CA2131415C - Vector for liver-specific gene therapy - Google Patents
Vector for liver-specific gene therapyInfo
- Publication number
- CA2131415C CA2131415C CA 2131415 CA2131415A CA2131415C CA 2131415 C CA2131415 C CA 2131415C CA 2131415 CA2131415 CA 2131415 CA 2131415 A CA2131415 A CA 2131415A CA 2131415 C CA2131415 C CA 2131415C
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- Prior art keywords
- vector
- liver
- promoter
- gene
- sequence
- Prior art date
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Abstract
A tissue specific, suitably liver specific vector for gene therapy of a diseased host, wherein the disease is suitably a diseased liver, comprising an insect virus that contains (i) a therapeutic DNA-sequence, (ii) a promoter for gene expression in the target tissue, suitably the liver tissue of the host, and optionally (iii) an establishment sequence, and processes for its preparation and administration to the host.
Description
Z1314~5 VECI'OR FOR LIVER-SPECI~lC GENE THERAPY
Field of the invention The invention relates to a vector for tissue-specific, suitably liver-specific gene therapy. It is particularly useful in medicine, and in genetic engineering.
Background of the invention Numerous methods and vectors have been developed in recent years for gene therapy. See, e.g. the survey by Mulligan, in Science, vol. 260, p. 926.
Many vectors are favored in this connection for gene therapy, particularly all those which are derived from le~lo~huses or from adenoviruses. Both types of viral vectors are relatively broadly useful, retroviral vectors being generally effective only in proliferating cells. Adenoviruses also infect nondividing cells.
Although both types of vectors are suitable for gene transfer in vitro into liver cells (hepatocytes), they can hardly be considered for use in in vivo gene therapy application in humans. Although a partial liver resection is necessary to stimulate cell division (regeneration) in the application of retroviral vectors, the adenoviral 2 l 3 l 4 l 5 213~S
gene transfer is not very stable because no gene integration into the genome takes place.
Alternative vectors with potential applicability for gene transfer to liver cells are based on liposomes or also on multicomponent particles with protein domains, which bind specifically to certain receptors of the liver, such as the asialoglycoprotein receptor and can be taken up in the cell due to receptor intern~li7~ion. These were su~ yed by Versland et al. in 1992, Seminars in L*er Disease 12, 332. An important disadvantage of these vectors is the endocytotic absorption pathway, which leads to a degration of a large portion ofthe vectors and their DNA in the endosomes, so that only little DNA capable of functioning, can reach the cell nucleus.
A solution to this problem was found for the in vitro applications, but that is not useful for in vivo use in patients. The principle is based on the simultaneous infection of the target cells with adenovirus, which leads to a dislu~lion of the endosomes and a release of vector (DNA), as described by Curiel, D.T., Agrawal, S., Wagner, E. and Cotten, M. 1991, PNAS 88, 8850 -8854.
It was proposed in our Ge~. patent application No. 4,339,922.3 to pack a therapeutic gene, which is coupled to a promoter and is able to correct agenetic defect for the rlice~ce that is to be treated, in a peptide coat and to couple it to a compon~-nt of the he~ati~ B (HBV) or other specific virus, thus fi~ ing an i ~ prerequisite for ll~ali lg genetic tlice~ces of the liver or other tissue.
Descli),lion of the drawing The invention is eYrl~ine~l also with re~,cllce to the sole drawing, showing 104 cells lysed in 50 ml of Iysis buffer (100 mM KH2PO4/K2HPO4, pH 7.8, 1%
Triton X-lO0* lmMDTT) and m~cllred in 180m~ re~rti~n buffer (25 mM
KH2POJK2~04, 4 mM of EGTA~ pH8, 15 mM of MgSO4, 1 mM DDT, 1 mM
ATP~ in a Bertheld lllm~tc (20 mM l"~;r~ .), wherein A iS infection with Bac-CMV-luci B is inf~ctifm with BAC-PP-luci, and 1 is the activity of the luciLl~sc per 104 cells.
