The invention relates to site-specific integrating expression vectors for therapy Hemophilia B and to methods of preparing them. A vector of the invention contains a human Factor IX gene in a vector constructed using as a chromosome targeting sequence a polynucleotide without any important physiological function-related gene homologous to DNA on the short arms of human group D and human group G chromosomes. The vector of the invention provides high stability of Factor IX expression, high expression efficiency, no immunogenicity and safety in use.

Description of the Invention

RELATED APPLICATIONS

This application is the national phase under 35 U.S.C. .sctn. 371 of PCT International Application No. PCT/CN01/01291 which has an International filing date of Aug. 29, 2001, which designated the United States of America.

FIELD OF THE INVENTION

The invention relates to a genetic drug for treatment of hemophilia B and its preparation.

BACKGROUND OF THE INVENTION

Hemophilia B, also called second-type Hemophilia is a kind of haemorrhage. It is an X-linked, recessive hereditary disease arising due to deficiency of fibrinogen IX (FIX). Its incidence in the male is 1/30,000. Human FIX is a single-stranded glycoprotein containing 415 amino acid residues, having a molecular weight of 56 kDa. FIX can be divided into four different functional domains; in order from the N-terminus of the protein: a Gla domain (.gamma.-carboxyl glutamic aciddomain), a growth factordomain, an activating peptide domain and a catalytic domain (or serineprotease domain). Human FIX is synthesized mainly in the liver. Its initial translation product includes a leader sequence with 46 amino acids at the N-terminus. FIX is processed into mature FIX by cleavage of the leader peptide, glycosylation and vitamin K-dependent .gamma.-carboxylation. FIX exists in blood in as a zymogen that is activated ("FIX") by a protease activity FIXa or by the FVII-tissue factor complex. The content of FIX in plasma in normal people is 5 .mu.g/ml and its half-life of activity is 24 hours (refer to Chuah MKL, DesireCollen & Vanden Driessche. Gene therapy for Hemophilia. J Gene Med 2001;3:3-20).

FIX is a necessary protein factor in the processes of the endogenous blood coagulation cascade response. A complex of FIX and regulatory protein accelerates the rate of the endogenous clotting cascade response thousands of times, which makes clotting process be finished just in a few minutes. Therefore, deficiency of FIX in the human body, can lead to endless spontaneous or minutely traumatic bleeding, serious joint distortion and lameness or death due to bleeding in the bowel or skull. The human FIX gene (hFIX) was identified in 1982 at chromosome Xq27.1. hFIX is composed of 8 exons, the coding sequence is 1.383 kb, encoding 415 amino acids. (Choo K H, Goule K G, Rees D J, et al. Nature 1982;299:178-180; Kurachi K, Davie E W. Proc Natl Acad Sci USA 1982;79:6461-6464.). The normal plasma concentration of FIX protein in normal plasma is 5 .mu.g/ml (Kaufman R J. Human Gene Therapy 1999; 10:2091-2107). Clinical treatment of Hemophilia B is confined to protein substitution treatment by blood transfusion, or by administering a replenishing FIX preparation to obtain the correct plasma concentration. However, because FIX's half-life in the body is only 24 hours, patients need repeated transfusion or administration of blood preparations toremain alive. Hemophilia B patients not only undertake heavy economic burden, but also confront a threat of infection by HIV, HBV and mad cow virus.

DETAILED DESCRIPTION OF THE INVENTION

How to introduce a normal FIX gene into a patient's body to substitute the deficient FIX gene is the key problem of gene therapy for Hemophilia B. At present, vectors used for FIX gene therapy research are mainly virus vectors, such as retrovirus vectors, adenovirus vectors and adeno-associated virus vectors.

