WO1996014837A1 - Therapie genique contre l'hypercholesterolemie - Google Patents

Therapie genique contre l'hypercholesterolemie Download PDF

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Publication number
WO1996014837A1
WO1996014837A1 PCT/US1995/014541 US9514541W WO9614837A1 WO 1996014837 A1 WO1996014837 A1 WO 1996014837A1 US 9514541 W US9514541 W US 9514541W WO 9614837 A1 WO9614837 A1 WO 9614837A1
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adenoviral
vector
apoe
fragment
plasma
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PCT/US1995/014541
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English (en)
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Alan Mcclelland
Susan C. Stevenson
Ba Bie Teng
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Genetic Therapy, Inc.
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Publication of WO1996014837A1 publication Critical patent/WO1996014837A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/775—Apolipopeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus

Definitions

  • This invention relates to hypercholesterolemia and the treatment thereof. More particularly, this invention relates to the treatment of hypercholesterolemia through the administration of adenoviral vectors including DNA encoding apolipoprotein E. BACKGROUND OF THE INVENTION
  • Apolipoprotein E (also known as Apo E) is a component of several plasma lipoproteins including chylomicrons, VLDL, and
  • HDL Receptor-mediated catabolism of lipoprotein particles is mediated through the interaction of Apo E with the LDL receptor or with the LDL receptor-related protein, LRP
  • Apo E-deficient mice demonstrate further the physiologic importance of Apo E in lipoprotein metabolism.
  • Apo E- deficient mice are severely hypercholesterolemic with average plasma cholesterol concentrations of 400 to 800 mg/dl on a regular chow diet. (Zhang, et al . , Science. Vol. 258, pgs. 468-471 (1992); Plump, Cell. Vol. 71, pgs. 343-351 (1992)). These mice also develop atherosclerotic lesions at approximately 11 weeks of age with the appearance of foam cells which progress to a more involved, complex lesion including cholesterol clefts and fibrous caps (Redderick, et al . , Arterioscler. Thromb. , Vol.
  • Figure 1 is a map of plasmid pCRII
  • Figure 2 is a map of plasmid pCRE
  • Figure 3 is a schematic of the construction of plasmid pHR
  • Figure 4 is a schematic of the construction of an adenoviral vector including an adenoviral ITR, an Ela enhancer sequence, a Rous Sarcoma Virus promoter, and an Adenovirus 5 tripartite leader sequence;
  • Figure 5 is a schematic of the construction of plasmid pAvS6;
  • Figure 6 is a plasmid map of plasmid pAvS6;
  • Figure 7 is a map of plasmid pAvS6E
  • Figure 8 is a schematic of the construction of AvlRE
  • Figure 9 is a map of plasmid pAvS6-nLacZ.
  • Figure 10 is a schematic of the construction of AvlLacZ4
  • Figure 11 is a Western blot of in vitro human apoE expression from HepG2 cells transduced with AvlLacZ4 or AvlRE;
  • Figure 12 is a Western blot of in vivo expression of human apoE in apoE-deficient mice treated with AvlLacZ4 or AvlRE;
  • Figure 13 is a graph of plasma cholesterol concentrations in apoE-deficient mice treated with AvlLacZ4 or AvlRE at 0 and 7 days post-inf ction;
  • Figure 14 is a graph of plasma lipoprotein distributions in apoE-deficient mice treated with AvlLacZ4 or AvlRE;
  • Figure 15 is a Western blot of human apoE distribution among mouse plasma lipoproteins in mice treated with AvlRE; and Figures 16A and 16B are graphs of total plasma cholesterol and plasma human apoE concentrations, respectively, in mice treated with AvlLacZ4 or AvlRE.
  • an adenoviral vector including a nucleic acid sequence encoding apolipoprotein E or a fragment or derivative thereof having the biological activity of apolipoprotein E.
  • Apolipoprotein E results in cholesterol lowering through an increase in the receptor mediated catabolism of apolipoprotein B containing lipoprotein particles.
  • fragment or derivative thereof as used herein means that the apolipoprotein E may be a protein which has deletion(s) of amino acid residues within the protein structure, and/or may be truncated at the C-terminal and/or the N-terminal, and/or may be mutated such that one or more amino acid residues normally present in the protein structure are replaced with other amino acid residues.
  • Such fragments and derivatives of apolipoprotein E retain the same biological activity as unmodified apolipoprotein E.
  • the nucleic acid sequence encoding apolipoprotein E or a fragment or derivative thereof may be the native nucleic acid sequence which encodes apolipoprotein E or a fragment or derivative thereof, or an allelic variant thereof.
