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Title:  Products and methods for gaucher disease therapy

United States Patent:  6,696,272

Issued:  February 24, 2004

Inventors:  Mahuran; Don J. (Toronto, CA); Clarke; Joe T. R. (Toronto, CA); Callahan; John W. (Mississauga, CA)

Assignee:  HSC Research & Development Limited Partnership (CA)

Appl. No.:  586216

Filed:  June 2, 2000

Abstract

The invention relates to products and methods for medical treatment of Gaucher disease and, in particular, an improved Gcc DNA for insertion into any applicable expression vector for gene therapy treatment. The invention includes an isolated Gcc DNA molecule, wherein nucleic acid molecules have been modified at cryptic splice sites to prevent or decrease splicing of mRNA produced from the DNA molecule, while preserving the ability of the DNA to express functional Gcc polypeptides.

SUMMARY OF THE INVENTION

The invention includes a modified Gcc cDNA insert that can be inserted into any mammalian expression vector for use in the medical treatment of Gaucher disease. In a preferred embodiment, the modified cDNA was inserted into a vector named pINEX2.0 which was then used to transfect mammalian cells. When pINEX2.0 containing the unmodified Gcc cDNA coding sequence, pINEX5'GCC3', was transfected into cells, their RNA purified from cell lysates and subjected to reverse transcription followed by the polymerase chain reaction (RT-PCR), two distinct major bands were observed after agarose gel electrophoresis. Isolation, purification and sequencing of the RT-PCR products identified a major aberrantly spliced mRNA species which encodes only a 19 amino acid peptide before encountering a STOP codon. Surprisingly, this aberrant splicing event occurred completely within the Gcc cDNA coding sequence, i.e. no vector sequences were involved. Site directed mutagenesis was performed to modify the nucleotide sequence in the region of aberrant mRNA splicing without affecting polypeptide coding. Modifications were aimed at disrupting the known consensus sequences for RNA-splicing (Krawczak et al. 1992). The effectiveness of these modifications were tested by transient transfection into CHO cells, followed by our human-specific immunoprecipitation assay for Gcc. Data (n=18) indicate a 5+1 (Std. Error)-fold increase in Gcc activity was achieved when the modified replaced the unmodified insert in the pINEX2.0 expression vector.

The invention relates to an isolated Gcc DNA molecule, wherein the DNA molecule has a modification in at least one nucleotide that disrupts a splicing consensus sequence and prevents splicing of mRNA produced from the DNA molecule, while preserving the ability of the DNA to express active Gcc. The modification impairs a consensus nucleotide sequence needed to induce splicing. The DNA molecule is preferably modified at two cryptic splice sites. The DNA preferably includes a mutation in the 3' junction site. In one embodiment, the mutation is as shown in the 3' junction site (SEQ ID NO.: 18) in Table 1, or a functionally equivalent mutation. In another embodiment, the DNA molecule includes a mutation in the 5' splice junction site.

Another aspect of the invention relates to a vector including a DNA molecule of the invention. The vector preferably includes a promoter that is functional in a mammalian cell.

The invention also includes mRNA produced from the DNA molecule or vector of the invention.

Another aspect of the invention relates to a method of medical treatment of Gaucher disease in a mammal, including administering to the mammal an effective amount of a nucleic acid molecule of the invention or a vector of the invention and expressing an effective amount of the polypeptide encoded by the nucleic acid molecule for alleviating clinical symptoms of Gaucher disease.

The invention includes a host cell, or progeny thereof, including a nucleic acid molecule of the invention. The host cell is preferably selected from the group consisting of a mammalian cell, a human cell and a Chinese Hamster Ovary cell. The invention also includes a method for producing a recombinant host cell capable of expressing a Gcc nucleic acid molecule, the method including introducing into the host cell a vector of the invention. The invention also includes a method for expressing a Gcc polypeptide in a host cell including culturing the host cell under conditions suitable for DNA molecule expression. Another aspect of the invention relates to a method for producing a transgenic cell that expresses elevated levels of Gcc polypeptide relative to a non-transgenic cell, including transforming a cell with a vector of the invention.

