TECHNICAL FIELD

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.

BACKGROUND OF THE INVENTION

There are presently several approaches being studied for delivering genes to humans.

These approaches have either removed lymphocytes, permanently transformed them in vitro using retrovirus vectors, and reinfused the transformed cells, or used adenoviruses to deliver the gene via the airways, again using retrovirus vectors. Such therapy has been designed for use with

patients having inherented deficiency of gene products, such as proteins, due to abnormalities of the gene development at an early age. Such therapies have also been used with diseases such as emphysema wherein it is thought that the disease develops as a result of a relative deficiency of an antiprotease over a long period of time. Further, diseases such as acute lung injury resulting in the adult respiratory distress syndrome (ARDS) is thought to involve a relative deficiency of antiprotease activity. Thus, the delivery of a gene which expresses a product, such as the delivery of a functioning alpha-1 antiprotease gene, to the lungs could be therapeutic in many human conditions characterized by injury of the lungs.

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.

SUMMARY OF THE INVENTION

In accordance with the present invention, there is provided 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.

FIGURES IN THE DRAWINGS

Other advantages of the present invention will be readily appreciated as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings wherein: 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; and

Figure 7 is a restriction enzyme site map list for the alpha-l antitrypsin.

DETAILED DESCRIPTION OF THE INVENTION

Generally, 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. Thusly, 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.

More specifically, 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.

In order to enhance the expression of the human alpha-l antitrypsin gene, 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. Applicant shows below that when the above described plasmid is complexed with liposomes and injected into rabbits, expression of the human alpha-l antitrypsin gene in the lungs can be demonstrated by several techniques, including Northern blots (demonstrating messenger RNA specific for and a precursor of the human protein) , Western blots of supernatants for organ cultures (demonstrating actual production and secretion of the protein) and immunohistoche ical staining of thin sections of the lungs taken from the transfected rabbits.

The specific use of the human alpha-l antitrypsin is significant because 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. Presently applicant shows the ability to transfect animals with the human alpha-l antitrypsin-pCMV4 construct thereby delivering a gene of physiological importance. In synthesizing the liposome-plasmid construct, applicant first constructed fusion genes in expression vectors. Applicant utilized the polymerase chain reaction (PCR) amplification and "linker primers" as shown in Figure 5 to insert the coding sequence of human alpha-l antitrypsin into the pCMV4 expression vector.

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.

After the primers were designed, 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. For the PCR reactions the buffer was: lOmM KCl lOmM (NHU) ₂ S04

20mM Tris-HCl (pH 8.8)

2mM MgS0 ₄

0.1% Triton X-100

2 units of Vent DNA polymerase

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.

After PCR amplification, the unique restriction sites were cleaved with the appropriate restriction enzymes. Restriction enzymes digest was conducted using the following:

Small Buffer 60mM Tris-HCl, pH 8.0 lOx Buffer 60mM MgCl ₂ 200 mM KCl 60 mM β-mercapto ethanol

1 mg/ml BSA

Note: Due to buffer incompatibility the DNA was precipitated with sodium acetate and EtOH between the digests. Clal Buffer lOOmM Tris-HCl, pH 7.5 lOOmM MgCl ₂

10mM (DDT) dithiothreitol . lmg/ml BSA 1.0 M NaCl 10-15 μl of each enzyme was used. Reactions were for 2 hours at 30°C for small and 37°C for clalC. The amplified gene was separated from the small fragments released by the action of the restriction enzymes and from unincorporated primers and nucleotides by gel filtration through an S-400 spin column. 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. In particular, 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. In addition, 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 development of the polymerase chain reaction procedure by Cetus proved to be of great importance in the development of the present invention. However, there were two major problems with PCR which had to be overcome. First, 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. However, this approach yields a piece of DNA with two "blunt" ends. Blunt end cloning is more difficult to accomplish and prevents directional cloning. This was overcome.in the above process by utilizing "linker-primers" as discussed above. Applicant designed specific restriction enzyme sites into the PCR primers such that applicant could achieve directional cloning of the amplified DNA.

After ligation, 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 Bioloσy 166:557-580, 1980.

The preparation of Competent E.Coli for one transformation was as follows:

1. Inoculate 10-20 ml of dYT medium with a single colony of the desired strain of E. coli. Prepare the plates at this time. When plasmids are used as vectors, the appropriate antibiotics should be used (ampicillin lOOμg/ml carbenicillin

lOOμg/ml. Incubate overnight at 37 ^β C in a. shaker set to 300 rpm.

2. Add 300 μl of E. coli. culture into 30 ml dYT medium. 3. Grow until A ₆₀₀ is between 0.45 and 0.55. Check O.D. after 1.5 hours.