Des.;l i~lion of the h~ llioll It is an object of the i~ llLioU to col,~lluct a vector, which targets tissue cells, suitably liver cells highly sl~e~;r;r~lly~ in vilro and in vivo, is effectively * tra~e-mar~
CA 0213141S 1998-0~-21 taken up by the cells, and can direct the therapeutic genes into the cell nucleus.
The vector is useful for gene thel~y in various m~mm~ n hosts.
The present invention comprises a tissue-specific vector, suitably a liver-specific vector for gene therapy of an animal host wherein an insect virus, S suitably a baculovirus, or a n~rle~r polyhedrosis virus, which co~ s (i) a therapeutic DNA sequence, (ii) a promoter for gene eA~lession in the target tissue, suitably the liver, and optionally (iii) an establishment sequence.
Although the present invention is described principally with referellce to the liver-specificity of the vector, it is to be understood that the present invention enc~mp~seC m~mm~ n animal tissue specificity more generally.
Baculoviruses belong to a family of large DNA viruses, the host s~ecllulll of which is naturally limited e Aclu~ively to arthropods. Its genome (80 kbp - 200 kbp) is p~rl-~l in a flexible nucleocapsid which permits the insertion of large amounts of foreign DN~ When it was recogr~ized that the use of chemical insecticides is potentially dangerous to people and the envilu~ ont~ the pronounced host specificity of the baculov.luses suggested their use for biological insect control.
._ It is a prerequisite for this use of baculoviruses that the baculovirus gene is not t;~lessed in human cells. It was observed in investigations of numerous human cell types that the treatment with baculoviruses did not result in any significant infections.
S The cDNA of a gene, which is defective, absent or was changed by mutation in the case of the disease to be treated, is used as therapeutic DNA
sequence for the vector of the present invention. Examples of such genes are the LDL receptor gene, the gene for the alpha-1-antitrypsin, for the blood clotting factors VIII and ~, for elyl~lro~)oietin, and for thymidine kinase. A portion of a genomic sequence can also be used which bridges a mutation in the target gene, and can be recombined substantially homologously with this mutation.
Strong viral promoters, preferably the very early promoters of the cytomegalovirus (CMV), can suitably be used as promoters. Suitable liver-specific promoters, such as promoters/enhancers of the hepatitis B virus (HBV), such as the combination of core promoter/enhancer II, can also be advantageously employed. In addition to their specificity, they are also sufficiently small so that they can be easily incorporated into an expression vector. Promoters of liver-2~31415 specific genes, such as albumin, phosphoenol pyruvate carboxykinase (PEPCK), or ornithine transcarbamylase (OTC) can also be suitably employed.
The optional establishment sequence has the task of ensu~ g the stabilization of the vector in the cell without integration in the genome. It is used S particularly in those cases where ~ ession is necessary over prolonged periods.
Suitable establishment sequences in accordance with the present invention include viral nucleus establishment sequences, such as those of the Epstein-Barr virus, or autonomous replication sequences from the m~mm~ n genome.
The new vectors are essentially prepared by (a) cloning the therapeutic DNA sequence together with the promoter in a recombination vector, (b) optionally inserting an establishment sequence before or after the cloning, (c) transfecting the conslluct obtained together with the DNA of an insect virus into insect cells, and (d) obtaining from the supernatant of the insect culture the vector, packaged in the insect cells.
The present invention thus provides a novel vector for tissue-specific, suitably liver-specific gene transfer. This new vector offers appreciable advantages over previously developed viral vectors based on retroviruses or adenoviruses.
21314~5 . .
These advantages include tissue-specificity, suitably liver-specificity, the almost unlimited possibility for incorporating foreign DNA, the infection of cells incapable of dividing, the absence of cytotoxicity, and the simple generation of high-titer recombinant viruses.
As will be readily apparent to skilled practitioners, the vector of the present invention can be ~ red to the host, in any suitable manner known per se, in a suitable dosage form for the desired method.
It is particularly ~ul~lising that the vector infects particularly hepatocytes highly specifically. The vector makes possible the introduction of a desired gene into the liver of a patient and optimally configures the path of this gene to the site of functioning, because, for example, the vector is produced and "ini~lered to a patient through the bloodstream, suitably through the portal vein of the liver, or through the alimentary system. A significant prerequisite is thus fulfilled for the treatment of genetic ~ e~ses of the liver.
