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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells

Definitions

• the present invention relates to the use of viral vectors in recombinant DNA manipulation and in therapeutic and analytical proced ures. More particularly, it relates to the use of modif ied mammalian long terminal repeat (LTR) vectors for obtaining selectable tissue exp ression of heterologous genes.
• LTR long terminal repeat
• This invention is directed to a process for the tissue specific exp ression of heterologous genes using modified LTR vectors.
• Any retroviral LTR can be synthetically modified in its enhancer region using the methods described herein and tissue specificity can thereby be achieved.
• This invention is also directed to novel tissue specific LTR vectors.
• tissue specific vectors are constructed, using a tissue specific enhance r(s) operatively positioned in the same sequence with a heterologous DNA segment corresponding to the polypeptide of interest, as well as a stop codon and polyadenylation sequence downstream (3 ') from that gene.
• the vector should also contain a replication origin, as is known in the art.
• the vector contains at least the segment of an enhancer which determines the tissue specificity of that enhancer, hereinafter referred to as the "tissue specificity determinant.”
• the vector preferably contains a complete viral enhancer which preferably includes the tissue specific determinant for the desired tissue.
• the promoter contained in the vector can be any of the known promoters which function to permit expression of the desired product in the host of choice.
• the promoter is a viral promoter from the same class of virus in the enhancer.
• the preferred class of virus is retrovirus, and the preferred viruses for use in conjunction with the invention are the Akv, SL3-3, and F riend viruses.
• tissue specific means that the vector operates to produce a g reater amount of desired product in the targeted tissue than it does in other tissues under normal culture conditions. Tissue specific vectors may produce 1.5 to 1,000 or more times as much expression product in the target tissue as in other tissues.
• tissue specific determinant can be homologous, meaning it came from the same virus as the promoter, or heterologous, in which case it is not from the same virus as the promoter.
• Heterologous tissue specific determinants can be excised f rom other viral systems, or can be synthesized using known techniques.
• Tissue specific determinants which are specific to the target tissue can be identified by assay techniques, where vectors encoding an indicator or marker compound, e.g., chloramphenicol acetyl transf erase (CAT) , an indicator which can be easily quantified as described below, to determine which vectors are effective in the tissue.
• an indicator or marker compound e.g., chloramphenicol acetyl transf erase (CAT)
• enhancer(s) from tissue specific vectors can be compared in DNA sequence to the enhancers which are not specific to the target tissue to determine its DNA sequence of the tissue specific determinant. Thereafter, an enhancer which contains the tissue specific determinant may be utilized in the desired vector containing the gene which it is desired to express, and the resulting tissue specific vectors utilized to express the desired product in the tissue of choice.
• Tissue specific expression is highly advantageous in the production of large quantities of desired product from the chosen tissue. It minimizes expression in other tissues. Such techniques can be used to characterize or identify tissue, to foster growth of some tissues over others, to minimize the amount of contaminants produced, and for other purposes.
• the vectors of the present invention can be in the form of plasmids, or viral vectors, such as those produced in accordance with the procedure described by Mann et al., Cell, 33: 153 (1983) .
• the vectors constructed in accordance with this invention can also be utilized in vivo, e.g., for the treatment of diseases caused by genetic or other defects wherein certain types of cells produce superabundant or damaging amounts of particular materials. In the latter case, such excess materials can be eradicated for example, by expressing the gene which codes for an enzyme or other material which reacts with the overproduced material. Other methods of genetic treatment utilizing the present invention are described below.
• the vectors used are preferably viral vectors as described above.
• Figures 1A, 1B, and 1C illustrate the isogenic constructs made when the U3 region of Akv (pAU3CAT) and SL3-3 (pU3CAT) were placed 5' to the chloramphenicol acetyl transf erase (CAT) gene;
• Figu res 2(a) - (d) illustrate the dramatic difference in the CAT activity, seen between the Akv and SL3-3 , LTR sequences;
• Figure 3 is a compa rison of the seq uence organization within the tandem repeat regions of the Akv (Van Beveren, C., et al., J. Virol., 41: 542 - 556 (1982) , SL3-3 (Lenz, J. , et al., J. Virol., 47: 317 - 328 (1983) and Fr-MuLV57 (Olif f, A.I., et al., J. Virol., 33: 475 - 486 (1980) ) LTRs. The sequence of a single 99 bp repeat element of Akv is shown at the top.