The invention includes an isolated polypeptide encoded by and/or produced from a nucleic acid molecule of the invention, or a vector of the invention.

The invention includes a method of producing a genetically transformed cell which expresses or overexpresses a Gcc polypeptide, including: a) preparing a Gcc nucleic acid molecule according to any of claims 1-18; b) inserting the nucleic acid molecule in a vector so that the nucleic acid molecule is operably linked to a promoter; c) inserting the vector into a cell. The invention includes a transgenic cell produced according to the method of the invention.

The invention also includes a pharmaceutical composition, including a carrier and (i) a nucleic acid molecule of the invention (ii) a vector of the invention or (iii) Gcc polypeptide produced from (i) or (ii), in an effective amount for reducing clinical symptoms of Gaucher disease. The carrier preferably carrier includes a liposome.

DETAILED DESCRIPTION OF THE INVENTION

The invention satisfies the need for a DNA (preferably a cDNA) that when inserted into any mammalian expression vector transcribes RNA that is resistant to aberrant processing in the transfected or transduced target cells and thus, is much more likely to translate the functional full length Gcc protein. Therefore, such a modified insert would improve the levels of Gcc expression when used in any vectors designed for in vivo or ex vivo gene therapy treatments of Gaucher disease. As well, when inserted into any efficient mammalian expression vector, such as pINEX2.0, the modified Gcc cDNA as compared to the unmodified cDNA will increase the production levels of recombinate Gcc polypeptide for use in enzyme replacement therapy for Gaucher disease. Thus, the modified insert directs a higher level of Gcc expression through a mechanism that is independent of the mammalian expression vector used whether in vivo or in vitro. The modified insert is safe as preferably no change in the amino acid sequence of Gcc is encoded by the nucleotide changes, and should confer a sustained and appropriate level of cell-specific expression for gene therapy when coupled with the appropriate vector and transfection or transduction methodologies. The expressed DNA insert is preferably a modified Gcc cDNA or a modified fragment of a Gcc cDNA that express a polypeptide having Gcc activity which is effective for treatment of Gaucher disease. The DNA is modified to prevent aberrant cellular splicing of its mRNA produced when expressed in mammalian cells. The modified DNA insert may be used with any expression vector to transfect or transduce any mammalian cell type, such as CHO cells for the expression of human Gcc for enzyme replacement. These would also include human stem cells for ex vivo gene therapy or macrophages for in vivo gene therapy.

The invention also includes the methods of making the modified DNA. The methods may be applied to a Gcc DNA from any source that requires modification to avoid undesirable splicing including humans, other mammals or synthetic DNA.

During the search to improve the efficiency of human Gcc expression it was determined that a major amount of the RNA transcribed from any vector was aberrantly spliced due to cryptic 5' and 3' splice sites contained in the human Gcc cDNA (FIGS. 1 & 2). Since this RNA species encodes only a 19 amino acid peptide, it is far less stable than the properly spliced product encoding the complete 536 residues of Gcc (Maquat 1996), and therefore transcribed at a much higher level than is indicated from our steady-state RT-PCR data. We modified the two cryptic sites in a manner that conserved the wild type amino acid sequence while destroying the consensus nucleotide sequences needed to induce splicing. Transient expression of this modified insert indicated a 5-fold increase in Gcc expression. Such an increase in expression efficiency is not only valuable for any gene therapy approach, but also useful in decreasing the cost of enzyme replacement since the enzyme source is now Gcc-transfected mammalian cells.

Treatment using any vector containing a modified insert to prevent aberrent transcript processing (by gene therapy or by administration of polypeptide produced from a vector) will lower the cost of the present enzyme replacement therapy (currently as much as about US$100,000 per yr. for a patient) by increasing the yield of functional Gcc protein.

The modified insert when used with any appropriate expression vector is also used to direct the expression of Gcc for use in research and characterization of the enzyme's function.