The present invention is described in greater detail by reference to the following specific example.
CA 0213141~ 1998-0~-21 Example Baculovlrus Vector for the Expresslon of Luclferase ln Hepatocytes 1. Constructlon of the baculovlrus transfer vector The PvuII fragment of the pCMV-Lucl, descrlbed by Mueller et al., ln Proc. Natl. Acad. Scl. U.S.A. vol. 91, p. 2945 (1994) contalns the cytomegalovlrus promoter, the luclferase reporter gene and the CMV polyadenylatlon slgnal as the expresslon cassette. It ls cloned lnto the SmaI restrlctlon slte of the pVL
1392. The resultlng (pVL-CMV-Lucl), purlfled by a Qlagen column*, ls used to prepare a recombinant baculovlrus.
The luclferase gene ls obtalned through ~amHI-Hlnd III
restrlctlon dlgestlon and fllllng up the end by Klenow enzyme from the plasmld T7-lol. After clonlng ln the SmaI restrlctlon slte of the pVL-1392, the luclferase gene ls under the control of the polyhedrln promoter (pVL-PP-Lucl). Thls construct serves as a negatlve control, to lllustrate the effect of the mammallan promoter ln the case of the hepatocyte lnfectlon.
* trade-mark The luciferase gene is replaced in an analogous col~Lluction by therapeutic genes, such as the LDL rece~lol gene. A liver-specific ~ n promoter (hepatitis) can be used incte~l of the CMV promoter. The polyhedrin sequenres, fl~nkinE~ the LA~ression cassette, can also be replaced by other nonessential baculovirus sequencec such as P94.
2. Preparation of recombinant viruses pVL-CMC-Luci or pVL-PP-Luci (3 ~g) and 0.5 ~g of baculovirus wild t~rpe DNA (R~yll~Gold~* PharMingen) are c~ recte-l by means of lipofectin (BRL) in 2 million Spodoptera fn~giperda insect cells (Sf9, ATCC). Due to homologous recombin~tion, bluu~l about by the polyhedrin sequences of the fer vectûrs, recombinant baculuvilu~es will result (Bac-CMV-Luci, Bac-PP-Luci), which carIy the lu~ sc gene under the control of the CMV, or polyhedrin promoter.
* trade-mark Z1314iS
Field of the invention The invention relates to a vector for tissue-specific, suitably liver-specific gene therapy. It is particularly useful in medicine, and in genetic engineering.
Background of the invention Numerous methods and vectors have been developed in recent years for gene therapy. See, e.g. the survey by Mulligan, in Science, vol. 260, p. 926.
Many vectors are favored in this connection for gene therapy, particularly all those which are derived from le~lo~huses or from adenoviruses. Both types of viral vectors are relatively broadly useful, retroviral vectors being generally effective only in proliferating cells. Adenoviruses also infect nondividing cells.
Although both types of vectors are suitable for gene transfer in vitro into liver cells (hepatocytes), they can hardly be considered for use in in vivo gene therapy application in humans. Although a partial liver resection is necessary to stimulate cell division (regeneration) in the application of retroviral vectors, the adenoviral 2 l 3 l 4 l 5 213~S
gene transfer is not very stable because no gene integration into the genome takes place.
Alternative vectors with potential applicability for gene transfer to liver cells are based on liposomes or also on multicomponent particles with protein domains, which bind specifically to certain receptors of the liver, such as the asialoglycoprotein receptor and can be taken up in the cell due to receptor intern~li7~ion. These were su~ yed by Versland et al. in 1992, Seminars in L*er Disease 12, 332. An important disadvantage of these vectors is the endocytotic absorption pathway, which leads to a degration of a large portion ofthe vectors and their DNA in the endosomes, so that only little DNA capable of functioning, can reach the cell nucleus.
A solution to this problem was found for the in vitro applications, but that is not useful for in vivo use in patients. The principle is based on the simultaneous infection of the target cells with adenovirus, which leads to a dislu~lion of the endosomes and a release of vector (DNA), as described by Curiel, D.T., Agrawal, S., Wagner, E. and Cotten, M. 1991, PNAS 88, 8850 -8854.
