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WO1992019730A1 - Structure d'adn servant a l'expression in vivo d'un gene humain - Google Patents

Structure d'adn servant a l'expression in vivo d'un gene humain Download PDF

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Publication number
WO1992019730A1
WO1992019730A1 PCT/US1992/002465 US9202465W WO9219730A1 WO 1992019730 A1 WO1992019730 A1 WO 1992019730A1 US 9202465 W US9202465 W US 9202465W WO 9219730 A1 WO9219730 A1 WO 9219730A1
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WO
WIPO (PCT)
Prior art keywords
plasmid
cells
antitrypsin
set forth
human alpha
Prior art date
Application number
PCT/US1992/002465
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English (en)
Inventor
Kenneth Brigham
Jon Conary
Angelo Canonico
Barbara Meyrick
Original Assignee
Vanderbilt University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Vanderbilt University filed Critical Vanderbilt University
Publication of WO1992019730A1 publication Critical patent/WO1992019730A1/fr

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    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8121—Serpins
    • C07K14/8125—Alpha-1-antitrypsin

Definitions

  • the present invention provides novel plasmids, liposomes, primers, and methods of using the same for gene therapy or gene therapeutics. More specifically, the present invention provides a means for delivery of genetic material capable of expressing a product which would otherwise be deficient in a target tissue and expression of the genetic material which will produce the product which was deficient in the target tissue.
  • the present invention provides means for the delivery of a gene capable of expressing a product to a target tissue. More specifically, the present invention provides means for providing the human alpha-1 antitrypsin gene to the lungs for expression of the human alpha-1 antitrypsin capable of alleviating the enzyme deficiency.
  • a plasmid consisting essentially of a small pCMV4 expression vector including a coding seguence of human alpha-1 antitrypsin incorporated therein.
  • the present invention further provides a mechanism for delivery of a gene to a patient, the mechanism comprising a liposome including an expression plasmid incorporated therein, the plasmid being capable of tissue specific targeting.
  • the plasmid is also capable of expression of the gene extrachromosomally in the cells of the target tissue and is unincorporable into the chromosomes of the cells of the target tissue.
  • the present invention also provides a primer for inserting a coding sequence of a protein into an expression vector, the primer including an oligonucleotide having a 3'end and a 5'end.
  • An oligonucleotide includes a restriction enzyme site about five to ten nucleotides downstream of the 5'end for insuring a three dimensional structure of the primer for a restriction enzyme.
  • the present invention further provides a method of treatment for a deficiency of a gene product in cells of a target tissue.
  • the method includes the steps of transfecting a liposome into cells of a target tissue, the liposome including an expression plasmid incorporated therein which codes for the deficient protein, the plasmid remaining extrachromosomal and being non-replicative in the cells, and expressing the deficient gene product in the cells.
  • Figure 1 is a diagram of the DNA construct made in accordance with the present invention.
  • Figure 2 is a chart showing the expression of a human alpha-1 antitrypsin gene in the organs of a rabbit;
  • Figure 3 shows two photomicrographs showing an immunohistochemical demonstration of human alpha-1 antitrypsin in the lungs of a rabbit two days after .in vivo transfection with pCMV4 containing the human alpha-1 antitrypsin gene;
  • Figure 4 is a photomicrograph of a Northern blot of RNA extracted from the organs of a rabbit two days after in vivo transfection with the pCMV4 plasmid including the human alpha-l antitrypsin gene;
  • Figure 5 is a schematic illustration of the primers used to insert the coding sequence of the human alpha-l antitrypsin into the pCMV4 expression vector;
  • Figure 6 is the DNA sequence for human alpha-l antitrypsin.
  • Figure 7 is a restriction enzyme site map list for the alpha-l antitrypsin.
  • the present invention provides a plasmid consisting essentially of a pCMV4 expression vector including a coded sequence of human alpha-l antitrypsin incorporated therein.
  • the plasmid can be incorporated into the liposome capable of targeting the specific tissue.
  • the plasmid is then capable of expression of the gene extrachromosomally in the cells of the target tissue and is unincorporable into the chromosome of the cells of the target tissue.
  • the liposome including the plasmid can be used in a method for treating a deficiency of the gene product in cells of the target tissue.
  • the pCMV4 expression vector including the coding sequence for the human alpha-l antitrypsin (the sequence being shown in Figure 6 and the restriction enzyme map list being shown in Figure 7 incorporated therein is shown in Figure l.
  • the diagram is of the DNA construct, the plasmid being a circular piece of DNA which can function to express the genes (pieces of the DNA) which have been inserted into the plasmid.
  • the plasmid shown, designated as small pCMV4 contains the cDNA for human alpha-l antitrypsin.