  • allelic variant as used herein means that the allelic variant is an alternative form of the native nucleic acid sequence which may have a substitution, deletion, or addition of one or more nucleotides, which does not alter substantially the function of the encoded apolipoprotein E or fragment or derivative thereof.
  • the nucleic acid sequence encoding apolipoprotein E or a fragment or derivative thereof encodes human apolipoprotein E, and in particular, human apolipoprotein E3.
  • the nucleic acid sequence preferably is a DNA sequence, which may be a cDNA sequence or a genomic DNA sequence.
  • the DNA sequence encodes the full length apolipoprotein E or may encode a fragment or derivative of apolipoprotein E, and the DNA sequence may further include a leader sequence or portion thereof, a secretory signal or portion thereof of the apolipoprotein E gene, and/or may further include a trailer sequence or portion thereof of the apolipoprotein E gene.
  • Applicants have found unexpectedly that, when animal hosts were infected with adenoviral vectors including a nucleic acid sequence encoding apolipoprotein E, that in such animal hosts, one obtains long term expression of the nucleic acid sequence encoding apolipoprotein E, as well as long term lowering of plasma cholesterol concentrations.
  • the adenoviral vector which is employed may, in one embodiment, be an adenoviral vector which includes essentially the complete adenoviral genome. (Shenk, et al., Curr. Top. Microbiol. Immunol.. (1984); 111(3) :l-39) .
  • the adenoviral vector may be a modified adenoviral vector in which at least a portion of the adenoviral genome has been deleted.
  • the vector comprises an adenoviral 5' ITR; an adenoviral 3' ITR; an adenoviral encapsidation signal; a DNA sequence encoding apolipoprotein E or a fragment or derivative thereof; and a promoter controlling the DNA sequence encoding apolipoprotein E or a fragment or derivative thereof.
  • the vector is free of at least the majority of adenoviral El and E3 DNA sequences, but is not free of all of the E2 and E4 DNA sequences, and DNA sequences encoding adenoviral proteins promoted by the adenoviral major late promoter.
  • the gene in the E2a region that encodes the 72 kilodalton binding protein is mutated to produce a temperature sensitive protein that is active at 32°C, the temperature at which viral particles are produced, but is inactive at 37°C, the temperature of the animal or human host.
  • This temperature sensitive mutant is described in Ensinger, et al., J.Virology. 10:328-339 (1972); Van der Vliet, et al., J.Virology. 15:348-354 (1975); and Friefeld, et al., Virology. 124:380-389 (1983); Englehardt, et al., Proc.Nat.Acad.Sci.. Vol. 91, pgs. 6196-6200 (June 1994); Yang, et al., Nature Genetics. Vol. 7, pgs. 362-369 (July 1994) .
  • Such a vector in a preferred embodiment, is constructed first by constructing, according to standard techniques, a shuttle plasmid which contains, beginning at the 5' end, the "critical left end elements," which include an adenoviral 5' ITR, an adenoviral encapsidation signal, and an Ela enhancer sequence; a promoter (which may be an adenoviral promoter or a foreign promoter); a tripartite leader sequence, a multiple cloning site (which may be as herein described) ; a poly A signal; and a DNA segment which corresponds to a segment of the adenoviral genome.
  • a shuttle plasmid which contains, beginning at the 5' end, the "critical left end elements," which include an adenoviral 5' ITR, an adenoviral encapsidation signal, and an Ela enhancer sequence; a promoter (which may be an adenoviral promoter or a foreign promoter); a tripartite leader sequence,
  • Such DNA segment serves as a substrate for homologous recombination with a modified or mutated adenovirus, and such sequence may encompass, for example, a segment of the adenovirus 5 genome no longer than from base 3329 to base 6246 of the genome.
  • the plasmid may also include a selectable marker and an origin of replication.
  • the origin of replication may be a bacterial origin of replication.
  • Representative examples of such shuttle plasmids include pAvS6, shown in Figure 6. A desired DNA sequence encoding a therapeutic agent may then be inserted into the multiple cloning site to produce a plasmid vector.
  • This construct then is used to produce an adenoviral vector.
  • Homologous recombination then is effected with a modified or mutated adenovirus in which at least the majority of the El and E3 adenoviral DNA sequences have been deleted.
  • Such homologous recombination may be effected through co- transfection of the plasmid vector and the modified adenovirus into a helper cell line, such as 293 cells, by CaP0 4 precipitation.
  • a recombinant adenoviral vector which includes DNA sequences derived from the shuttle plasmid between the Not I site and the homologous recombination fragment, and DNA derived from the El and E3 deleted adenovirus between the homologous recombination fragment and the 3' ITR.
  • the homologous recombination fragment overlaps with nucleotides 3329 to 6246 of the adenovirus 5 (ATCC VR-5) genome.