• SL3-3 is an N tropic, ecotropic, potent leukemogenic virus isolated f rom a spontaneous T cell lymph oma of an AKR mouse.
• SL3-3 resembles the endogenous, N tropic Akv virus in both viral genome structure and replication properties in tissue culture, SL3-3 is capable of inducing T cell leukemias in a wide variety of mouse strains, whereas Akv is non-leukemogenic.
• Virus produced upon transfection of NIH 3T3 with the SL3-3 and Akv recombinant proviruses was tested for the ability to induce disease.
• One such recombinant a virus that contains all of the Akv structural genes flanked by SL3-3 LTR sequences, induced T cell leukemias in several strains of mice.
• Nucleotide sequence analysis of the SL3-3 LTR revealed that the only difference distinguishing the LTR of the SL3 from that of the avirulent Akv virus, was located within the repeat element present in the U3 region, (with the exception of a single nucleotide change near the 5 ' end of the LTR) .
• This repeat region known to function as an enhancer element in related viruses, may permit viral gene expression to occur in the appropriate cellular context. For this reason the ability of the LTR sequences of SL3-3 and Akv to function as transcriptional elements in several cell types were tested.
• Recombinant viruses that possess the LTR region of SL3-3 also replicate to high titers in thymocytes, but not in marrow or spleen cells of infected animals.
• the avirulent Akv virus itself does not replicate to high titers in any of these tissues.
• the cell type specificity of leukemias induced by viruses containing different LTR seq uences is due in part to the ability of the virus tp replicate in the approp riate cellular environment (see, DesG roseillers, L. , et al., PNAS USA, 80: 4203-4207 (1983) ; Chatis, P. A. , et al., PNAS USA, 80 : 4408- 4411 (1983) ) .
• LTR encodes tissue permissive transcriptional elements. It has been discovered that there are differences in the transcriptional activity of the SL3-3 and Akv LTR sequences in different murine cell types, and that the sequences present in the LTR of SL3-3 exhibit significantly enhanced transcriptional activity in T cells as compared to the cor responding region of the Akv LTR. The results suggest that transcriptional elements are primary determinants of cell tropism and of leukemogenicity of these viruses.
• plasmids were introduced into cultured cells using either the calcium phosphate (see, Wigler, M. , etal., Cell, 14: 725-731 (1978) ) or the DEAE-dextran methods (see, Stafford, J. et al., Natu re, 306: 77-79 (1983) ) . After a transient expression period of 44-50 hours, cell extracts were prepared, and the level of CAT enzymatic activity was measured (Gorman et al., s upra) .
• the level of CAT gene expression of the plasmid containing the Akv LTR sequences is th ree times that of the plasmid containing the corresponding region of the SL3-3 virus.
• a similar result was obtained using mouse L cells, another murine fibroblast cell line. F rom these results we conclude that in some murine fibroblasts, the Akv LTR seq uences are more potent than those of SL3-3 in promoting CAT gene exp ression.
• LTR sequence between SL3-3 and Akv are located within the tandemly duplicated region located 221 to 443 bp from the RNA start site (Lenz et al., Natu re, s upra) , a region shown to include enhancer element functions of other murine retroviruses (see, Spandidos et al., supra; Blair, D. G., et al.,PNAS USA, 77: 3504-3508 (1980) ; Blair, D. G., et al.,
• the portion of the SV40 early region promoter that governs start site specificity was positioned 5' to the CAT gene (designated as pSVIXCAT-AB3) , and the region harboring the tandem repeat element of either SL3-3 or Akv was linked in cis, in both orientations, at a site about 2 k ilobases from the SV40 early start sites ( Figure 1b) .