  • This circular piece of DNA including the coded sequence for the human alpha- 1 antitrypsin is driven by a cytomeg lovirus (CMV) derived promoter.
  • CMV cytomeg lovirus
  • the construct also includes a short transcription augmenter sequence 5' to the coded region of the human alpha-l antitrypsin.
  • the augmenter sequence increases the amount of translation per unit messenger RNA.
  • the construct also includes a human growth hormone (hGH) 3* untranslated region (UTR) . The hGH 3' UTR stabilizes the message.
  • alpha-l antitrypsin is a normally produced antiprotease which is important in protecting the lungs against emphysema.
  • the adult respiratory distress syndrome (ARDS) is thought to involve a relative deficiency of antiprotease (alpha-l antitrypsin) activity. Therefore, the delivery of a functioning alpha-l antiprotease gene to the lungs can be therapeutic in many human conditions characterized by injury of the lungs.
  • the liposome-plasmid complex made in accordance with the present invention offers many advantages over prior art approaches discussed above.
  • the plasmid made in accordance with the present invention does not replicate in eukaryotic cells. Therefore, the increased expression of the gene is transient.
  • the plasmid is not readily incorporated into the host DNA. Both of these characteristics are important for safety in human administration because the characteristics avoid permanent alterations in gene expression, do not interfere with the normal functioning of the host genome, and do not have malignant potential which is possible with the use of retroviruses.
  • the preferred liposome used in accordance with the present invention is Lipofectin (Bethesda Research Laboratories) which provided applicant with the means to transfect cells or animals with the plasmid without the manipulation and potential harm inherent to such procedures as electroporation or CaP0 4 precipitation. Applicant demonstrated first with cells and then with mice that a plasmid construct a containing a reporter gene could be transfected by Lipofectin and that the transfected gene could be expressed. Additional studies confirm that non-reporter genes (human alpha-l antitrypsin) could be expressed in endothelial cells after Lipofection.
  • non-reporter genes human alpha-l antitrypsin
  • the method consisted of synthesizing two oligonucleotide primers of twenty to thirty nucleotides in length.
  • One nucleotide was homologous to the 5' untranslated region immediately upstream (5') of the initiation codon.
  • the second oligonucleotide was complementary to the 3' untranslated region immediately downstream (3 1 ) of the stop transcription codon.
  • Both oligonucleotides have a one or two base substitution which creates a unique and different restriction enzyme site in the untranslated regions of the amplified gene.
  • the 5' and 3' oligonucleotides were designed such that the created restriction enzyme site is approximately eight nucleotides downstream from the 5' end of the oligonucleotide. Both of these requirements were critical, the former to insure a restriction enzyme site which was recognizable and cleavable and the latter to insure that the reading frame of the gene was not altered.
  • the primers were synthesized by the Vanderbilt University Molecular Biology Core Laboratory.
  • the reading frame of the gene of interest was amplified using Vent DNA polymerase, 100 ng of target DNA, a programmable temperature cycler, and standard reaction conditions.
  • the buffer was: lOmM KCl lOmM (NHU) 2 S04
  • each primer 200mM (for each dNTP, dATP, dCTP, dGTP, dTTP) l.OmM each primer.
  • Denaturing conditions were at 93.5° C, annealing at 56° C and extension at 75° C.
  • Vent DNA polymerase was used because it has a 3' to 5' proofreading activity in addition to enhanced stability at high temperature and a highly specific and processive 5• to 3' DNA polymerase activity.
  • Restriction enzymes digest was conducted using the following:
  • the amplified genes which had cloning sites on each end were ligated into pCMV4 which had been previously cleaved with the same restriction enzymes which were utilized to prepare the cloning sites on the amplified gene.
  • Applicant had selected the pCMV4 vector as the expression construct because it represents a "state of the art" expression vector.
  • this vector provides a stable mRNA through the use of growth hormone termination and poly-adenylation signals.
  • This vector also provides sequences from the alpha Mosiac virus 4 which act as a transription augmenter by decreasing the requirement for initiation factors in protein synthesis.
  • this construct also contains a polylinker region and the promoter-regulatory region of the human cytomegalovirus major immediate early gene, as shown in Figure 1.
  • the original PCR method utilized Taq DNA polymerase as manufactured by Cetus.
  • the Taq DNA polymerase is an enzyme purified from Thermatus aquaticus. While this enzyme performs well in the role of DNA amplification, it does not have any proofreading capability (i.e. 3' to 5' exonuclease) .
  • the lack of proofreading ability and the reasonably high rate of base misincorporation makes it difficult to clone a PCR product and be assured of an authentic copy.
  • the use of the Vent DNA polymerase manufactured by New England Biolabs provided a different high temperature DNA polymerase which had proofreading activity thereby alleviating the aforementioned problems.