  • a vector which includes an adenoviral 5' ITR, an adenoviral encapsidation signal; an Ela enhancer sequence,- a promoter; a tripartite leader sequence; a DNA sequence encoding apolipoprotein E or a fragment or derivative thereof; a poly A signal; adenoviral DNA free of at least the majority of the El and E3 adenoviral DNA sequences,- and an adenoviral 3' ITR.
  • This vector may then be transfected into a helper cell line, such as the 293 helper cell line (ATCC No. CRL1573) , which will include the Ela and Elb DNA sequences, which are necessary for viral replication, and to generate infectious viral particles. Transfection may take place by electroporation, calcium phosphate precipitation, microinjection, or through proteoliposomes.
  • the vector hereinabove described may include a multiple cloning site to facilitate the insertion of DNA sequence(s) encoding lipoprotein(s) into the cloning vector.
  • the multiple cloning site includes "rare" restriction enzyme sites; i.e., sites which are found in eukaryotic genes at a frequency of from about one in every 10,000 to about one in every 100,000 base pairs.
  • An appropriate vector is thus formed by cutting the cloning vector by standard techniques at appropriate restriction sites in the multiple cloning site, and then ligating the DNA sequence encoding a lipoprotein(s) into the cloning vector.
  • apolipoprotein E or fragment or derivative thereof is under the control of a suitable promoter.
  • suitable promoters which may be employed include, but are not limited to, adenoviral promoters, such as the adenoviral major late promoter; or heterologous promoters, such as the cytomegalovirus (CMV) promoter,- the Rous Sarcoma Virus (RSV) promoter; inducible promoters, such as the MMTV promoter, the metallothionein promoter,- heat shock promoters,- the albumin promoter; the ApoE promoter,- and the ApoAI promoter. It is to be understood, however, the scope of the present invention is not limited to specific foreign genes or promoters.
  • the adenovirus may be constructed by using a yeast artificial chromosome (or YAC) containing an adenoviral genome according to the method described in Ketner, et al . , PNAS. Vol. 91, pgs. 6186-6190 (1994), in conjunction with the teachings contained herein.
  • the adenovirus yeast artificial chromosome is produced by homologous recombination in vivo between adenoviral DNA and yeast artificial chromosome plasmid vectors carrying segments of the adenoviral left and right genomic termini.
  • a DNA sequence encoding apolipoprotein E or a fragment or derivative thereof then may be cloned into the adenoviral DNA.
  • the modified adenoviral genome then is excised from the adenovirus yeast artificial chromosome in order to be used to generate infectious adenoviral particles as hereinabove described.
  • the infectious viral particles may then be administered in vivo to a host.
  • the host may be an animal host, including mammalian, non-human primate, and human hosts.
  • the particular target cell or tissue to which the particles are targeted is dependent upon the ligand with which the particle is engineered.
  • the viral particles are administered in an amount effective to treat hypercholesterolemia in a host, i.e., the administration of the viral particles provides for the lowering of cholesterol levels in the blood of the host.
  • the viral particles may be administered in an amount of from 1 plaque forming unit to about 10 14 plaque forming units, preferably from about 10 6 plaque forming units to about 10 13 plaque forming units.
  • Hypercholesterolemia often is associated with cardiovascular disease, such as, for example, atherosclerosis, which is the blocking of arteries as a result of cholesterol deposits.
  • cardiovascular disease such as, for example, atherosclerosis, which is the blocking of arteries as a result of cholesterol deposits.
  • the adenoviral vectors of the present invention are useful in the prevention and/or treatment of cardiovascular disease, wherein such vectors are administered in the amounts hereinabove described.
  • the infectious vector particles are administered systemically, such as, for example, by intravenous administration (such as, for example, portal vein injection or peripheral vein injection) , or intramuscular administration.
  • intravenous administration such as, for example, portal vein injection or peripheral vein injection
  • intramuscular administration such as, for example, intramuscular administration.
  • the viral particles may be administered in combination with a pharmaceutically acceptable carrier suitable for administration to a patient.
  • the carrier may be a liquid carrier (for example, a saline solution) , or a solid carrier, such as, for example, microcarrier beads.
  • the viral particles may be administered to apolipoprotein-deficient animals (such as, for example, apoE-deficient mice) in order to use such animals as models for studying hypercholesterolemia and the treatment thereof.
  • apolipoprotein-deficient animals such as, for example, apoE-deficient mice
  • an adenoviral vector containing the apoE gene may be given to apoE-deficient mice.
  • the mice are evaluated for expression of apolipoprotein E. From the results of such a study, one then may determine how such adenoviral vectors may be administered to human patients for the treatment of apolipoprotein-E deficiency.
  • the adenoviral vectors of the invention are useful as scientific reagents in the study of hypercholesterolemia.