• plasmids were transfected into murine T cells (the AKSL3 cell line) , and the levels of CAT activity were determined relative to pRSVCAT and pSVIXCAT-AB3) (Figu re 2C) , Table 2) .
• the level of CAT activ ity directed by the plasmid that contains the SL3-3 repeat region was more than 30 times that of the pSVlXCAT-AB3 plasmid that lacks murine LTR seq uences.
• the increased rate of CAT gene transcription directed by the SL3-3 LTR element in T cells permits an explanation of the T cell tropism and leukemogenic potential of the SL3-3 LTR containing viruses.
• Replication of retroviruses proceeds via transcription from integrated proviruses. Tissue specific differences in the rate of transcription of the magnitude observed here, 10 to 60-fold, may have a profound effect on the extent of viral replication, as such differences might be expected to be magnified as a power function on successive rounds of replication.
• the present inventors speculate that an additional, if not simultaneous, characteristic of the LTR of a T cell leukemogenic virus. is to provide strong enhancer element function sufficient to transcriptionally activate adjacent cellular loci.
• the level of CAT gene expression on the plasmid that contained the Akv LTR sequence is three times that observed by the plasmid that contains the corresponding region of the SL3-3 virus.
• a similar result was obtained using mouse L cells, another murine fibroblast cell line.
• T cell lines established from murine T lymphomas were used as recipients and include ; SL3, a cell line derived from a premature thymic T cell tumor of an AKR mouse that had been injected with the SL3-3 virus (see, Hayes, E.F., et aI., J. Natl.
• AKSL3 a cell line established from a spontaneous tumor of an AKR mouse, the cell line from which the SL3-3 virus was isolated (see, Nowinski, R.C., et al., J. Virol., 27: 13 - 18 (1978); Laimins, L.A., et al., PNAS U SA, 79: 6543 - 6547 (1982); Oskarsson, M., et al., Science.
• the level of CAT activity directed by the plasmid that contains the SL3-3 LTR sequences is much higher than that which contains the corresponding Akv sequences in all the T cell lines tested.
• the ratio of activities in SL3-3/Akv ranges f rom 0.3 to 0.6 in fibroblasts and f rom 6 to 20 in T lymphocytes.
• the level, of activity of these two plasmids is roughly comparable in all cell types used, and activ ity of the viruses that contain the murine LTR. sequences are expressed relative to the level CAT activity obtained in transfection experiments with pRSVCAT and pSV2 CAT plasmids.
• Each element's activity is normalized relative to the absolute activity of the 3 ' LTR of RSV in pRSVCAT within each cell line. Absolute activity for each plasmid in every cell line was calculated f rom the slope of the line in the initial velocity of the CAT enzymic assay for cell extracts containing 400 fg of protein as depicted in Figure 2.
• the absolute activ ity in any given cell line transfected with each plasmid is expressed in terms of percent conversion/hour in the in vitro assays, and may be obtained quantitatively by multiplying the relative value in the Table by the absolute activity value of the same cell line transfected with pRSVCAT.
• NIH3T3 (2.06%/hr)
• L (0.64%/hr)
• M12 (2.5%/hr)
• AKSL3 (4.8%/hr)
• SL3 B 2.04%/hr
• L691 0.4%/hr
• AND L691.SL3-3 (2.37%/hr) .
• murine leukemia viruses Upon inj ection of susceptible strains of mice, murine leukemia viruses exhibit characteristic tissue specificity, latency period, and disease phenotypes. Recent findings demonstrate that the ability to induce T cell leukemia is associated with the non-coding long terminal repeats (LTRs) of these viruses (Lenz, supra; Chatis, P. A. , et al., PNAS U SA. 80: 4408 (1983) ; and Des Groseillers, supra) . To test if the LTR sequences might determine the preferred organ site of replication and disease phenotype, a series of isogenic recombinant viruses that differ only in the LTR regions were constructed. Viruses used included the Akv virus (Rowe, W.
• the ability of these viruses to replicate efficiently in the thymus or spleen and to induce thymic or erythro leukemias is determined by the LTR sequences. Additionally, it has been discovered that recombinant MCF viruses are not detected in animals infected with these viruses.
