. This 586 base pair Ncol/Hindlll natural CD4 fragment was also inserted into pBg into the place of the

Ncol/Hindlll fragment of the lacZ gene to result in the plasmid pCd. The 339 base pair PvuII/Hindlll SV40 early promoter fragment was then inserted into both pCb and pCd in the unique Nrul site to generate the plasmids pSvCb and pSvCd. To generate the final pG1BgSvCb and pG1BgSvCd vectors, the Sall/Xhol fragment of pSvCb containing the SV40 promoted

CD4/Btx gene or the similar Sall/Hindlll fragment of pSvCd were individually ligated to the large

Sall/Xhol fragment of pG1Bg. The cloning strategy for generating retroviral vector pG1BgSvCb is shown in Figure 5.

The cloning strategy for three other vectors, pGlN2Sv12, pG1N2Sv111, and pG1N2SvI11 provide further examples of the utility of the pGene/pG1 system. All of these vectors may be easily derived directly from pGlN2SvBg, described above, with the gene for β-gal replaced by one for interleukin-2 (IL-2),

interleukin-1β (IL-1β), or tumor necrosis factor-α (TNFα), respectively. The IL-2 gene is derived from the plasmid HT-5.1 (ATCC #59396). The 1.0 kb BamHI fragment is isolated from this plasmid and then truncated down to a 445 base pair HgiAI/Dral

fragment. To restore the authentic 5' coding sequence, a 100 base pair linker is constructed including the entire 20 amino acid coding region of the amino-terminal end of IL-2, and then a 40 base pair stretch identical in sequence to that of pGene between the Bglll and Ncol sites is added as a 5' leader. A SnaBI site is added 5' to the Bglll, permitting direct insertion of this reconstructed IL-2 fragment into pBg which has been digested with SnaBI and Hindlll (the Hindlll blunted with the

Klenow polymerase). From this resulting pI2 plasmid, a 550 base pair Bglll/Clal fragment is isolated and then inserted into Bglll/Clal digested pGlN2SvBg in the place of the lacZ gene.

The vector pGlN2SvIll is constructed using a commercially available IL- 1β gene obtained from Beckman (catalogue number 267408). The gene is isolated as a 499 base pair NcoI/EcoRI fragment and inserted in the place of the lacZ gene in Ncol/EcoRI digested pBG. An 87 base pair oligomer containing the rat growth hormone secretion signal is then inserted into the Ncol site. The resulting gene can then be removed as a 586 base pair Bglll/BamHI fragment, filled in with Klenow polymerase, and inserted into pGlN2SvBg. The lacZ gene of pGlN2SvBG is removed by digestion with Bglll and Xhol, followed by filling in with Klenow polymerase. These few, simple steps thus yield the final pG1N2SvIll final vector.

As a final example, pGlN2SvT11 is constructed similarly. Starting with a commercially available plasmid (Beckman catalogue number 267430), the TNF gene is isolated as a 521 NcoI/EcoRI fragment, inserted in the place of the lacZ gene in Ncol/EcoRI digested pBG and has added the identical rat growth hormone secretion signal described above. The resulting gene is then removed as a 608 base pair Bglll/BamHI fragment, and inserted into pGlN2SvBg digested with Bglll and Xhol as described above.

This emphasizes how, by using pBG as an intermediate, several different genes can rapidly and identically be inserted into the same vector backbone, in this case to generae pGlN2SvT11.

Thus, by a simple process involving minimal steps, fragments of plasmids may rapidly be exchanged and new vectors can be constructed. If regulation by a different promoter is desirable, a variety of strategies would be available. In the instance of pSvCb, the SV40 promoter could be removed and

replaced by an alternative. Also, an entire series of promoters could be inserted into the identical Nrul site of pCb, or the Ncol/Xhol fragment

containing the CD4/Btx gene could be removed from pCb and put in the place of a gene running off a

different promoter (eg., β-galactosidase regulated by the Cytomegalovirus promoter, exchanging CD4/Btx for β-galactosidase). This last method requires, for maximal efficiency, that the gene with the different promoter is in the pGene backbone.

EXAMPLE 2

To create an example of a retroviral vector system which provides for more accurate regulation of internal genes a derivative of pG1 called pG2 was constructed. A construction strategy for pG2 is shown in Figure 6. The difference between pG1 and pG2 is a series of sequence alterations in the 3' LTR which eliminate all enhancer and promoter function without altering the overall structure of the LTR. When a vector is introduced into a target cell, viral replication duplicates the U3 and R portions of the 3' LTR to both the 5' and 3' ends of the proviral integrant. The U3 portion of the LTR is 449 base pairs in length and incorporates several regions of strong enhancer activity. Also in the U3 region are sequences capable of binding several transcriptional regulatory proteins, as well as sequences of the consensus distal and proximal promoter signal regions (i.e., the CAAT and TATA boxes). The R portion of the LTR is 70 base pairs in length and contains the signal for polyadenylation of transcribed mRNAs. In addition, the R portion is a region of strong

ribosomal binding and is the region where

transcription regulated by the promoter in the U3 portion is initiated. As such, the 5' end of R represents the "cap site" for the transcribed viral RNAs. Since the 5' LTR is now lacking its own regulatory sequences, introduced genes are completely under the regulation of their own promoters. This enables the construction of vectors with gene

