WO1996004397A1

WO1996004397A1 - Gene therapy using homologous recombination

Info

Publication number: WO1996004397A1
Application number: PCT/SE1994/001038
Authority: WO
Inventors: Maria Anvret; Margareta BLOMBÄCK; Zhiping Zhang
Original assignee: Maria Anvret; Blombaeck Margareta; Zhiping Zhang
Priority date: 1994-08-05
Filing date: 1994-11-03
Publication date: 1996-02-15
Also published as: AU1285495A; SE9402642D0

Abstract

The present invention is related to a method to correct the mutation(s) behind a genetic disease in the genome of somatic cells derived from an individual afflicted with this disease by contacting said cells with the corresponding non-mutant DNA-fragment to allow it to undergo homologous recombination with and, thus, replace a DNA sequence of the somatic cell genome, wherein said mutation(s) is(are) located. The cells obtained according to the invention can be administered to an individual as a treatment of the said disease. The present invention is also related to a DNA-liposome suspension comprising the said non-mutant DNA-fragment. The said suspension can be used as a DNA-vehicle in the above process. In addition, it can be administered to an individual to obtain correction of mutation(s) by in vivo integration of the said DNA into a mutated gene by homologous recombination.

Description

GENE THERAPY USING HOMOLOGOUS RECOMBINATION

The present invention is generally related to gene therapy as a means for treatment of genetic diseases. More specifically, the invention is related to correction of gene mutation(s) occurring in a gene of an individual and being responsible for a genetic disease. Although, in a broad sense the expression gene therapy is concerned with the use of genetically modified cells as a system for long-term production and delivery of a desired protein having a therapeutic activity in an individual, the present invention is concerned with gene therapy in a more restricted and commonly adopted sense, viz. the modification of an individual's gene(s) in order to treat a genetic disease. In principal, gene therapy could be applied to germ cells. However, since such therapy would not be appropriate for the treatment of humans based on ethical as well as practical criteria, the present invention is merely concerned with somatic cell gene therapy.

The primary strategies for somatic cell gene therapy include the deployment of the following general steps: removing cells from an individual to be treated, culturing the cells in vitro, transfecting the cells with foreign DNA, such as a complete gene encoding a desired substance, usually a therapeutic protein, whereafter transfected cells usually are selected and reintroduced into said individual.

These individual steps can be carried out in different ways. For instance, transfection of the cells with foreign DNA is often accomplished with the aid of a vector which carries the DNA into the cell. Presently available approaches to gene therapy make use of infectious vectors, such as retroviral vectors. However, use of an infectious, e.g. retroviral, vector is associated with disadvantages, i.a. an increased risk of occurrence of a tumorigenic insertional event, possibly leading to tumorgenesis; the potential of generating replication-competent viruses during vector production; and short-lived in vivo expression of the product of interest.

The use of vectors to carry the DNA into the cell inherently would introduce into the cell exogenous DNA originating from the vector. However, in therapy it is appreciated if introduction of exogenous DNA other than the exogenous DNA encoding the desired gene product could be avoided.

The present invention is concerned with gene therapy on individuals afflicted with a genetic disease caused by mutation(s), said mutation(s) involving one or a few nucleosi- des, which is(are) deleted, inserted or substituted, in the gene behind said disease. mutation(s) in the genome of somatic cells derived from an individual being in need of such therapy by introduction of an exogenous DNA sequence comprising the correspon¬ ding normal, i.e. non-mutant, DNA-fragment into said cell and causing said DNA sequence to undergo homologous recombination with and, thus, replace a DNA sequence of the somatic cell genome, wherein the mutant gene is located.

It is appreciated that according to the present invention introduction of the exogenous DNA sequence and stable integration thereof in the host cell genome can be achieved without the need of an infectious vector, such as a retroviral vector. For instance, introduction of said DNA into the host cells can be accomplished by con- ventional liposome fusion technique, a liposome suspension being used as DNA vehicle. Besides, a variety of other techniques are known for introduction of exogenous DNA into recipient cells without the aid of an infectious vector, e.g. electroporation, microinjection, (modified) calcium phosphate or polybrene precipitation and receptor- mediated DNA delivery, which also could be used in accordance with the present invention.