• the ability of virus to replicate in thymus, spleen, and marrow cells was determined by flushing the cells from the organ, and measuring the number of infected cells using an infectious center, XC assay.
• Table 2 shown that the titers of Akv virus remained low in the thymus, spleen and marrow of animals infected with Akv for the duration of the experiments, 20-40 weeks.
• the virus titers in the thymus of animals inoculated with RECAS115 were high in the range of 1-5 X 10 5 by 6 weeks post- inoculation.
• the titers were low in the spleen and marrow, generally less than 5 X 10 2 throughout the duration of the experiment.
• the phenotype of the cells was also examined. These studies demonstrated that the majority of the cells infected by viruses that contain the SL3-3 LTR sequences were adherent neither to nylon wool nor to plastic, were theta antigen positive, IgG negative, Ly1+, and Lt2-. About 20% of infected cells in the spleens of these animals were theta negative and, IgG positive, indicating that B cells, or B cell precursor might also be infected. Previous studies show viruses that induce T cell leukemia also infect B cells at a low but measurable frequency.
• the organotropism of the Friend recombinants differs dramatically from those with Akv or the S13-3 LTR recombinants (Table 4) . These viruses replicate to high titers in the spleen but not the thymus. Viral titers are at least two orders of magnitude greater in the spleens, and about two orders of magnitude less in the thymus relative to Akv and RECAS115.
• Recombinants between inoculated viruses and cellular sequences that encode MCF envelope genes are often detected in the preleukemic and leukemic stages of disease induced by both thymic and erythro leukemia viruses (Teich, N.M., et al., Cell, 12: 937 - 982 (1977) ; Coffin, J., Endogeneous Viruses in RNA Tumor Viruses. Cold Spring Harbor Laboratory (1982) ; Famulari, N.G., et al., J. Vi rol.. 40: 971 - 976 (1981)) .
• infected thymocytes or spleen cells were tested for the presence of MCF viruses.
• the ability of the infected cells to produce foci in mink cells Hartley, J. W. et al., P roc. Natl. Acad. Sci .
• results presented here demonstrate that the ability of viruses to replicate efficiently in either thymus or spleen of NFS mice is a property of the LTR and is not determined by the gag, pol or env genes of the virus.
• tissue specificity of replication of the viruses examined herein is not attributable to formation of M FC recombinants, as no such recombinants were formed upon infection of NFS mice for either of these viruses. For the same reason, it is not believ ed that induction of the thymic leukemia of erythroleukemia requires formation of M FCs.
• the sequence, of the LTR of Akv, SL3-3, Fr-MuLV and Mo-MuLV are shown in Figure 3.
• the only difference in the sequence of the Akv and SL3-3 viruses are located in the tandem repeat sequence of the U3 region of the LTR (Lenz, supra) .
• the sequence of the U3 region of Fr-MuLV differs from that of Akv and SL3-3 in a similarly placed tandem repeat region.
• the sequence of the LTR of Fr-MuLV also differs from that of the Akv and SL3-3 virus in the region 3' to the tandem repeat elements.
• the sequence of the Friend LTR is very similar to that of the thy motropic Mo- MuLV (Koch, 1983) .
• the tandem repeat elements of Mo-M uLV, Akv, and SL3-3 hav e been shown to function as transcriptional enhance r elements capable of di recting high levels of t ranscription of heterologous promoters (Lev inson, B., et al., Natu re, 295: 568-572 (1982) ; Laimins, su pra; Oskarsson, M. , et al., Science.
• the plasmid subclones harboring the 5 '-LTR of Akv and SL3-3 (pLTR-A4 and pLTR-S3, respectively) were provided by J. Lenz.
• the Pstl-Aval fragment of the LTRs comprise the U3 and a small f raction of the R regions, extending from -443 to +31, relativ e to the viral RNA start site (designated as +1) for both viruses.
• This fragment was isolated f rom each LTR ⁇ ubclone where. the natural Aval site was converted to a Hindlll site using Hindlll synthetic linkers, and the natural PstI site was converted to a blunt end.