expression optimized for particular target cell types. This differs from previous attempts to disable retroviral regulatory sequences in that other efforts relied on large deletions of the LTR to eliminate the retroviral enhancer and/or promoter. Such substantial disruption of LTR structure likely contributed to the substantial decreases in vector titer that have been observed with such vectors, since LTR structure is critical for efficient

integration of the provirus into the cellular genome. By utilizing sequence alterations instead of

deletions, the elimination of retroviral regulatory sequences with the maintenance of LTR structure and high titer are combined making the G2 vector series much more amenable to clinical applications where high titer is critical. The details of the construction are as follows: Starting with the pGO plasmid, we subcloned from this a 619 base pair Clal/Smal fragment from base pairs 7676 to 8295 of MoMuLV from the 3' end of env to just inside R. This fragment was further digested with Sau3A, and a 235 base pair ClaI/Sau3A fragment representing the 5' most portion, ending 12 base pairs 5' to the first long enhancer repeat within U3, was isolated. The remainder of the original

Clal/Smal fragment was reconstructed with a series of twelve overlapping oligonucleotide fragments. These fragments maintained the overall length of the original LTR, but incorporated sequence alterations which eliminated the recognition sequences for enhancers or promoters. The overall result of this reconstruction was to generate a new Clal/Smal fragment equal in length and general structure to the native fragment, but with all enhancers, distal promoter and TATA regions altered to

non-functionality. A diagram of the natural MoMuLV LTR and the region of the altered sequence is shown in Figure 7. The altered sequence is shown in Figure 8. This new Clal/Smal fragment was then restored into the remaining Smal/Clal fragment from pGO.

Then, the same EcoRI fragment containing the 5'

MoMuSV LTR as used to make pG1 was inserted into the unique EcoRI site of the altered pGO to yield the vector backbone pG2. This vector backbone is

identical to pG1 up to the first Sau3A 3' of the unique Clal site. Then, the 384 base pairs of the altered 3' LTR, and finally normal LTR sequence, including integration signals.

This vector combines all the cloning advantages of pG1, in that the multiple cloning site is

compatible with that of the pGene plasmid. In addition, it incorporates changes, but not deletions, of the 3' LTR such that inactiviation of retroviral regulatory sequences occurs in the integrated

provirus without sacrifice of integration efficiency and its reflection in maintenance of high titer. The pG2 vector provides a useful backbone for the

introduction and expression of genes which require regulation of maximal accuracy. This enables one to correct genetic defects involving genes under very precise physiological regulation.

It is to be understood, however, that the scope of the present invention is not to be limited to the specific embodiments described above. The invention may be practiced other than as particularly described and still be within the scope of the accompanying claims.

Claims

WHAT IS CLAIMED IS:

1. A retroviral vector, said vector including at least four different restriction enzyme sites, and wherein at least two of said at least four sites have an average frequency of appearance in eukaryotic genes of less than once in 10,000 base pairs.

2. The retroviral vector of Claim 1 wherein said at least two of said at least four sites are selected from the group consisting of Notl, SnaBI, Sall, and Xhol.

3. The retroviral vector of Claim 1 wherein said vector includes the Notl, SnaBI, Sail, BamHI, Xhol, Hindlll, Apal, and Clal cloning sites.

4. The retroviral vector of Claim 3 wherein said retroviral vector is pG1.

5. The retroviral vector of Claim 1 wherein at least the promoter sequence of the 3' LTR is mutated such that said promoter sequence becomes

non-functional.

6. A cloning system for the manipulation of genes in retroviral vectors, comprising:

a retroviral vector including at least four different restriction enzyme sites, wherein at least two of said at least four sites have an appearance in eukaryotic genes of less than once in 10,000 base pairs; and

a shuttle cloning veptor including at least two cloning sites compatible with at least two cloning sites selected from the group consisting of Notl, SnaBI, Sall and Xhol, of said retroviral vector, and said shuttle cloning vector including at least one desired gene sequence capable of being transferred from said shuttle cloning vector to said retroviral vector.

7. The cloning system of Claim 6 wherein said first retroviral vector includes the Notl, SnaBI, Sall, BamHI, Xhol, Hindlll, and Apal, and Clal cloning sites.

8. The cloning system of Claim 7 wherein said second vector includes the Sphl, Notl, SnaBI, Sall, SacII, Accl, Nrul, Bglll, Ncol, Xhol, Hindlll, Apal, and Smal cloning sites.

9. A retroviral vector produced by

transferring said at least one desired gene from said shuttle vector of Claim 6 to said retroviral vector of Claim 6.

10. A retroviral vector including a 3' LTR, wherein at least the promoter sequence(s) of the 3' LTR is mutated such that said promoter sequence becomes non-functional.

11. The retroviral vector of Claim 10 wherein said vector further includes a mutation(s) of the enhancer sequence(s) of the 3'LTR such that said enhancer sequence(s) becomes non-functional.

12. The retroviral vector of Claim 10 wherein the integrator sequence is maintained.

13. A retroviral vector, said vector including a heterologous gene, said retroviral vector having been prepared from the vector of Claim 1.

14. A packaging cell transfected with the retroviral vector of Claim 13.

15. Infectious viral particles generated from the retroviral vector of Claim 13.

16. A eukaryotic cell transfected with the infectious viral particles of Claim 15.