Other useful DNA inserts include a nucleic acid molecule having at least about: 50%, 60%, 70%, 80%, 90%, 95%, 99% or 99.5% sequence identity to the modified Gcc nucleic acid molecule wherein the molecule having sequence identity has a modification in at least one nucleotide (preferably two nucleotides) that disrupts a splicing consensus sequence and prevents splicing of mRNA while it encodes a polypeptide having Gcc activity. Changes in the Gcc nucleotide sequence which result in production of a chemically equivalent (for example, as a result of redundancy of the genetic code) or chemically similar amino acid (for example where sequence similarity is present), may also be made to produce high levels of unspliced transcript from the Gcc cDNA for therapeutic use. The DNA molecule or DNA molecule fragment may be isolated from a native source (in sense or antisense orientations) and modified or synthesized (with or without subsequent modification). It may be a mutated native or synthetic sequence or a combination of these in order to prevent or decrease aberrently spliced transcripts.

Selection of Vector

Separating the Gcc activity derived from transfected human cDNA from the endogenous Gcc activity of the host cells, e.g. CHO, was done to determine the efficiency of expression vectors. A high level of expression is needed not only for any in vivo or ex vivo gene therapy approach, but also for the efficiency of producing Gcc for enzyme replacement therapy now being done in transfected cells (Grabowski et al. 1995). We have developed an immuno-precipitation assay that is specific for the human enzyme and have used it to evaluate several expression vectors. The vector producing the highest level of Gcc in transiently transfected CHO cells was pINEX2.0 from INEX Pharmaceuticals. The vector contains a CMV-based promoter and a potential intron prior to the initiating ATG of the Gcc cDNA. In general our results indicated that a CMV-based promoter gave the highest level of expression and that the placement of the vector's intron at the 5' end of the insert was superior to placing it at the 3' end. Other suitable vectors will be apparent to a skilled person.

After some initial modifications to the 5' untranslated end of the cDNA construct to ensure a match with the consensus sequences for protein initiation (Kozak 1987) and the 3' end to eliminate most of the untranslated nucleotides prior to the vector's polyadenylation signal, a lysate from transiently transfected CHO cells still produced low levels of Gcc specific activity, requiring our immunoprecipitation assay to detect the increase in human activity in the cells' total Gcc pool. A line of permanently-transfected CHO cells was prepared in order to analyzed the sequence(s) of the Gcc mRNA(s) being transcribed from the expression vector.

Cells Containing a Vector of the Invention

The invention relates to a host cell (isolated cell in vitro or a cell in vivo, or a cell treated ex vivo and returned to an in vivo site) containing a vector and modified Gcc sequence of the invention. The preparation of transformed cells is done according to known techniques (see Materials and Methods for example of CHO cells containing a vector). The invention includes methods of expressing Gcc in the cell.

Pharmaceutical Compositions

The pharmaceutical compositions of this invention used to treat patients having Gaucher Disease could include an acceptable carrier, auxiliary or excipient. Polypeptides may be administered in pharmaceutical compositions in enzyme replacement therapy or in gene therapy.

The pharmaceutical compositions can be administered by ex vivo and in vivo methods such as electroporation, DNA microinjection, liposome DNA delivery, and virus vectors that have RNA or DNA genomes including retrovirus vectors, lentivirus vectors, Adenovirus vectors and Adeno-associated virus (MV) vectors. Dosages to be administered depend on patient needs, on the desired effect and on the chosen route of administration. The vectors may be introduced into the cells or their precursors using in vivo delivery vehicles such as liposomes or DNA or RNA virus vectors. They may also be introduced into these cells using physical techniques such as microinjection or chemical methods such as coprecipitation. The vector may be introduced into any mammalian cell type, such as CHO cells or human cells.

The pharmaceutical compositions can be prepared by known methods for the preparation of pharmaceutically acceptable compositions which can be administered to patients, and such that an effective quantity of the vector or polypeptide is combined in a mixture with a pharmaceutically acceptable vehicle. Suitable vehicles are described, for example in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA).

On this basis, the pharmaceutical compositions could include an active compound or substance, such as a polypeptide, in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and isoosmotic with the physiological fluids. The methods of combining the vectors with the vehicles or combining them with diluents is well known to those skilled in the art. The composition could include a targeting agent for the transport of the active compound to specified sites within the mammalian cells.

Method of Medical Treatment of Gaucher Disease

Any vectors containing the DNA molecules of the invention may be administered to mammals, preferably humans, in gene therapy using techniques described below. The polypeptide produced from the modified inserts may also be administered to mammals, preferably humans, in enzyme replacement therapy.