  • the second problem with cloning PCR products is the lack of an easily clonable piece of DNA after PCR amplification.
  • the amplification process routinely leaves the 3' overhang of either an A or T nucleotide.
  • the overhanging bases can be removed by the action of one of several DNA exonucleases.
  • this approach yields a piece of DNA with two "blunt” ends. Blunt end cloning is more difficult to accomplish and prevents directional cloning.
  • Applicant designed specific restriction enzyme sites into the PCR primers such that applicant could achieve directional cloning of the amplified DNA.
  • the pCMV4-AAT (alpha-l antitrypsin) construct was transfected into fresh competent bacteria (E.coli NM522) .
  • the competent bacteria were prepared by standard methods as disclosed by Hanahan, D. J. Molecular Biolo ⁇ y 166:557-580, 1980.
  • Competent E.Coli for one transformation was as follows:
  • the appropriate antibiotic should be added to the LB agar and top agar (amp. 100 ⁇ g/ml) .
  • Steps 1 and 2 need to be done quickly before the top agar begins to harden. After melting, keep the top agar in a 50°C water bath until it is used. SOB Medium OTYL/L
  • the bacteria was plated out on plates containing carbenicillin, an ampicillin analog. This analog provided selection pressure for bacteria which contained the pCMV4 construct.
  • the plasmid carries the gene for ampicillin resistance. After the bacteria which harbored the plasmid were grown into distinct colonies, several of the colonies were grown up as individual 5 ml liquid cultures. An aliquot of the liquid cultures was stored and the rest was processed as a "mini" preparation.
  • the plasmid DNA mini prep was conducted as follows: 1. Inoculate 5 ml of dYT
  • the isolated plasmid contained the inserted piece of DNA by performing both a dot blot analysis and by releasing the inserted piece of DNA by performing a restriction enzyme digest.
  • the restriction enzyme digest yielded both the linearized plasmid and the original piece of DNA (as demonstrated by ethidium bromide staining of the DNA after electrophoresis to separate the two species in a 1% agurose gel. 1XTAE buffer 5V/cm.)
  • the restriction enzyme map ( Figure 7) shows that sequences specific for restriction enzyme used are only in the novel premises and not in the unmodified sequence.
  • plasmid was purified by lysis of the bacteria and precipitation of the plasmid was accomplished with polyethylene glycol. Then the plasmid was purified an additional time by ultracentrifugation in an isopynic CsCL solution. After ultracentrifugation for 40 hours at 45,000 rpm, the purified plasmid was withdrawn through the side of the tube and the ethidium bromide was removed by extraction with TE saturated butanol. Finally, the isolated plasmid was precipitated with ethanol and resuspended in sterile water.
  • Lipofection was performed by the following method.
  • the DNA was administered at a dose of 500 mg/kg as a complex with Liopfectin.
  • 500 mg of DNA were brought up to a volume of 2.5 ml of sterile water and combined with 2.5 ml of Lipofectin at a ratio of 1:5 DNA to Lipofectin.
  • the DNA/Lipofectin mixture was gently mixed and allowed to equilibrate for 10 to 15 minutes. Extreme care was exercised to prevent any negative pressure on the mixture because this would tend to result in the precipitation of the complexed DNA/Lipofectin.
  • the DNA/Lipofectin complex was then slowly administered to the animal by intravenous injection. The success of the transfection was demonstrated by analyzing the animals tissue for the presence of the RNA synthesized from the plasmid and by immunohistochemical staining.
  • FIG. 1 shows the results of studies in living animals of Lipofection of the
  • FIG. 2 shows the production of human alpha-l antitrypsin by organs removed from a rabbit four days following in vivo transfection with pCMV4-AAT.
  • the alpha-l antitrypsin activity was measured by ELISA using a human specific antibody.
  • Figure 2 shows production of the human alpha-l antitrypsin predominantly in lungs.
  • the localization in lungs by this method is particularly significant with regard to the gene therapy use of the present invention against emphysema and other related alpha-l antitrypsin activity diseases as discussed above. Applicant studied histological sections of lungs removed from the transfected rabbits.
  • Figure 3 shows a immunohistochemical demonstration of human alpha- l antitrypsin in the lungs of a rabbit two days after in vivo transfection with pCMV4-AAT.
  • the section on the left of Figure 3 shows red staining of the airway epithelium and some lung parenchymal cells resulting from immunohistochemical staining using an antibody specific for human alpha-l antitrypsin.
  • the control section on the right of Figure 3 was treated identically, except the antibody was omitted and there was no red staining.
  • mRNA was demonstrated for the introduced gene by Northern blot as shown in Figure 4.
  • RNA extracted from the organs of a rabbit two days after in vivo transfection with pCMV4-AAT is shown.
  • the blot was probed with a cDNA specific for human alpha-l antitrypsin.
  • the lane on the left was from lung, the middle lane was from liver and the right lane from kidney.
  • the arrow points to the alpha-l antitrypsin specific band.
  • the blot shows mRNA for human alpha-1 antitrypsin in the lung and liver with greatest expression in the lung.