WO 93/09222 is related to gene therapy, wherein somatic cells of vertebrate, particularly mammalian, origin is transfected with exogenous DNA, said exogenous DNA encoding, or being itself, a desired product. Although transfection of host cells including DNA targeting by homologous recombination is disclosed, no clear description of stable integration of exogenous DNA comprising a complete gene into the cell recipient genome is contained therein. Knock-out, replacement or repair of a resident DNA sequence, e.g. an entire gene, is vaguely indicated but not experimentally supported.

It is previously known to use gene knock-out to produce transgenic mice by introducing subtle mutation(s) into the wild type allele of the targeting gene by homolo- gous recombination (Mansour, S.L., Thomas, K.R. , Deng, C.X., and Capecchi, M.R. (1990), "Introduction of a lacZ reporter gene into the mouse int-2 locus by homologous recombination", Proc. Natl. Acad. Sci. USA 87:7688-7692).

WO 91/19796 is directed to methods using homologous recombination for modifying endogenous genes in a chimeric or transgenic animal or plant. Methods are disclosed therein which permit production of animal or plant cells, which contain a desired gene which has been inserted into a predetermined gene sequence by homologous recom¬ bination. Animal and plant cells can be produced which have subtle and precise modifica¬ tions of a gene sequence and expression thereof. The methods disclosed therein can be used to produce chimeric or transgenic animals or plants having defined, and specific, gene alterations, suitably mutations in medically or clinically significant heterologous genes. Although, it is stated therein that the disclosed methods could be used to replace a genetic lesion causing a disease, with a gene sequence encoding a preferred gene product there is no experimental support for this allegation. Furthermore, the efficiency of gene knock-out is low (Jasin M. et al, Genes Devel. 2, 1353-1363 (1988)) and, thus, such methods would be expected to have limited clinical use.

According to the present invention it has unexpectedly been found that it is possible to provide a method for treatment of genetic diseases caused by gene mutation(s) by correcting such mutation(s) by "mutation knock-out", which method is convenient, efficient and well suited for adaption to routine use.

"Mutation knock-out" is used herein to define a type of gene knock-out wherein a mutant sequence is knocked out, i.e. replaced, by a corresponding non-mutant sequence, in order to "repair", i.e. correct, the mutant sequence. Previously, the opposite has been done (Jasin, M. et al., loc. cit.). According to the present invention replacement of the endogenous DNA sequence is accomplished by homologous recombination. This is a well known natural cellular process, by which cells can transfer a DNA sequence from one DNA molecule to another DNA-molecule, provided that these molecules possess a region of ho ology with respect to one another, between which regions the DNA transfer may occur. (For further details, see Watson, J.D. in Molecular Biology of the Gene, 3rd Ed., W.A. Benjamin, Inc., Menlo Park, CA (1977)).

Cells, that could be repaired according to the present invention, are all somatic cells, which can be cultured and from which stable cell lines can be established, e.g. lymphocytes, hematopoietic cells, endothelial cells, fibroblasts, myoblasts, epithelial cells and liver cells. Thus, preferably specialized, differentiated cells are used.

The present invention is generally applicable to treatment of genetic diseases caused by mutation(s) in a specific gene, especially by point mutations, small deletions or small insertions. Often more than one mutation is responsible for the disease. According¬ ly, if e.g. two mutations are responsible for a disease and these are located within the same region, the disease can be treated with one single non-mutant DNA fragment.

Diseases for which the responsible mutations have been identified and which, thus, could be treated according to the present invention are autosomal and X-linked genetic disorders, such as von Willebrand's disease, sickle-cell anemia, β-thalassaemia, haemophilia A and B, and cystic fibrosis. A specific embodiment of the present invention is concerned with von Wille- brand's disease type III.