  • the adenoviral vector may be employed to transduce eukaryotic cells in vitro, whereby the eukaryotic cells will produce apolipoprotein E in vitro.
  • Eukaryotic cells which may be transduced include any eukaryotic cell type, and, in particular, include hepatocytes, endothelial cells, and primary cells, such as primary nucleated blood cells.
  • Such apolipoprotein E then may be obtained from the culture of transduced eukaryotic cells, and may be administered to a host for the treatment of hypercholesterolemia.
  • the eukaryotic cells which were transduced with the adenoviral vector in vitro, may be administered to a host in an amount effective to treat hypercholesterolemia in a host.
  • Apolipoprotein E (ApoE) cDNA was constructed using gene overlap extension PCR methods (Horton, et al. , Biotechni ⁇ ues. Vol. 8, pgs. 528-535 (1990)), using Pfu DNA polymerase (Stratagene, La Jolla, CA) in the presence of 10% DMSO.
  • the 5' end of the apoE cDNA, nucleotides-39 to 292 was generated using human liver cDNA (Clonetech, Palo Alto, CA) as template with the following primers:
  • the primer Pi was designed to start at the major transcription initiation site in Exon 1 (Paik, et al . , Proc. Nat. Acad. Sci.. Vol. 82, pgs. 3445-3449 (1985).
  • the PCR reaction was carried out using the following conditions: 95°C for 10 min., followed by 30 cycles of 95°C for 30 seconds, 60°C for 1 minute, 72°C for 2 minutes, and finally a 72°C extension for 10 minutes.
  • the 3' end of the apoE cDNA was amplified from a cloned apoE fragment, EB4 (Wallis, et al . , EMBO J. ⁇ Vol. 2, pgs. 2369-2373 (1983)) obtained from Dr. Steven Humphries using the primers P3 (5'- GCCTACAAATCGGAACTGGAGGAA- 3 ' ) and P4 (5' - AGGCTTCGGCGTTCAGTGATTGT-3' ) to produce a 696 bp fragment.
  • the 5' and 3' PCR generated apoE fragments were gel purified. The PCR reaction was set up using equal volumes of the melted fragments and the end primers PI and P4.
  • the apoE cDNA sequence includes a leader sequence of 60 bp 5' to the ATG start codon, a secretion signal peptide coding region, the apoE3 coding region, and a trailer sequence of 93 bp 3' to the stop codon. This sequence corresponds to bp 2 to bp 1,026 of the apoE3 sequence of Genbank accession #K00396.
  • Several clones were screened by restriction enzyme analysis and then were sequenced. A clone having a perfect match with the expected sequence (Genbank accession #K00396) was selected.
  • the adenoviral construction shuttle plasmid pAvS6 was constructed in several steps using standard cloning techniques including polymerase chain reaction based cloning techniques.
  • Ad-dl327 (Thimmappaya, et al., Cell. Vol. 31, pgs.
  • Adenovirus 543-551 (83), incorporated herein by reference) is identical to Adenovirus 5 except that an Xbal fragment including bases 28591 to 30474 (or map units 78.5 to 84.7) of the Adenovirus 5 genome, and which is located in the E3 region, has been deleted.
  • the complete Adenovirus 5 genome is registered as Genbank accession #M73260, incorporated herein by reference, and the virus is available from the American Type Culture Collection, Rockville, Maryland, U.S.A. under accession number VR-5.
  • Ad-dl327 was constructed by routine methods from Adenovirus 5 (Ad5) . The method is outlined briefly as follows and previously described by Jones and Shenk, Cell 13:181-188, (1978).
  • Ad5 DNA is isolated by proteolytic digestion of the virion and partially cleaved with Xbal restriction endonuclease. The Xbal fragments are then reassembled by ligation as a mixture of fragments. This results in some ligated genomes with a sequence similar to Ad5, except excluding sequences 28591 bp to 30474 bp.
  • This DNA is then transfected into suitable cells (e.g. KB cells, HeLa cells, 293 cells) and overlaid with soft agar to allow plaque formation. Individual plaques are then isolated, amplified, and screened for the absence of the 1878 bp E3 region Xbal fragment.
  • This plasmid was designated pHR. ( Figure 3) .
  • the ITR, encapsidation signal, Rous Sarcoma Virus promoter, the adenoviral tripartite leader (TPL) sequence and linking sequences were assembled as a block using PCR amplification ( Figure 4) .
  • the ITR and encapsidation signal (sequences 1-392 of Ad-dl327 [identical to sequences from Ad5, Genbank accession #M73260] , incorporated herein by reference) were amplified (amplification 1) together from Ad-dl327 using primers containing NotI or Ascl restriction sites.