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Abstract

Un plasmide est constitué essentiellement par un petit vecteur d'expression de pCMV4 comprenant une séquence de codage d'antitrypsine alpha-1 qui lui est intégrée. L'invention décrit, de plus, un procédé servant à introduire le gène chez un patient, le processus incluant un liposome dans lequel est intégré le plasmide d'expression; le plasmide peut effectuer l'expression extrachromosomique du gène dans les cellules d'un tissu cible, mais ne peut pas s'intégrer au chromosome des cellules du tissu cible. L'invention décrit, de plus, une amorce servant à introduire la séquence de codage de la protéine dans le vecteur d'expression. Enfin, elle décrit un procédé de traitement de la déficience d'un produit génétique dans les cellules d'un tissu cible au moyen du nouveau liposome.
PCT/US1992/002465 1991-04-24 1992-03-27 Structure d'adn servant a l'expression in vivo d'un gene humain WO1992019730A1 (fr)

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US690,283 1991-04-24

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998017321A1 (fr) * 1996-10-24 1998-04-30 Vanderbilt University Apport et expression geniques dans des zones inaccessibles a l'apport direct de proteines
US6638909B1 (en) 1996-11-01 2003-10-28 Ethicon, Inc. Wound healing compositions containing alpha-1-antitrypsin

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CELL, Volume 41, issued June 1985, G. CILIBERTO et al., "Cell-Specific Expression of a Transfected Human alpha 1-Antitrypsin Gene", pages 531-540. *
JOURNAL OF PHARMACEUTICAL SCIENCES, Volume 72, No. 2, issued February 1984, R.M. ABRA et al., "Liposome Disposition in Vivo VI: Delivery to the Lung", pages 203-206. *
NEURON, Volume 1, issued June 1988, A.F. RUSSO et al., "Neuronal Expression of Chimeric Genes in Transgenic Mice", pages 311-320. *
VIROLOGY, Volume 171, issued 1989, C.M. GORMAN et al., "The Human Cytomegalovirus Major Immediate Early Promoter can be Trans-Activated by Adenovirus Early Proteins", pages 377-385. *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998017321A1 (fr) * 1996-10-24 1998-04-30 Vanderbilt University Apport et expression geniques dans des zones inaccessibles a l'apport direct de proteines
AU732447B2 (en) * 1996-10-24 2001-04-26 Vanderbilt University Gene delivery and expression in areas inaccessible to direct protein delivery
US6365575B1 (en) 1996-10-24 2002-04-02 Vanderbilt University Gene delivery and expression in areas inaccessible to direct protein delivery
US6638909B1 (en) 1996-11-01 2003-10-28 Ethicon, Inc. Wound healing compositions containing alpha-1-antitrypsin

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