Kay et al carried out experiments of gene therapy on a dog with Hemophilia B. (refer to Kay M A, Rothenberg S, Landen C N, et al. In vivo gene therapy of hemophilia B: sustained partial correction in factor IX deficient dogs. Science 1993; 262: 117-119). The research suggests that the retrovirus (RV) transferred gene in the liver of big animals possibly has a long expression time, but the amount of expression is low. It was also found that just 0.1%-1% of liver cells of mouse are transformed by RV inexperiments performed in mice (refer to Kay M A, Li Q T, Liu J J, et al. Hepatic Gene Therapy: Persistent expression of human a 1 antitrypsin in mice after direct gene delivery in vivo. Hum Gene Ther 1992; 3:641-647). This is mainly because retroviruses do not integrate into the genome of non-dividing cells. So it is necessary to do perform partial hepatic removal to induce the remaining hepatic cells to divide when hepatic cells are used as target cells for gene therapy. Relatively low gene transfer efficiency of RV in vivo and low retransplanting efficiency of cultured cells transformed in vitro lead to low FIX expression that cannot thoroughly correct the phenotype of Hemophilia B. (refer to Lieber A. Peters M J, Gown A, et al. A modified urokinase plasminogen activator induces liver regeneration without bleeding. Hum Gene Ther 1995; 6:1029-1037; Bowles N E, Eisensmith R C, Mohuiddin R, et al. A simple and efficient method for the concentration and purification of recombinant retrovirus for increased hepatocyte transduction in vivo. Hum Gene Ther 1996; 7:1735-1742; Bosch A, McCray P B, Jr. Chang SMW, et al. Proliferation induced by keratinocyte growth factor enhances in vivo retroviral-mediated gene transfer to mouse hepatocytes. J Clin invest 1996; 98:2683-2687). Though retroviral vectors can integrate stably into the genome of target cells, retroviruses only infectdividing cells and further have the potential danger of insertional mutagenesis.

Recombinant Adeno-associated virus (rAAV) vectors are more efficient among viral vectors used for gene therapy of Hemophilia B. rAAV vectors not only express FIX cDNA s efficiently and stably in the receiving cell, but many researchers appreciate that such vectors do not contain viral genes and so are unlikely give rise to a cytotoxic T Lymphocyte response (refer to Jooss K, Yang Y, Fishe K J, et al. Transduction of dendritic cells by DNA viral vectors directs the immune response to transgene products in muscle fibers. J Virol 1998;72:4212-4223). Kay and his colleagues carried out pre-clinical research on mice and a Hemophilia B dog and clinical experiments in some patients (refer to Kay M A, Mannno C S, Ragni M V, et al. Evidence for gene transfer and expression of factor IX in Hemophilia B patients treated with an AAV vector. Nat Genet 2000; 24:257-261). Nevertheless rAAV vectors require further research for human gene therapy because of low titer, complicated preparation and poorlong-term expression. In recent years, nonviral vectors have been developed and used such as liposomes and microcapsules (refer to Hortelano G, Xu N, Vandenberg A, et al. Persistent delivery of factor IX in mice: gene therapy for Hemophilia using implantable microcapsules. Hum Gene Ther 1999;10(8);1281-1288).

In recent ten years, many effective measures in gene therapy for Hemophilia B have been taken, but no safe and stable genetic drug with one-off therapy is available. The key problem lies in that there are no safe, non immunogenic vectors that are highly efficient in transforming the target cell and and provide long expression of the therapeutic gene in the target cell.

One objective of invention is to offer a safe genetic drug for Hemophilia B therapy that provides stable expression of the therapeutic gen in a target cell.

Another objective of invention is to offer a method for preparation of the above-mentioned genetic drug.

Gene drug for Hemophilia B therapy offered by this invention contains vector-FIX recombinant whose leading sequence of objective gene is DNA sequence without important physiological function-related gene on short arm of human group D, G chromosomes or its homologous DNA sequence.

The Inventor has found two families with normal phenotype carrying additional bisatellite microchromosome (BM) that is stably inherited among two and three generations in two families, respectively, and causes no harm to a human body. The present invention is made by dissecting the BM origins and constructing a human-originated gene vector. To date, 17 such families have been reported, both in China and elsewhere, but nobody has put forward any similar idea of using BM as a gene vector.

In this study, the Inventor confirmed using the Fluorescence In Situ Hybridization technique that the BM came from the short arms of human group D chromosomes (chromosomes 13, 14 and 15) and G chromosomes (chromosomes 21 and 22), which are include abundant ribosome DNA (rDNA) that is abundant in the nucleolus organelle and that have polymorphism in length (different contents of rDNA). Gene transcription of the rDNA section during interphase of cell division is very active.