More specifically, the present invention is related to a method to correct muta¬ tion^) in a gene of animal or human cells by restoring the normal non-mutant DNA- sequence of the gene in order to obtain cells capable of expressing the normal gene product, said method comprising a) incubating said cells with an exogenous DNA-molecule comprising the normal, non- mutant DNA-fragment and, optionally, flanking sequences, e.g. intron sequences, on each side, said DNA-molecule having a sufficient length to permit said DNA-molecule to undergo homologous recombination with the gene comprising mutation(s) b) causing the exogenous DNA-molecule to be introduced into the cells, c) permitting the introduced DNA-molecule to undergo homologous recombination with the gene comprising mutation(s), cells being produced, wherein a fragment of the said gene comprising the mutation(s) has been replaced by the exogenous DNA-molecule, said cells being able to express the normal gene product; and d) recovering the cells.

Suitably, the cells are derived from an individual, preferably a human, suffering from a genetic disease, as discussed above, and the cells from step d) are administered to said individual to cure or alleviate the disease. Although it has been reported in the literature that the efficiency of gene knock¬ out is low, in accordance with the present invention it has been shown that it is possible to increase the said efficiency by repeated gene delivery. Thus, by repeated (e.g. 10-15 times) feeding of a culture of cells, from an individual to be treated, with the exogenous DNA comprising the normal DNA-fragment the amount of repaired, i.e. non-mutant, cells will increase. By repeated feeding at least about 15 times, an amount of repaired cells of at least 0.5 %, suitably 2-5 % or more, of total cells can be achieved.

Since the "repaired" cells obtained according to the present invention virtually are normal somatic cells they will express the normal protein forever (as long as the cells survive) and the expression thereof will be regulated as in normal cells. Thus, if such cells are injected back into the individual to be treated, they can be expected to express the functional protein, i.e. the normal cell product, to increase, at least to some extent, the concentration thereof in said individual.

The exogenous DNA, which is introduced into the cells and stably integrated into their genome, is preferably comprised of a non-mutant DNA fragment covering at least the entire exon, which is intended to be repaired in order to restore the function of the gene comprising said exon. Suitably said fragment also covers flanking intron sequences on both side of the exon to facilitate targeting of the homologous recombination. Such fragments can be obtained from cells from individuals not carrying the mutation(s), amplified and cloned by conventional methods. If the DNA sequence has been determined, it is possible, although less convenient, especially for long sequences, to chemically synthesize the desired DNA fragment.

Although the said exogenous DNA usually is comprised of a fragment of a gene, the present invention is not limited to the use of such DNA but an entire gene may constitute the said DNA. If an entire gene is used as exogenous DNA, preferably it has a short DNA-sequence. Moreover, the DNA-fragment could comprise less than an entire exon provided that the length of said fragment is sufficient to allow said homologous recombination to occur.

Liposomes have been shown to be non-toxic in short- and long-term animal tests (San, H. et al., Hum. Gene Therapy 4, 781-788 (1993) and Stewart, M.J. et al., Hum. Gene Therapy 3, 267-275 (1992)). Accordingly, one aspect of the present invention is concerned with treatment of an individual afflicted with a genetic disease, the mutation(s) behind the disease being known, by administration, e.g. by venous injection, of an . exogenous DNA liposome-suspension, wherein the exogenous DNA is the normal non- mutant DNA fragment corresponding to the endogenous DNA fragment carrying the mutation(s), to achieve in vivo integration of the exogenous DNA into cell genomes by homologous recombination and, thus, correction of the mutated gene. Cells comprising the corrected gene will express the normal gene product and, thus, the symptoms of the disease can be expected to be reduced or even eliminated. The following examples are provided to further illustrate the invention through reference to specific embodiments thereof, which are related to the von Willebrand's disease.

On the accompanying drawings, Figure 1 is a diagrammatical representation of the mechanism through which the mutation knock-out is achieved; Figure 2 A-D illustrate the efficiency of the mutation knock-out in lymphocytes; and Figure 3 shows the breakpoint sequence for the homologous recombination.

The von Willebrand's disease (vWD) is a congenital bleeding disorder related to abnormalities in the von Willebrand Factor (vWF), a blood factor that plays a key role in platelet adhesion, thrombus formation and coagulation. The vWF has two main hemostatic functions, viz. as a carrier protein of Factor VIII (FVIII) and as a promotor of platelet interaction with damaged vessel walls. As a carrier of FVIII, vWF protects FVIII, which exerts procoagulant activity in the blood coagulation system. In its capacity as a promoter of platelet interactions, vWF mediates platelet adhesion to damaged vessel walls and platelet aggregation, and, thus, thrombus formation.