• the plasmid that served as the recipient vector of the LTR fragments contains the entire CAT gene transcription unit (Gorman et al., Mol. Cell. Biol ., su pra) (CAT coding sequences followed by the simian virus 40 (SV40) t- intron and polyadenylation signals) under the control of a SV40 early region promoter mutant that carries a deletion of nearly the entire 72 bp tandem repeat element (designated as pSVIXCAT, see infra) .
• CAT gene transcription unit Gibman et al., Mol. Cell. Biol ., su pra
• SV40 simian virus 40
• the SV40 early region promoter bounded by a synthetic Xhol site (5') and a natural Hindlll site (3') , was removed f rom pSVIXCAT by successive digestion with Xhol, T4 DNA polymerase, and Hindlll enzymes in a fashion whereby the Xhol site was converted to a blund end.
• the LTR fragments were ligated to the vector fragment containing the CAT gene transcription unit to generate plasmids in which the CAT gene is under the control of the transcriptional elements in the U3 region of Akv (pAU3CAT) or SL3-3 (pSU3 CAT) .
• the Akv and SL3-3 U3 regions are identical except for the presence of a single base insertion at nucleotide position 65 (designated by an asterisk) in SL3-3 relative to Akv and for the sequences within the repetitive elements (designated by arrows) (see, Lenz, et al., Natu re, supra) .
• a plasmid that ca rries the CAT gene under the control of the SVB40 early region promoter (designated as pSV2 CAT (Gorman et al., Molec. Cell. Biol., su pra) was digested with AccI and SphI to remove the majority of the 72 bp repeat region.
• the natu ral Acl and SphI sites were converted to Xhol sites using T4 DN A polymerase and an Xhol synthetic leader.
• the resulting plasmid contains the CAT gene unde r the control of SV40 promoter sequences extending f rom -127 to +65 (relative to the 5 ' most early start site) (Benoist et al., su pra) .
• the structure of this promoter deletion mutant is shown relative to the entire SV40 early region promoter found in pSV2 CAT.
• the repetitive elements designated by arrows) , the origin of replication (ori) , the TATA element (denoted at A/T) and early start site(s) (designated as +1) are illustrated.
• the SV40 promoter seq uences present in pSVIXCAT are deficient in enhancer element activity; however, this activity can be restored by linkage of sequences containing enhancer activity to pSVIXCAT at a variety of sites relative to the SV40 early start site(s) (unpublished observations) .
• pSVIXCAT-AB3 a variant of SVIXCAT (designated as pSVIXCAT-AB3) was constructed in which the natural Apal site (see, Fiers, W. , et al., Nature. 273: 113-120 (1978) ) located greater than 2.1 kilobases from the SV40 early start site(s) was converted to a Bglll site using T4 DNA polymerase and a synthetic Bglll linker.
• the U3 region sequences present in the 620 bp Sau3A f ragment consists of sequences extending f rom -443 to -106 (relative to the viral LTR start site in Figure la) ., and contains the entire tandem repeat region of each virus.
• CAT assays for plasmids containing either the Akv U3 region abbreviated Au3; SL3-3 U3 region, abbreviated SU3; the 3'-LTR of Rous sarcoma virus, abbreviated RSV; or no eukaryotic promoter element, abbreviated SVO, linked 5' to the CAT gene transcription unit transfected in parallel into cultured cells is shown in Figs. 1(a) -1(c) for NIH3T3 fibroblasts (a) and AKSL3 T cells (b).
• CAT assay of enhancer element function for plasmids containing either the SV40 promoter sequences lacking enhancer linked sequences abbreviated SVIX-AB3; the Akv repeat region inserted at the Bglll site 2 kilobases away from the SV40 early start site(s) in the same and opposite transcriptional sense as CAT gene transcription, abbreviated AE(+) and AE(-), respectively; the SL3-3 repeat region inserted at the Bglll site and in the same and opposite transcriptional sense as CAT gene transcription, abbreviated SE(+) and SE(-), respectively; and the 3' LTR of Rous sarcoma virus, abbreviated RSV, linked to the CAT gene and transfected in parallel into cultured cells is illustrated for AKSL3 T cells (c).