Gene Therapy

Gene therapy to replace Gcc expression (Nolta et al. 1992; Tsai et al. 1992; Sidransky et al. 1993; Schuening et al. 1997; Dunbar et al. 1998) may be useful to modify the development or progression of Gaucher disease. The invention includes methods for providing gene therapy for treatment of diseases, disorders or abnormal physical states characterized by insufficient Gcc expression or inadequate levels or activity of Gcc polypeptide.

The invention includes methods and compositions for providing a nucleotide sequence encoding Gcc or biologically functional equivalent nucleotide sequence to the cells of an individual such that expression of Gcc in the cells provides the biological activity or phenotype of Gcc polypeptide to those cells. Sufficient amounts of the nucleotide sequence are administered and expressed at sufficient levels to provide the biological activity or phenotype of Gcc polypeptide to the cells. For example, the method can involve a method of delivering a gene encoding Gcc to the cells of an individual having a disease, disorder or abnormal physical state, comprising administering to the individual a vector comprising DNA encoding Gcc wherein the DNA has modified sites to prevent undesirable splicing. The method may also relate to a method for providing an individual having a disease, disorder or abnormal physical state with biologically active Gcc polypeptide by administering DNA encoding Gcc. The method may be performed ex vivo or in vivo. Gene therapy methods and compositions are demonstrated, for example, in U.S. Pat. Nos. 5,672,344, 5,645,829, 5,741,486, 5,656,465, 5,547,932, 5,529,774, 5,436,146, 5,399,346 and 5,670,488, 5,240,846.

The method also relates to a method for producing a stock of recombinant virus by producing virus suitable for gene therapy comprising modified DNA encoding Gcc. This method preferably involves transfecting cells permissive for virus replication (the virus containing modified Gcc) and collecting the virus produced.

Typically, a male or female is treated with the vector containing the invention (subject age will typically range from 1 to 60 years of age). At the time of treatment, he typically will have bone involvement, bone thinning and bone pain and will have an enlarged spleen and liver. The vector containing the invention is administered intravenously in order to achieve a desired level of enzyme in the patient. Treatments are repeated as deemed appropriate by a physician to ameliorate the clinical symptoms of Gaucher disease. Such treatments may be life-long.

Patients report significant improvement in bone involvement, pain and thinning, with reduction in frequency and/or intensity of pain episodes, or complete disappearance of skeletal pain often within the first six months of treatment. Patients also show improvement in cortical bone thickness. Enlargement of the spleen and liver are reduced. One of the disease markers, the enzyme chitotriosidase, shows a dramatic reduction during the course of a year.

Administration of Gcc Polypeptide

The Gcc polypeptide is administered in pharmaceutical compositions in enzyme replacement therapy, examples of which are described above (Beutler et al. 1991; Barton et al. 1992; Fallet et al. 1992; Brady et al. 1994; Grabowski et al. 1995; Rosenthal et al. 1995).

Typically, a male or female is treated with the polypeptide of the invention (subject age will typically range from 1 to 60 years of age). At the time of treatment, he typically will have bone involvement, bone thinning and bone pain and may have an enlarged spleen and liver. The polypeptide of the invention is administered intravenously at about 30 U/kg every 2 weeks in order to achieve a desired level of enzyme in the patient.

Patients report significant improvement in bone involvement, pain and thinning, with reduction in frequency and/or intensity of pain episodes, or complete disappearance of skeletal pain often within the first six months of treatment. Patients also show improvement in cortical bone thickness. Enlargement of the spleen and liver are reduced. One of the disease markers, the enzyme chitotriosidase, shows a dramatic reduction during the course of a year.

Claim 1 of 17 Claims

We claim:

1. An isolated and modified glacocerebrosidase DNA molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:12, wherein said DNA molecule comprises a disruption at a donor splicing site residing in SEQ ID NO: 2 or an acceptor splicing site residinig in SEQ ID NO:6 wherein said disruption prevents splicing of a RNA produced from said DNA molecule, while preserving the ability of said mRNA to encode active glucocerebrosidase.



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