A severe form of this disease is vWD type III. Patients suffering from vWD type III have very low or even undetectable plasma levels of vWF due to mutations in the gene coding for vWF. Either homozygous, i.e. similar, mutations or compound heterozygous, i.e. different, mutations (Anvret, M. et al., Hum. Genet. 89, 147-154 (1992), Zhang, Z.P. et al., Genomics 21, 188-193 (1994); and Zhang, Z.P. et al., Hum. Mol. Genet. 1, 767-768 (1992)) have been established for such patients.

In the following examples it is shown that such mutations can be corrected in lymphocytes. Other somatic cells, such as endothelial cells and megakaryocytes, that normally express vWF and are easily accessible from the patient to be treated can also be used in accordance with the present invention. Example 1.

Lymphocytes from a patient with a homozygous nonsense mutation (R1659X/R1659X) in exon 28 of the vWF gene were used to establish Epstein Barr virus (EBV) transformed lymphocytes in a manner known per se (Elisabeth V. Walls and Dorothy H. Crawford, "Generation of human B Lymphoblastoid cell lines using Epstein- Barr virus", 149-162 in Lymphocytes a practical approach, edited by G.G.B. Klaus, IRL Press Limited, Oxford 1987).

A non-mutant DNA fragment comprised of the whole exon 28 and parts of introns 27 and 28 and corresponding to the mutant region, was amplified from normal individuals, cloned and used to feed a culture of the above mentioned lymphocytes. Transfer of the non-mutant DNA fragment into the lymphocytes was mediated by lipofectamine, a positively charged liposome, which can surround the DNA to form small liposome-DNA suspensions (Feigner, P.L. et al., Proc. Natl. Acad. Sci. USA 84, 7413- 7417 (1987)). Every time the tested cells were fed with DNA-liposome suspension (once a week), an aliquot was saved for analysis of the repair efficiency.

In Figure 1, the mechanism behind the correction of the mutant gene region is illustrated, an intron being shown as a line and an exon as an open box. The mutant DNA sequence (R1659X) is designated A and the non-mutant DNA fragment is designated B. To amplify the non-mutant DNA-fragment the primers Vi27u (5'-TGTGGGAATATGGAAGTCATTG-3') and Vi28- Id (5 ' -GTATCTTGGC AGATGCATGTAGC-3 ' ) were used and the fragment was directly cloned into the pCT™II vector (obtained from Invitrogen Corporation, San Diego, CA, USA). The sequencing of the cloned fragment was shown to be correct (Stewart, M.J. et al, Hum. Gene Therapy 3, 267-275 (1992)). The primers

Ve28-2u (5 ' -ATGGTTCTGGATGTGGCGTTC-3 ' ), Vi28-3d (S'-CAGAGGTΓACCTTGAAGGCA-S') and Vi28-2d (5ΑGGCAAGTTTCAACCAAGGC-3') were used in Example 2 to analyze the efficiency of the mutation knock-out.

Example 2.

In this example the efficiency of the cell repair after repeated feeding with the non-mutant DNA-fragment liposome suspension was analyzed. a) As shown in Figure 1, the mutant sequence comprises a Dde I restriction site. Accordingly the mutant sequence CTGAG is recognized and cleaved by the restriction enzyme Dde I, whereas the corresponding non-mutant sequence CCGAG is not cleaved by Ddel. This indicates that digestion of DNA fragments with Ddel can be used to check whether or not the mutation R1659X has been corrected. More specifically, as shown in Figure 2A the 943 bp fragment amplified with the primers Ve28-2u and Vi28-3d, is cleaved by Dde I into two fragments of 491 bp and 452 bp in the presence of the muta¬ tion, the uncleaved fragment of 943 bp representing the non-mutant sequence. To estimate the accuracy of the efficiency of this method, different ratios of non-mutant DNA/mutant DNA (from lymphocytes with mutation R1659X/R1659X) (1:25, 1 :50, 1: 100, 1:200, 1:400, 1:800, 1 : 1600, 1:3200, 1:6400, 1: 12800) were used as standard controls. See Figure 2A, wherein the following designations are used: a: lOObp ladder, m: mutant