  • the Rous Sarcoma Rous Sarcoma Virus promoter, the adenoviral tripartite leader (TPL) sequence and linking sequences were assembled as a block using PCR amplification ( Figure 4) .
  • the ITR and encapsidation signal (sequences 1-392 of Ad-dl327 [identical to sequences from Ad5, Genbank accession #M73260] ,
  • Virus LTR promoter was amplified (amplification 2) from the plasmid pRC/RSV (sequences 209 to 605; Invitrogen, San Diego,
  • CA CA
  • DNA products from amplifications l and 2 were joined using the "overlap" PCR method (amplification 3) (Horton, et al.,
  • the ITR-encapsidation signal-TPL fragment was then purified, cleaved with NotI and Xbal and inserted into the NotI, Xbal cleaved pHR plasmid.
  • This plasmid was designated pAvS6A' and the orientation was such that the NotI site of the fragment was next to the T7 RNA polymerase site ( Figure 5) .
  • the SV40 early polyA signal was removed from SV40 DNA as an Hpal-BamHI fragment, treated with T4 DNA polymerase and inserted into the Sail site of the plasmid pAvS6A- ( Figure 5) to create pAvS6 ( Figures 5 and 6).
  • the apoE cDNA fragment was obtained from pCRE ( Figure 2) after digestion with EcoRV and Ecll36 restriction enzymes. The apoE cDNA fragment was then ligated into the EcoRV- linearized pAvS6 to generate pAvS6E ( Figure 7) , in which the apoE cDNA is placed downstream of the RSV promoter. pAvS6E was linearized with Kpnl restriction enzyme and co-transfected with the large Clal fragment of Ad5dl327. This transfection produced the recombinant adenovirus containing the apoE cDNA and is referred to as AvlRE.
  • a schematic diagram of AvlRE is shown in Figure 8.
  • adenovirus titers were determined spectrophotometrically (Weiden, Proc. Nat. Acad. Sci.. Vol. 91, pgs. 153-157 (1994); Halbert, J. Virol. , Vol. 56, pgs. 250-257 (1985) and compared to the biological titer (pfu/ml) . The ratio of total particles to infectious particles (particles/pfu) usually was 100 or less.
  • AvlLacZ4 The infectious, replication-deficient, AvlLacZ4 was assembled in 293 cells by homologous recombination. To accomplish this, plasmid pAvS6-nLacZ was linearized by cleavage with Kpnl. Genomic adenoviral DNA was isolated from purified Ad-dl327 viruses by Hirt extraction, cleaved with Clal, and the large (approximately 35 kb) fragment was isolated by agarose gel electrophoresis and purified. The Clal fragment was used as the backbone for all first generation adenoviral vectors, and the vectors derived from it are known as Avl.
  • IMEM-2 IMEM plus 2% FBS, 2mM glutamine (Bio Whittaker 046764)
  • IMEM-10 Improved minimal essential medium (Eagle's) with 2x glutamine plus 10% vol./vol. fetal bovine serum plus 2mM supplemental glutamine (Bio Whittaker 08063A) and incubated at 37°C for approximately three days until the cytopathic effect, a rounded appearance and "grapelike" clusters, was observed. Cells and supernatant were collected and designated as CVL-A.
  • AviLacZ4 vector (a schematic of the construction of which is shown in Figure 10) was released by three cycles of freezing and thawing of the CVL-A. Then, a 60 mm dish of 293 cells was infected with 0.5 ml of the CVL-A plus 3 ml of IMEM-10 and incubated for approximately three days as above. Cells and supernatant from this infection then were processed by three freeze/thaw cycles in the same manner. AviLacZ4 also is described in Yei, et al., Human Gene Therapy. Vol. 5, pgs. 731-744 (1994); Trapnell, Advanced Drug Delivery Reviews. Vol. 12, pgs. 185-199 (1993), and Smith, et al., Nature Genetics. Vol. 5, pgs. 397-402 (December 1993), which are incorporated herein by reference. E. Transduction of HepG2 cells.
  • HepG2 cells were cultured in Eagle's minimum essential medium (EMEM) containing 10% fetal bovine serum (FBS) .
  • EMEM Eagle's minimum essential medium
  • FBS fetal bovine serum
  • Transductions of the cells with AvlRE or AvlLacZ4 were carried out in EMEM containing 2% FBS, 100 units/ml penicillin, and lO ⁇ g/ml streptomycin, and were carried out when the cells had reached approximately 90% confluency, and at multiplicities of infection (MOD of 10 or 100.
  • the adenoviral vector was diluted in 0.5 ml of the transduction medium, and was placed on the cell monolayer for 1.5 hours at 37°C. The medium was removed and 1 ml of fresh transduction medium then was added.