The Inventor infers that a gene in these sites will have high, stable and harmless expression if special DNA fragments isolated from BM can be used as a leader sequence of a therapeutic gene. The Inventor further infers that the therapeutic gene can be site-directed into the nucleolus organelle if sections of the short arms of human group D, G chromosomes are used in constructing a gene vector. The examples following provide proof of principle of this invention.

The inventor at first constructed a pUC19 library of BM specific DNA fragments through micro-dissection and micro-cloning techniques, from which single copy fragments from the BM and p arms of human group D and G chromosomes were selected by Southern blotting and confirmed by the FISH technique. A 120 kb specific DNA fragment (BMSF) was acquired using single copy fragments as probes to screen a human PAC genome and DNA library. The fragments also come from the BM and short arms of human group D and G chromosomes as proved by the FISH technique (FIG. 1 (see Original Patent)). Analysis of the sequence of the BMSF did not identify any important physiologic function and thus it was concluded that it is safe to use the BMSF sequence as a target site for insertion of a desired DNA fragment. The Inventor further used specific DNA fragments of 120 kb or even smaller to construct a gene vector.

The Inventor considers it within the scope of the invention to use DNA sequences lacking important physiologic function-related genes on the short arm of human group D and G chromosomes or a homologous DNA sequence as a targeting sequence of a desired gene. Preferred embodiments of the gene vector target the therapeutic gene in site-directed fashion into the short arms of human group D and/or G chromosomes and the therapeutic gene can thus be expressed with high efficiency.

A "genetic drug" or "gene drug" according to the invention can also comprise reagents for assisting introduction of the therapeutic gene vector into a host cell, and especially in a site-directed fashion, such as liposomes and proteins particular to targeting the host cell. According to the targeting sequence of the therapeutic gene, different forms of vectors can be constructed by the existing technique. The methods of constructing vectors and introducing therapeutic genes into vectors are common.

On the base of obtaining a therapeutic gene leader sequence, different vectors can be constructed. The methods for constructing vectors are routine.

Example 1 provides a vector-FIX recombinant as shown in SEQ NO.1, in which a targeting sequence of 3.8 kb is obtained from BMSF. A Neo.sup.r gene for positive selection is inserted into a site at nucleotide 1500 of the leader sequence of the therapeutic gene, which is divided into two arms of 1.5 kb and 2.3 kb, respectively, using thymidine kinase (TK) as a negative screening gene. The Insertion position of the FIX therapeutic gene is at nucleotide 5910. A therapeutic gene may be inserted in either the forward or reverse direction, and the example of invention below adopts the latter. FIG. 2 (see Original Patent) is construction of vector-FIX recombinant.

The Example provides detailed description of the process of constructing an embodiment of the vector using specific DNA sequence of 3.8 kb isolated from short arms of human group D and G chromosomes as a targeting sequence of a desired gene. The vector-FIX recombinant obtained was deposited in the China Typical Culture Conservation Center (China.Wuhan Wuhan University, post code: 430072) on 29.sup.th of Sep., 2000, numbered: CCTCC M2000031. The deposit is named Escherichia coli JM109/JH/pNS FIX.

The vector carrying a therapeutic gene can be transferred into cultured human cells, such as fibroblast cells and blood stem cells, which also can be used as a gene therapy agent for treatment of hemophilia B. The Example of this invention describes an embodiment of such a delivery method using existing techniques.

In summary, to correct clinical symptoms caused by a deficient gene, cultured human cells transfected with a therapeutic gene the drug composition comprising the therapeutic gene vector can be introduced into a patient's body by means of hypodermal embed (infusion), electroporation or intravenous injection or liposome packaging and thereby the therapeutic gene can be stably expressed.
 

Claim 1 of 14 Claims

1. A plasmid vector comprising: a) a marker gene providing for selection for the vector in a mammalian host cell; b) a polynucleotide segment comprising nucleotides 2841-4341 of SEQ ID NO: 1; c) a polynucleotide segment comprising nucleotides 8678-11032 of SEQ ID NO: 1; d) a polynucleotide segment providing for replication of the vector in a bacterial host cell; and e) a polynucleotide segment providing for selection for the vector in a bacterial host cell.
 

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