It was proposed in our Ge~. patent application No. 4,339,922.3 to pack a therapeutic gene, which is coupled to a promoter and is able to correct agenetic defect for the rlice~ce that is to be treated, in a peptide coat and to couple it to a compon~-nt of the he~ati~ B (HBV) or other specific virus, thus fi~ ing an i ~ prerequisite for ll~ali lg genetic tlice~ces of the liver or other tissue.
Descli),lion of the drawing The invention is eYrl~ine~l also with re~,cllce to the sole drawing, showing 104 cells lysed in 50 ml of Iysis buffer (100 mM KH2PO4/K2HPO4, pH 7.8, 1%
Triton X-lO0* lmMDTT) and m~cllred in 180m~ re~rti~n buffer (25 mM
KH2POJK2~04, 4 mM of EGTA~ pH8, 15 mM of MgSO4, 1 mM DDT, 1 mM
ATP~ in a Bertheld lllm~tc (20 mM l"~;r~ .), wherein A iS infection with Bac-CMV-luci B is inf~ctifm with BAC-PP-luci, and 1 is the activity of the luciLl~sc per 104 cells.
Des.;l i~lion of the h~ llioll It is an object of the i~ llLioU to col,~lluct a vector, which targets tissue cells, suitably liver cells highly sl~e~;r;r~lly~ in vilro and in vivo, is effectively * tra~e-mar~
CA 0213141S 1998-0~-21 taken up by the cells, and can direct the therapeutic genes into the cell nucleus.
The vector is useful for gene thel~y in various m~mm~ n hosts.
The present invention comprises a tissue-specific vector, suitably a liver-specific vector for gene therapy of an animal host wherein an insect virus, S suitably a baculovirus, or a n~rle~r polyhedrosis virus, which co~ s (i) a therapeutic DNA sequence, (ii) a promoter for gene eA~lession in the target tissue, suitably the liver, and optionally (iii) an establishment sequence.
Although the present invention is described principally with referellce to the liver-specificity of the vector, it is to be understood that the present invention enc~mp~seC m~mm~ n animal tissue specificity more generally.
Baculoviruses belong to a family of large DNA viruses, the host s~ecllulll of which is naturally limited e Aclu~ively to arthropods. Its genome (80 kbp - 200 kbp) is p~rl-~l in a flexible nucleocapsid which permits the insertion of large amounts of foreign DN~ When it was recogr~ized that the use of chemical insecticides is potentially dangerous to people and the envilu~ ont~ the pronounced host specificity of the baculov.luses suggested their use for biological insect control.
._ It is a prerequisite for this use of baculoviruses that the baculovirus gene is not t;~lessed in human cells. It was observed in investigations of numerous human cell types that the treatment with baculoviruses did not result in any significant infections.
S The cDNA of a gene, which is defective, absent or was changed by mutation in the case of the disease to be treated, is used as therapeutic DNA
sequence for the vector of the present invention. Examples of such genes are the LDL receptor gene, the gene for the alpha-1-antitrypsin, for the blood clotting factors VIII and ~, for elyl~lro~)oietin, and for thymidine kinase. A portion of a genomic sequence can also be used which bridges a mutation in the target gene, and can be recombined substantially homologously with this mutation.
Strong viral promoters, preferably the very early promoters of the cytomegalovirus (CMV), can suitably be used as promoters. Suitable liver-specific promoters, such as promoters/enhancers of the hepatitis B virus (HBV), such as the combination of core promoter/enhancer II, can also be advantageously employed. In addition to their specificity, they are also sufficiently small so that they can be easily incorporated into an expression vector. Promoters of liver-2~31415 specific genes, such as albumin, phosphoenol pyruvate carboxykinase (PEPCK), or ornithine transcarbamylase (OTC) can also be suitably employed.
The optional establishment sequence has the task of ensu~ g the stabilization of the vector in the cell without integration in the genome. It is used S particularly in those cases where ~ ession is necessary over prolonged periods.