  • Vector derived human apoE had an identical molecular weight of 34,000 daltons compared to both purified human apoE ( Figure 11, lanes 1 and 2) and the endogenous apoE synthesized by HepG2 cells ( Figure 11, lanes 3 and 4) .
  • mice Adenoviral Expression of Human APOE in vivo C57BL/6J apoE-deficient mice were obtained from the Jackson Laboratory (Bar Harbor, Maine) . These mice were homozygous for the disruption of the apoE gene (Piedrahita, et al . , Proc. Nat. Acad. Sci.. Vol. 89, pgs. 4471-4475 (1992); Zhang, et al . , Science. Vol. 258, pgs. 468-471 (1992) ) . The mice were fed Purina mouse chow and allowed to acclimate approximately 3 weeks prior to vector administration. Each mouse then was treated by tail vein injection with 5x10" recombinant AvlRE or AvlLacZ4 adenovirus vector particles in 400 ⁇ l Hank's buffered saline solution (HBSS) .
  • HBSS Hank's buffered saline solution
  • Plasma cholesterol concentrations were determined before and after treatment using enzymatic methods (Sigma, St. Louis, Missouri) . Blood was collected either from the retro- orbital plexus or the tail vein, and was transferred immediately to heparinized tubes . Plasma was collected following centrifugation at 7000 xg for 5 minutes. EDTA, Pefabloc, and apoprotinin were added to all plasma samples at final concentrations of 2 mM, 1 mM, and 10 ⁇ g/ml, respectively.
  • PVDF polyvinylidene difluoride
  • Biorad Hercules, CA
  • Molecular weight markers used ranged from 200 kDa to 14 kDa (Biorad) .
  • the PVDF membrane was blocked in 10 mM Tris, pH 7.4, containing 150 mM NaCl, 2 mM EDTA, 0.04% Tween 20 and 5% milk.
  • the blocked membrane was first incubated for l hour at room temperature in a 1:3000 dilution of the primary antibody, anti-human apoE monoclonal, clone 3H1 (obtained from Dr. Y. Marcel, University of Ottawa) .
  • the membrane was developed with a secondary goat anti-mouse IgGl-horseradish peroxidase (HRPO) conjugated antibody (Southern Biotechnology Associates, Inc., Birmingham, AL) using an enhanced chemiluminescence system (Amersham Lifesciences, Arlington Heights, ID .
  • HRPO horse anti-mouse IgGl-horseradish peroxidase
  • the membrane was exposed to film for approximately 1 to 10 sec.
  • Purified human apoE Calbiochem, La Jolla, CA was used as a positive control on all Western analysis.
  • the column was equilibrated in 10 mM Tris, pH 7.4 containing 150 mM NaCl, 2 mM EDTA, and 0.02% sodium azide at a flow rate of 0.35 ml/min and 0.5 ml fractions were collected. Cholesterol was determined on 100 ⁇ l of each fraction by enzymatic methods. Purified human VLDL and HDL (Calbiochem) were used for calibration of the column.
  • Human apoE in mouse plasma was measured using a sandwich type ELISA utilizing a mouse anti-human apoE monoclonal, 9- H8, as the capture antibody and a goat polyclonal as the detecting antibody. Values were determined using a standard curve obtained by including varying amounts of purified recombinant human apoE3 into plasma from apoE-deficient mice. Microtiter plates (Immulon 4, Dynatech) were coated with the 9-H8 antibody (5 ⁇ g protein/ml) (Cappel, Durham, NO in 100 mM sodium bicarbonate, pH9.6 overnight at room temperature.
  • the monoclonal antibody solution was removed and the plate was washed 5 times using phosphate buffered saline (PBS) containing 0.05% Tween-20. Unless otherwise noted all subsequent incubations were carried out for one hour at room temperature. The nonspecific protein binding sites in each well were blocked using PBS containing 10% non-fat dry milk. The blocking buffer was removed and the plates were washed as described previously. The standards and samples were diluted in PBS containing 2% bovine serum albumin (BSA) and allowed to incubate overnight. A 100 ⁇ l aliquot of each sample or standard dilution was placed in individual wells and allowed to incubate.
  • PBS phosphate buffered saline
  • the plates were washed as described and were then incubated with 100 ⁇ l of a 1:4000 dilution of the secondary anti-human apoE goat polyclonal antibody (Calbiochem, La Jolla, CA) in PBS containing 2% BSA. After washing, the plates were incubated with 100 ⁇ l of a 1:3000 dilution of the tertiary, swine anti-goat IgG-HRPO polyclonal antibody (Caltag, San Francisco, CA) .
  • the reaction was developed using 100 ⁇ l of 0.2 mg/ml 3,3',5,5', - tetramethylbenzidine (TMB) , 0.01% H 2 0 2 and was stopped by the addition of 100 ⁇ l of 1M phosphoric acid.