4. Put cells into a 50 ml polypropylene tube on ice for 10-15 minutes.

5. Centrifuge the cells for 10 minutes at 3000 rpm.

6. Pour out the supernatant and add 1 ml of ice cold transformation buffer (TFB) . Carefully resuspend the cells with a pipet.

7. Add 9 ml TFB. Swirl gently and place on ice for 10 minutes.

8. Centrifuge the cells for 10 minutes at 3000 rpm.

9. Resuspend the cells in 2ml TFB.

10. Add 70 μl DMSO (fresh HPLC grade). Leave on ice for five minutes

11. Add 70 μl 2.25 M DDT/40 mM KAc [pH 6.0]. Leave on ice for five minutes.

12. Add 70 μl DMSO. Leave on ice for five minutes.

Transformation of E. Coli. with vectors containing insert DNA.

1. Add a 1/3 of ligation mixture. Mix gently. The volume added should be approximately 100 μl or less. Incubate for 45 minutes on ice. (The cells need not be kept on ice after this.)

2. Heat shock the mixture for 210 seconds (3.5 minutes) at 40°C. Put samples on ice for 1-2 minutes. 3. Add dYT to 1 ml final vol.

4. Shake gently for one hour at 37°C.

Note: When the vector used is plasmid DNA, the appropriate antibiotic should be added to the LB agar and top agar (amp. 100 μg/ml) .

Plating the Bacteria.

1. From the 1.0 ml of cells add: take alignots of 50, 100 and 200 μl. Plate each aliquot (spread with a flame sterilized glass spreader) on a different plate (LB agar and carbenicillin) .

2. Incubate at 37°C overnight.

Note: 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

Tryptone 20 g Yeast Extract 5 g NaCl 2 ml (5M) KCl 0.625 ml (4M)

Aliquot into 2-500 ml bottles. Autoclave, let cool and add 5 ml of the following solution to each bottle.

1 part 2M MgCl ₂ : 1 part 2M MgS0 ₄ (sterile filtered)

o , .

Sterile filter and store at 4°C.

Ran Med-iu-m

20 mM glucose SOB

1.8ml 20% glucose 1100 ml SOB

2.25 MDTT/40mMKOAc pH 6

dYT Medium Quantity/1

Typtone 16 g

Yeast Extract 10 g NaCl 5 g

After transfection, 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

(carbenicillin containing medium) and grow to saturation.

2. Centrifuge , (1.5 ml of step 1) remove supernatant, resuspend in (DDM) TE, and incubate 5' at room temperature.

3. Add 200μl of (NaOH/SDS) vortex and incubate 5' on ice. (0.2M NaOH, 1% SDS fresh).

4. Add 150 ml (potassium acetate), vortex incubate 5 ¹ on ice. 296 g potassium acetate, 70 ml 90% formic acid, pH to pH 4.8.

5. Centrifuge and transfer supernatant to a fresh tube.

6. Add 0.9 ml of 100% ethanol. Icubate 2 minutes at room temperature. Centrifuge for 10 minutes.

7. Wash to 70% ethanol.

8 . Dry

9. Resuspend in 20 μl of TE or sterile H ₂ 0.

Applicant then confirmed that 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.) Also, 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.

Once a colony had been isolated which had contained the piece of cloned DNA, a large scale plasmid preparation was grown. The 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. It appears that the 2 step purification (polyethylene glycol and CsCl) is critical to the process. It is essential to remove endotoxin (lipopolysaccharide A from gram negative bacterial) from the plasmid. Current analysis demonstrates that we have 45 pg of endotoxin/5μg of plasmid. Without the 2-step purification endothelial cells were consistently killed by Lipofection (presumably due to endotoxin contamination.

Lipofection was performed by the following method. For in vivo studies in rabbits, 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.

Figure 2 shows the results of studies in living animals of Lipofection of the

Lipofection/plasmids construct made in accordance with the present invention. Figure 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.

demonstrating the gene product by immunohistochemical staining as shown in Figure 3. Specifically, 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. Specifically referring to Figure 4, Northern blot of 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.

The above data shows successful transfection of both cells and animals with a construct made in accordance with the present invention. Related prior art as set forth by Felgener, P.L. and Rhodes, G. Nature 349:351- 352, 1991 disclosed examples of direct delivery of a gene in vivo. The vast majority of the examples disclosed were reporter genes and the only genes demonstrating expression after liposome mediated delivery were reporter genes. Applicant has demonstrated above not only the direct delivery of a gene in vivo but the production of immunoreactive protein which was found in the appropriate tissue (airway) despite the fact that the gene was given by intravenous injection.

The invention has been described in an illustrative manner, and it is to be understood that the terminology which has been used is intended to be in the nature of words of description rather than of limitation.

Obviously, many modifications and variations of the present invention are possible in light of the above teachings. It is,

therefore, to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described.