Suitable establishment sequences in accordance with the present invention include viral nucleus establishment sequences, such as those of the Epstein-Barr virus, or autonomous replication sequences from the m~mm~ n genome.
The new vectors are essentially prepared by (a) cloning the therapeutic DNA sequence together with the promoter in a recombination vector, (b) optionally inserting an establishment sequence before or after the cloning, (c) transfecting the conslluct obtained together with the DNA of an insect virus into insect cells, and (d) obtaining from the supernatant of the insect culture the vector, packaged in the insect cells.
The present invention thus provides a novel vector for tissue-specific, suitably liver-specific gene transfer. This new vector offers appreciable advantages over previously developed viral vectors based on retroviruses or adenoviruses.
21314~5 . .
These advantages include tissue-specificity, suitably liver-specificity, the almost unlimited possibility for incorporating foreign DNA, the infection of cells incapable of dividing, the absence of cytotoxicity, and the simple generation of high-titer recombinant viruses.
As will be readily apparent to skilled practitioners, the vector of the present invention can be ~ red to the host, in any suitable manner known per se, in a suitable dosage form for the desired method.
It is particularly ~ul~lising that the vector infects particularly hepatocytes highly specifically. The vector makes possible the introduction of a desired gene into the liver of a patient and optimally configures the path of this gene to the site of functioning, because, for example, the vector is produced and "ini~lered to a patient through the bloodstream, suitably through the portal vein of the liver, or through the alimentary system. A significant prerequisite is thus fulfilled for the treatment of genetic ~ e~ses of the liver.
The present invention is described in greater detail by reference to the following specific example.
CA 0213141~ 1998-0~-21 Example Baculovlrus Vector for the Expresslon of Luclferase ln Hepatocytes 1. Constructlon of the baculovlrus transfer vector The PvuII fragment of the pCMV-Lucl, descrlbed by Mueller et al., ln Proc. Natl. Acad. Scl. U.S.A. vol. 91, p. 2945 (1994) contalns the cytomegalovlrus promoter, the luclferase reporter gene and the CMV polyadenylatlon slgnal as the expresslon cassette. It ls cloned lnto the SmaI restrlctlon slte of the pVL
1392. The resultlng (pVL-CMV-Lucl), purlfled by a Qlagen column*, ls used to prepare a recombinant baculovlrus.
The luclferase gene ls obtalned through ~amHI-Hlnd III
restrlctlon dlgestlon and fllllng up the end by Klenow enzyme from the plasmld T7-lol. After clonlng ln the SmaI restrlctlon slte of the pVL-1392, the luclferase gene ls under the control of the polyhedrln promoter (pVL-PP-Lucl). Thls construct serves as a negatlve control, to lllustrate the effect of the mammallan promoter ln the case of the hepatocyte lnfectlon.
* trade-mark The luciferase gene is replaced in an analogous col~Lluction by therapeutic genes, such as the LDL rece~lol gene. A liver-specific ~ n promoter (hepatitis) can be used incte~l of the CMV promoter. The polyhedrin sequenres, fl~nkinE~ the LA~ression cassette, can also be replaced by other nonessential baculovirus sequencec such as P94.
2. Preparation of recombinant viruses pVL-CMC-Luci or pVL-PP-Luci (3 ~g) and 0.5 ~g of baculovirus wild t~rpe DNA (R~yll~Gold~* PharMingen) are c~ recte-l by means of lipofectin (BRL) in 2 million Spodoptera fn~giperda insect cells (Sf9, ATCC). Due to homologous recombin~tion, bluu~l about by the polyhedrin sequences of the fer vectûrs, recombinant baculuvilu~es will result (Bac-CMV-Luci, Bac-PP-Luci), which carIy the lu~ sc gene under the control of the CMV, or polyhedrin promoter.