  • the absorbance at 450 nm was measured using 405nm as reference.
  • the average concentrations in mouse plasma 7 days after vector administration was 1.2 ⁇ g/ml, which is approximately 4% of normal human apoE levels.
  • the mean cholesterol value in apoE-deficient mice prior to vector treatment was 794 mg/dl (Zhang, 1992; Plump, Cell. Vol. 71, pgs. 343-353 (1992); Nakashima, et al . , Arterioscler. Thromb.. Vol. 14, pgs. 133-140 (1994),- Ishibashi, et al . , Proc. Nat. Acad. Sci., Vol. 91, pgs. 4431-4435 (1994)).
  • mice that were given AvlLacZ4 had similar levels of plasma cholesterol before and after treatment, indicating that the reduction in plasma cholesterol concentrations was due to the expression of human apoE.
  • the plasma lipoprotein distribution of the AvlRE and AviLacZ4 treated mice 7 days after vector administration is shown in Figure 14.
  • Pooled plasma from each treatment group was fractionated using a Superose 6 gel filtration column as hereinabove described, and cholesterol content was measured in each fraction across the elution profile.
  • the open circles denote plasma from the AvlLacZ4 treated group
  • the dark circles denote plasma from the mice treated with AvlRE.
  • the lipoprotein elution profile of the AvlLacZ4-treated group showed that the majority of the cholesterol eluted in the VLDL/LDL region. (Zhang, et al .
  • the plasma lipoprotein distribution of human apoE in the AvlRE-treated mice was confirmed by Western analysis of the fractionated plasma samples (Figure 15) .
  • the FPLC fraction numbers from Figure 14 are listed above each lane.
  • the majority of the human apoE was associated with the VLDL/LDL fraction, although a smaller proportion of the apoE was detected in the HDL fraction.
  • FIGs 16A and 16B show total plasma cholesterol and plasma human apoE concentrations after adenoviral vector administration. Open circles denote the AvlLacZ4-treated mice, and black circles denote the AvlRE-treated mice. Plasma human apoE concentrations were determined by ELISA as hereinabove described. As shown in Figure 16A, the reduction in total plasma cholesterol levels in the AvlRE-treated apoE- deficient mice persisted for at least 21 days after administration of the adenoviral vector. The mean plasma cholesterol concentrations in the AvlRE-treated apoE- deficient mice were approximately 150 mg/dl over the first 21 days although a greater variation was found between individuals at 14 and 21 days.
  • the plasma cholesterol concentrations in the AvlRE-treated group increased to approximately 550 mg/dl, but this was lower than that seen in the control vector group. No significant change in the plasma cholesterol concentrations was observed over the course of the study in AvlLacZ4-treated control mice.
  • results in this example showed that adenovirus mediated expression of human apoE in hypercholesterolemic apoE-deficient mice resulted in a transient phenotypic reversion of the hypercholesterolemic state normally found in this mouse strain.
  • the levels of human apoE expression were sufficient to produce a significant lowering of plasma cholesterol concentrations for at least 21 days after vector administration.
  • correction of hypercholesterolemic conditions by direct in vivo gene transfer may be employed for the treatment of hyperlipidemias due to apoE deficiency (Schaefer, et al., J. Clin. Invest.. Vol. 78, pgs. 1206-1219 (1986)) or dysfunction (Rail, et al . , J. Internal Med.. Vol. 231, pgs. 653-659 (1992)).
  • ApoE-deficient mice have both intestinal- and liver-derived remnant lipoprotein particles that accumulate in plasma and result in elevated plasma cholesterol concentrations (Zhang, et al . , 1992; Plump, et al . , 1992) . These remnant particles are normally cleared from the circulation via the interaction of apoE with either the LDLR and/or the LRP (Ishibashi, et al . , Proc. Nat. Acad. Sci.. Vol.
  • ADDRESSEE Carella, Byrne, Bain, Gilfillan, Cecchi, Stewart & Olstein
  • MOLECULE TYPE oligonucleotide
  • MOLECULE TYPE oligonucleotide
  • MOLECULE TYPE oligonucleotide
  • MOLECULE TYPE oligonucleotide

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Abstract

La présente invention concerne un vecteur adénoviral comprenant une séquence d'acides nucléiques codant pour l'apolipoprotéine E ou bien un fragment ou un dérivé de celle-ci. Ces vecteurs adénoviraux peuvent être administrés à un hôte dans le cadre d'un traitement de l'hypercholestérolémie par thérapie génique.