• Results are expressed as percentage conversion of 14C-chloramphenicol to 14C-chloramphenicol acetate by extracts containing 400ug protein in (a) and (b) and extracts containing 600 ug protein in (c).
• the insets show typical auto-radiograms of CAT assays at 30 minutes (a), 80 minutes (b), and 30 minutes (c).
• the upper two spots correspond to the two isomers of 14C-chloramphenicol monoacetate while the spot is the un reacted substrate, 14 C-chloramphenicol.
• Each murine cell line was transf ected with an equivalent amount of each plasmid DNA per transfection cocktail.
• the amount of DNA used for each experiment was within the liner range of optimal CAT activity.
• the NIH3T3 cell transf ections were carried out as described previously (Wigler, et al., su pra) .
• the L cell and lymphoid cell transf ections were done as described previously (Stafford et al., sup ra) .
• the CAT activity of each plasmid is normalized relative to the absolute activity of the 3'- LTR of RSV in pRSVAT within each cell line. Absolute activity for each plasmid in every cell line was calculated from the slope of the line in the initial velocity of CAT enzymic assay for cell extracts containing 400 ug of protein.
• the absolute activity in any given cell line transfected with each plasmid is expressed in terms of % conversion/hour in the in vitro assays, and may be obtained quantitatively by multiplying the relative value in Table 8 by the absolute activity value of the same cell line transfected with pRSVCAT.
• NIH3T3 2.06%/hr
• L 0.64%/h4
• M12 2.5%/hr
• AKSL3 4.87%/hr
• SL3 B 2.04%/hr
• L691 0.84%/hr
• L691.SL3-3 2.37%/hr
• NFS/N thymus cell 0.03%/hr
• T cell lines established f rom murine T lymphomas were used as recipients and include: AKSL3, a cell line established f rom a spontaneous tumor of an AKR mouse (the cell line from which the SL3-3 virus was isolated) (see, Nowinski, R. C, et al., J . Virol . , 27: 13-18 (1978) ) ; SL3 B, a cell line derived from a thymic T cell tumor of an AXR mouse that had been injected with the SL3 virus (see. Hays, E. F., et al., J. Natl. Cancer Institute, 69: 1077-1082
• L691 the uninfefcted murine T cell line, L691 (see, McGrath, M. S., et al., in: Contempora ry Topics in Immunobiology. Vol. II, ed. N. L. Warner, Plenum Press, New York, 157-184 (1980) ); L691.SL3-3, L691 cells infected in vitro with SL3-3 virus and freshly eluted primary thymus cells isolated f rom NFS/N mice (see, Now inski, R. C , et al., Vi rology. 81: 363-370 (1977)) .
• the murine B cell line, M12 was also used as a recipient.
• the CAT activity of each plasmid is normaliz ed relative to the absolute activity of the 3 '-LTR in RSVCAT within the T cell line AKSL3. Absolute activ ity was determined as described in Table 8. Results shown here are an average of 3 transf ection experiments and CAT assay s performed using extracts containing 600 ug of protein. The range of activ ities around a particular value varied not more than 30% of that value.
• Figu re 1 indicates the viral sequences which are present in the recombinant plasmids.
• the solid lines depict the regions of the SL3-3 LTR that is present in the respective recombinant genomes. Regions corresponding to the SL3-3 LTR are shown in black and cross-hatching respectively.
• the strategy taken to construct the recombinant genomes PR- 8+ RE CAS 115 has been previously described (Lenz, (1982) , su pra) .
• RE CAS 115 contains the coding sequences of Akv virus and the LTR plus 75 nucleotides of the untranslated leader sequence of SL3-3 virus.
• the recombinant genomes SF24 and AF226 contain the Fr-MuLV U3 region plus part of H and the coding sequence of SL3-3 and Akv virus respectively.
• An aproximately 450 bp Pstl-Kpnl fragment encompassing the U3 region and part of R was removed from the Akv (LTR ⁇ 4) and SL3 LTR (LTRS3) ⁇ ubclones.