* trade-mark Z1314iS
3. Reproduction and purification of the recombinant baculoviruses Sf9 insect cells are infected in a bottle or spinner culture with an MOI
of 1 for 3 days with the Bac-CMV-Luci or Bac-PP-Luci. The viruses formed are separated from the insect cells by cellllirugation and subsequently pelletized at 12,000 g for 45 minutes. A band of purified virus is obtained by sucrose density gradient cen~lirugation for 4 hours at 100,000 g and with a gradient of 24~o to 62%. This is trall~Çelled by pelletizing twice into the application buffer (isotonic salt solution or salt culture medium).
of 1 for 3 days with the Bac-CMV-Luci or Bac-PP-Luci. The viruses formed are separated from the insect cells by cellllirugation and subsequently pelletized at 12,000 g for 45 minutes. A band of purified virus is obtained by sucrose density gradient cen~lirugation for 4 hours at 100,000 g and with a gradient of 24~o to 62%. This is trall~Çelled by pelletizing twice into the application buffer (isotonic salt solution or salt culture medium).
4. Investigations of the specificity of the infection or the luciferase expression of the CMV-Luci in liver cells l~mm~ n cell lines of different tissues and species and primary human (phH) as well as primary murine (pmH) hepatocytes are infected with Bac-CMV-Luci or Bac-PP-Luci (MOI 100) to investigate the cell specificity of thebaculovirus infection. The luciferase ~ression is measured after 1.5 days and the results show the specificity of the Bac-CMV-Luci in relation to the hepatocyte infection. Under control of the polyhedrin promoter (b: Bac-PP-Luci infection ofdiLlerent cell lines), the luciferase gene is active exclusively in insect cells.
S
S
5. Indications of a protein receptor-induced endocytosis of the baculovirus in hepatocytes (Huh7) A protease treatment of the hepatocytes decreases the number of ~rolein receptors, so that there is a reduction in the luciferase activity after Bac-S CMV-Luci infection.
Enzyme Concentration % Reduction in Luciferase Activity Trypsin 2 mg/mL 50.4 Papain 1 mg/mL 36.9 Pronase E 1 m~/mL 52.6 10The foregoing table shows that Huh7 cells were treated for 15 minutes with the proteases specified. After the proteases were inactivated, the cells were infected for 1 hour with Bac-CMV-Luci (MOI 100). After the 1 hour virus infection, Huh7 cells were treated similarly for 3 hours (rerelellce value: 100%).
The reduction in the luciferase activity by a measurement after 1.5 days, arises 15out of the ratio of the luciferase value of the hepatocytes previously treated with protease to the reference value.
Chloroquine and colchicine intervene in the endocytosis pathway of the virus or in its migration into the hepatocyte nucleus. The luciferase activity is also decreased by the ~ ation of either chemical.
Concentration% Reduction of Luciferase Activity Chloroquine 0.5 mM 100 0.1 mM 98.4 0.03 mM - 92 0.01 mM 81 S Colchicine 18 mg/mL 69.4 6 mg/mL 56 2 mg/mL 55 As shown in the foregoing table, Huh7 cells were infected with BAc-CMV-Luci (MOI 100), with chloroquine or colchicine being ~ ered at the same time. The reduction in the luciferase activity is related to a reference value, for which the two chemicals were given in each case 12 hours after the infection.
The expression of the luciferase was measured after 1.5 days.
Enzyme Concentration % Reduction in Luciferase Activity Trypsin 2 mg/mL 50.4 Papain 1 mg/mL 36.9 Pronase E 1 m~/mL 52.6 10The foregoing table shows that Huh7 cells were treated for 15 minutes with the proteases specified. After the proteases were inactivated, the cells were infected for 1 hour with Bac-CMV-Luci (MOI 100). After the 1 hour virus infection, Huh7 cells were treated similarly for 3 hours (rerelellce value: 100%).
The reduction in the luciferase activity by a measurement after 1.5 days, arises 15out of the ratio of the luciferase value of the hepatocytes previously treated with protease to the reference value.
Chloroquine and colchicine intervene in the endocytosis pathway of the virus or in its migration into the hepatocyte nucleus. The luciferase activity is also decreased by the ~ ation of either chemical.
Concentration% Reduction of Luciferase Activity Chloroquine 0.5 mM 100 0.1 mM 98.4 0.03 mM - 92 0.01 mM 81 S Colchicine 18 mg/mL 69.4 6 mg/mL 56 2 mg/mL 55 As shown in the foregoing table, Huh7 cells were infected with BAc-CMV-Luci (MOI 100), with chloroquine or colchicine being ~ ered at the same time. The reduction in the luciferase activity is related to a reference value, for which the two chemicals were given in each case 12 hours after the infection.