PCT/US1995/014541 1994-11-09 1995-11-08 Therapie genique contre l'hypercholesterolemie WO1996014837A1 (fr)

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US5770442A (en) * 1995-02-21 1998-06-23 Cornell Research Foundation, Inc. Chimeric adenoviral fiber protein and methods of using same
WO2000023587A2 (fr) * 1998-10-16 2000-04-27 Introgene B.V. Therapie genique de la maladie d'alzheimer par administration d'une apolipoproteine e codee
US6057155A (en) * 1995-11-28 2000-05-02 Genvec, Inc. Targeting adenovirus with use of constrained peptide motifs
US6127525A (en) * 1995-02-21 2000-10-03 Cornell Research Foundation, Inc. Chimeric adenoviral coat protein and methods of using same
US6913922B1 (en) 1999-05-18 2005-07-05 Crucell Holland B.V. Serotype of adenovirus and uses thereof
US6929946B1 (en) 1998-11-20 2005-08-16 Crucell Holland B.V. Gene delivery vectors provided with a tissue tropism for smooth muscle cells, and/or endothelial cells
US6951755B2 (en) 1994-09-08 2005-10-04 Genvec, Inc. Vectors and methods for gene transfer
US6974695B2 (en) 2000-11-15 2005-12-13 Crucell Holland B.V. Complementing cell lines
US7235233B2 (en) 2000-09-26 2007-06-26 Crucell Holland B.V. Serotype 5 adenoviral vectors with chimeric fibers for gene delivery in skeletal muscle cells or myoblasts
US7309606B2 (en) 2004-09-07 2007-12-18 The Trustees Of Boston University Methods and compositions for treating hypercholesterolemia
US7749493B2 (en) 1998-07-08 2010-07-06 Crucell Holland B.V. Chimeric adenoviruses

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WO1994010322A1 (fr) * 1992-10-29 1994-05-11 Board Of Regents, The University Of Texas System Transfert et ciblage du gene recepteur ldl induit par l'adenovirus

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, Vol. 200, No. 1, issued 15 April 1994, FAZIO et al., "Accumulation of Human Apolipoprotein-E in Rat Plasma After in Vivo Intramuscular Injection of Naked DNA", pages 298-305. *
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Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6951755B2 (en) 1994-09-08 2005-10-04 Genvec, Inc. Vectors and methods for gene transfer
US6127525A (en) * 1995-02-21 2000-10-03 Cornell Research Foundation, Inc. Chimeric adenoviral coat protein and methods of using same
US6153435A (en) * 1995-02-21 2000-11-28 Cornell Research Foundation, Inc. Nucleic acid that encodes a chimeric adenoviral coat protein
US5770442A (en) * 1995-02-21 1998-06-23 Cornell Research Foundation, Inc. Chimeric adenoviral fiber protein and methods of using same
US6057155A (en) * 1995-11-28 2000-05-02 Genvec, Inc. Targeting adenovirus with use of constrained peptide motifs
US6329190B1 (en) 1995-11-28 2001-12-11 Genvec, Inc. Targetting adenovirus with use of constrained peptide motifs
US6649407B2 (en) 1995-11-28 2003-11-18 Genvec, Inc. Targeting adenovirus with use of constrained peptide motifs
US7749493B2 (en) 1998-07-08 2010-07-06 Crucell Holland B.V. Chimeric adenoviruses
WO2000023587A2 (fr) * 1998-10-16 2000-04-27 Introgene B.V. Therapie genique de la maladie d'alzheimer par administration d'une apolipoproteine e codee
WO2000023587A3 (fr) * 1998-10-16 2000-08-03 Introgene Bv Therapie genique de la maladie d'alzheimer par administration d'une apolipoproteine e codee
US6929946B1 (en) 1998-11-20 2005-08-16 Crucell Holland B.V. Gene delivery vectors provided with a tissue tropism for smooth muscle cells, and/or endothelial cells
US6913922B1 (en) 1999-05-18 2005-07-05 Crucell Holland B.V. Serotype of adenovirus and uses thereof
US7250293B2 (en) 1999-05-18 2007-07-31 Crucell Holland B.V. Complementing cell lines
US7270811B2 (en) 1999-05-18 2007-09-18 Crucell Holland B.V. Serotype of adenovirus and uses thereof
US7235233B2 (en) 2000-09-26 2007-06-26 Crucell Holland B.V. Serotype 5 adenoviral vectors with chimeric fibers for gene delivery in skeletal muscle cells or myoblasts
US6974695B2 (en) 2000-11-15 2005-12-13 Crucell Holland B.V. Complementing cell lines
US9228205B2 (en) 2000-11-15 2016-01-05 Crucell Holland B.V. Complementing cell lines
US7309606B2 (en) 2004-09-07 2007-12-18 The Trustees Of Boston University Methods and compositions for treating hypercholesterolemia

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