DNA, b to : 1/25, 1/50, 1/100, 1/200, 1/400, 1/800, 1/1600, 1/3200, 1/6400, 1/12800 (non-mutant/mutant DNA), n: non-mutant DNA. b) In Figure 2B and 2C the results from incubation of the lymphocytes from Example 1 and the liposome suspension of the non-mutant DNA fragment (Example 1 , Figure 1) and subsequent analysis of the genomic DNA are shown. The following designations have been used: a: 100 bp ladder, m: mutant DNA, n: non-mutant DNA, l 14: aliquot of manipulated cells fed with DNA-liposome suspension after 1 to 14 times, respectively.

3-5xl0⁶ manipulated lymphocytes were washed with Opti-MEM I medium (Gibco, BRL, Life Technologies). 2.0 μg of non-mutant DNA fragment (shown in Figure 1) was mixed with 30 μl of lipofectamine in 100 μl of Opti-MEM I medium for 40 minutes to form the DNA-liposome suspension. After feeding the washed cells with DNA- liposome mixtures in 1 ml of Opti-MEM I serum-free medium for 8 hours (Figure 2B) or 24 hours (Figure 2C), 2 ml of fresh medium with 20 % foetal calf serum (FCS) was added and regular feeding was performed with medium containing 20 % FCS. Repeated feeding was performed once a week of an aliquot containing 3-5xl0⁶ manipulated cells. The remaining cells were used to isolate genomic DNA

1 μg of genomic DNA was cleaved with 20 unites of Dde I and used for PCR (Polymerase Chain Reaction) (Mullis, K.B., and Faloone, F.A. (1987), "Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction", Methods Enzymol. 155:335- 350) amplification with the primers Ve28-2u and Vi28-3d in 100 μl of reaction. The PCR conditions included incubation at 95°C for 4 minutes, and 35 step cycles, i.e. 95°C 1 minute, 60°C 1 minute and 72°C 1.5 minutes, and then a final cycle with extension at 72°C for 10 minutes. 17 μl of the PCR products were cleaved by 20 units of Dde I and separated on a 3 % NuSieve GTG (obtained from FMC Bioproduct, Rockland, ME, USA) agarose gel. As the primer Vi28-3d is located downstream of the non-mutant fragment, contamination with the non-mutant fragment in the genomic DNA will not interfere, c) In order to confirm that the uncleaved fragment (934 bp) was not due to incom- plete activity by the restriction enzyme Dde I, the uncleaved fragment was cut out from the agarose gel and reamplified by PCR with the same primers (Ve28-2u and Vi28-2d). The PCR products (17 μl) were digested with 20 units of Dde I. The results are shown in Figure 2 D wherein the following designations have been used: a: 100 bp ladder, m: mutant DNA, n: non-mutant DNA, b and c: reamplified uncleaved fragment. The results obtained in Example 2b) and c) are illustrated in Figure 2B-D and

Figure 3. As is obvious from Figures 2B and 2C, after incubation of the cell line with the DNA-liposome suspension for one week, analysis of the genomic DNA showed a visible uncleaved fragment of 943 bp. Re-amplification of the uncleaved fragment (943 bp) confirmed that the majority of the fragment was non-mutant DNA and only a small portion was mutant (Figure 2D). Furthermore, sequencing (Sanger, F. , Nicklen, S. , and Coulson, A.R. (1977), " DNA sequencing with chain-terminating inhibitors" , Proc. Natl. Acad. Sci. USA 74:5463-5467) of the uncleaved fragment showed that the sequence of the repaired fragment was identical with that of the non-mutant sequence (Figure 3). The homologous recombination was precise as no breakpoints were detected, except where the mutation was replaced. This result suggested that the mutation (R1659X) in some tested cells (roughly 1/4000) was replaced by the corresponding normal DNA fragment (Figure 2B, 2C), one or both mutant alleles having been corrected in these cells. In other words, a small population of the cells became heterozygous or homozygous for the non-mutant exon 28. It is evident that the amount of the uncleaved band gradually increased with repeated feeding of the cells with the DNA-liposome suspension (Figure 2B, 2C). After the cells were fed 14 times, the "repaired" cells amounted roughly to 0.5 % of the total tested cells.