The expression of the luciferase was measured after 1.5 days.
Claims (16)
1. A tissue specific vector for gene therapy of a host having a diseased liver, comprising an insect virus that contains (i) a therapeutic DNA sequence, (ii) a promoter for gene expression in the liver of the host, and optionally (iii) an establishment sequence.
2. The tissue specific vector of claim 1, wherein said insect virus is a baculovirus, or a nuclear polyhedrosis virus.
3. The vector of claim 1, wherein said therapeutic DNA
sequence is the cDNA sequence of a gene that is defective, absent, or was changed by mutation by the disease to be treated.
sequence is the cDNA sequence of a gene that is defective, absent, or was changed by mutation by the disease to be treated.
4. The vector of claim 1, wherein said therapeutic DNA
sequence comprises a portion of a genomic sequence from a gene which is not defective, bridging a mutation in a corresponding defective gene, and which can be recombined substantially homologously therewith.
sequence comprises a portion of a genomic sequence from a gene which is not defective, bridging a mutation in a corresponding defective gene, and which can be recombined substantially homologously therewith.
5. The vector of claim 1, wherein said therapeutic DNA
sequence comprises cDNA for the LDL receptor gene, the gene for alpha-1-antitrypsin, blood clotting factors VIII and IX, erythropoletin, or for thymidine kinase.
sequence comprises cDNA for the LDL receptor gene, the gene for alpha-1-antitrypsin, blood clotting factors VIII and IX, erythropoletin, or for thymidine kinase.
6. The vector of claim 1, wherein the promoter is a strong viral promoter.
7. The vector of claim 6, wherein said strong viral promoter is the very early promoter of the CMV, or a liver-specific promoter.
8. The vector of claim 7, wherein said liver specific gene promoter is derived from HBV, or a combination of core-promoter/enhancer II.
9. The vector of claim 7, wherein said liver-specific gene promoter is albumin, PEPCK, or OTC promoter.
10. The vector of claim 1, wherein said establishment sequence is a viral nucleus establishment sequence.
11. The vector of claim 10, wherein said viral nucleus establishment sequence is derived from the Epstein-Barr virus, or an autonomous replication sequence.
12. A method for producing the vector of claim 1, which comprises (a) cloning said therapeutic DNA sequence with said promoter in a recombinantionvector to obtain a construct, (b) optionally inserting said establishment sequence before or after said cloning, (c) transfecting said construct with the DNA of aninsect virus into insect cells, and (d) obtaining from the supernatant of the insect culture the vector packaged in the insect cells.
13. The method of claim 12, further comprising converting the vector into a dosage form that is suitable for administration to the host.
14 14. Use of the vector prepared by the method of claim 13 delivered intravenously for correcting a genetic defect of the liver.
15. Use of the vector according to claim 14, wherein said promoter is derived from HBV and the vector is delivered directly to the liver through the portal vein of the liver of the host.
16. Use of the vector prepared by the method of claim 13 for correcting a genetic defect of the liver, wherein the vector is absorbed by the alimentary system of the host.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP4407858.5 | 1994-03-04 | ||
| DE4407858 | 1994-03-04 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2131415A1 CA2131415A1 (en) | 1995-09-05 |
| CA2131415C true CA2131415C (en) | 1999-01-05 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2131415 Expired - Fee Related CA2131415C (en) | 1994-03-04 | 1994-09-02 | Vector for liver-specific gene therapy |
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| CA (1) | CA2131415C (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2003286198A (en) * | 2002-03-29 | 2003-10-07 | Japan Science & Technology Corp | Therapeutic medicine using protein hollow nanoparticle expression growth factor, and the like |
| JP2003286199A (en) * | 2002-03-29 | 2003-10-07 | Japan Science & Technology Corp | Therapeutic medicine for hepatic disease using protein hollow nanoparticle |
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1994
- 1994-09-02 CA CA 2131415 patent/CA2131415C/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CA2131415A1 (en) | 1995-09-05 |
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