• a similar Psti-Kpnl fragment encompassing the corresponding region of Fr-MuLV57 was isolated from a proviral clone (Oliff, et al., sup ra) and ligated to the pLTR-S3 and pLTRA4 ⁇ ubclones.
• the resulting ⁇ ubclones were cleaved with PVUI- EcoRI and ligated to restriction f ragments of the Akv and SL3 genome that lack the LTR sequences. Structure of the recombinant genomes was confirmed by digestion with restriction enzymes unique to the individual LTRs. Recombinant genomes containing a single LTR were linearized at their PstI sites and ligated to form concatamers containing two LTRs. Transf ection of recombinant proviral DNA into NIH 3T3 cells to yield infectious virus has been previously described (Lenz, et al., (1983) , su pra ; and Lenz, et al., (1982) , su pra) .
• Figu re 1C illustrates a comparison of the sequence organization within the tandem repeat regions of the Akv (Van Beveren, et al., su pra) , SL3-3 (Lenz, et al., (1983) , supra and Fr-MuLV57 (Koch, (1983) , supra) LTRs.
• the sequence of a single 99 bp repeat element of Akv is shown at the top.
• the solid lines below represent identical sequences present in the 72 bp by repeats and 66 bp repeats of SL3-3 and Fr-MuLV57 respectively.
• the number after the bracketed region indicates how many times their sequence is repeated.
• the actual sequences in the askerisked areas are shown in (b) .
• the LTR vectors of the present invention can be employed in gene therapy or gene repair.
• an infectious viral vector carrying the tissue specific enhancer is inserted within the 3 ' region of the genome of the present invention between the env gene and the LTR.
• An animal can then be inf ected with a virus that contains this modified LTR vector suitable for targeting to a particular tissue. Inf ection unde r these conditions would lead to a pref erential infection on a massive level, in the specific tissue controlled by the selection of the enhance r.
• the gene can be pref erentially directed into matu re thy mocytes, or eryth roblasts, using SL3 or Friend virus respectively.
• th is concept can be employed to (1) determine, using a CAT assay, other tissue specific enhancers; and (2) for the bu ilding of appropriate tissue specif ic LTR vectors.
• An other utility of the LTR vectors of the present invention is in cancer therapy .
• Each cancer cell, or each general type of cancer has different transcriptional properties which are recognizable by different enhance r elements.
• enhancer seq uences for particular types of cancer cells (as determined using the CAT assay) can be made.
• two approaches can be used. First, an infectious route wherein the LTR with an engineered enhancer is built onto a virus which carries a gene, or is built onto a non- replicating infector virus. In either case, the viruses could be used to infect patients suffering the particular cancer, and because of the tissue specific enhancer, the viruses would preferentially infect the tumor cells.
• the cancerous cells can be removed f rom the patient, e.g./ bone marrow, thereafter infected with the virus, and replaced into the patient.
• a lethal product can be expressed, which would kill the tumor cells, or a protein could be expressed, which in coating the cell surface, could provide a recognition site for a monoclonal or polyclonal antibody.
• hemagluten flu or ricin A chain genes could be inserted into the vectors of the present invention, either in a replicating or non- replicating cell.
• tissue specific LTRs of the present invention represents the first pharmaceutical use of these species.
• the viral LTRs may be combined with an appropriate pharmaceutical carrier for delivery of the tissue specific entity to a mammal.
• tissue specific vectors may be employed in the delivery of antitumor or like pharmaceutical agents, such as those described in the Physician' s Desk Reference. 38th ed.. Medical Economics Co. , Oradell, NJ (1984) , the disclosure of which is incorporated herein by reference.
• tissue specific enhancers can be used to advantage with non-viral promoters to provied tissue specific production in uninf ected mammalian tissue.
• Tissue specific viral enhancers may be provided and the tissues for which they are specific identified, by providing virus with the enhancer sequence to be tested, preferably also providing the viral genome with a detectable marker, infecting a mammal with such virus, and analyzing the various mammalian tissues for presence of the virus and the marker product (s) .

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