Example 3 This example is directed to the correction of a mutation in the vWF gene due to deletion of one single cytosine in exon 18, which in Sweden is the most common mutation in the vWF gene and is present in 50 % of mutant chromosomes. Methods equivalent to those disclosed in Example 1 were used to clone a non-mutant DNA fragment covering the exon 18 of the vWF gene, which non-mutant DNA fragment can be used to correct the said mutation in the same way as in Example 1.

In the above examples endothelial cells from a patient, which cells normally express the vWF, may substitute the lymphocytes. Corrected cells that express the functional vWF protein can then be used in therapy by injection of these cells back into the patient.

Claims

1. A method to correct in vitro, mutation(s) in the genome of somatic cells derived from an individual afflicted with a genetic disease, said mutation(s) occurring in the gene behind the said disease, said method comprising a) contacting said cells with an exogenous DNA sequence comprising the corresponding normal, i.e. non-mutant, DNA-fragment to introduce said DNA into said cells and b) causing said DNA sequence to undergo homologous recombination with and, thus, replace a DNA sequence of the somatic cell genome, wherein the said mutant gene is located.

2. The method of claim 1, wherein mutation(s) in a gene of animal or human cells is (are) corrected by restoring the normal non-mutant DNA-sequence of the gene in order to obtain cells capable of expressing the normal gene product, said method comp¬ rising a) incubating said cells with an exogenous DNA-molecule comprising the normal, non- mutant DNA-fragment and, optionally, flanking sequences on each side, said DNA- molecule having a sufficient length to permit said DNA-molecule to undergo homologous recombination with the said gene comprising mutation(s), b) causing the exogenous DNA-molecule to be introduced into the cells, c) permitting the introduced DNA-molecule to undergo homologous recombination with the gene comprising mutation(s), cells being produced, wherein a fragment of the said gene comprising the mutation(s) has been replaced by the exogenous DNA-molecule, said cells being able to express the normal gene product; and d) recovering the cells.

3. The method of claim 1 or 2, wherein the exogenous DNA is introduced into the cells by liposome fusion technique, suitably a liposome suspension being used as the

DNA vehicle.

4. The method of any preceding claim, wherein the exogenous DNA is repeated¬ ly, suitably 10-15 times or more, brought into contact with the cells to increase the rate of cells, wherein the said mutation(s) is (are) corrected.

5. The method of any preceding claim, wherein the exogenous DNA originates from cells, which comprise the normal non-mutant gene corresponding to the mutant gene intended to be corrected, preferably of the same species origin as the recipient cells.

6. The method of any preceding claim, wherein the mutation(s) to be corrected is (are) responsible for an autosomal or X-linked genetic disorder, such as von Willebrand's disease, sickle-cell anemia, J-thalassaemia, haemophilia A and B, or cystic fibrosis.

7. The method of any preceding claim, wherein the cells obtained are intended for subsequent reintroduction into the same individual from which they have been derived.

8. A method to treat a genetic disease by correction of mutation(s) in the genome of somatic cells of an individual afflicted with a genetic disease, said mutation(s) occur¬ ring in the gene behind the disease, said method comprising introduction, e.g. by venous injection, of an exogenous DNA-liposome suspension, wherein the exogenous DNA is the normal non-mutant DNA fragment corresponding to the endogenous DNA fragment carrying the mutation(s), to achieve in vivo integration of the exogenous DNA into cell genomes by homologous recombination and, thus, correction of the mutated gene.

9. A DNA-liposome suspension, wherein the DNA comprises a normal non- mutant DNA fragment corresponding to a mutant DNA fragment which mutant DNA fragment is responsible for a genetic disorder.

10. Use of cells, wherein mutation(s) has (have) been corrected in accordance with the method of any of claims 1-6 for treatment of a genetic disorder, e.g. selected from von Willebrand's disease, sickle-cell anemia, /3-thalassaemia, haemophilia A and B, and cystic fibrosis.