Therapeutic Gene

Details Therapeutic Gene Therapeutic Gene

1999-06-21

2003-04-01

Guzo, David (Department: 1636)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S320100, C435S455000, C435S456000, C435S069700, C435S325000, C435S183000, C536S023100, C536S023200, C536S023400, C536S023500, C536S024100, C424S093100, C424S093200, C424S093600

Reexamination Certificate

active

06541219

ABSTRACT:

This invention relates to polynucleotide sequences encoding fusion proteins, for use in gene therapy in particular for Parkinson's disease. The invention also relates to vectors carrying the polynucleotide sequences, in particular retroviral vectors, and to their use in gene therapy.
Parkinson's disease (PD) is a common neurodegenerative disorder that afflicts the growing population of elderly people. Patients display tremor, cogwheel rigidity and impairment of movement. It is generally thought to be an acquired rather than inherited disease in which environmental toxins, metabolic disorders, infectious agents and normal ageing have all been implicated. PD is associated with the degeneration of nigrostriatal neurons which have their soma located in the substantia nigra. They send axonal projections to the basal ganglia and they use dopamine as their neurotransmitter. Some features of the disease can be controlled by the administration of L-DOPA, the metabolic precursor to dopamine, which diffuses across the blood brain barrier more effectively than dopamine itself. Unfortunately as the disease progresses the side effects of this treatment become unacceptable.
PD is an ideal candidate for gene therapy for several reasons. The clinical efficacy of systemic administration of L-DOPA suggests that restoration of neuronal circuitry is not essential for disease management. Therefore genetic manipulation of brain cells to provide local production of L-DOPA from tyrosine may be a realistic strategy for treatment. The biosynthesis of L-DOPA from tyrosine involves a single step suggesting that provision of tyrosine hydroxylase (TH) by genetic means may be sufficient and some success has been achieved using this strategy in small animals and in cell culture (Kaplitt et al., 1994 Nature Genetics 8, 148; During et al., 1994 Science 266, 1399; Horellou et al., 1994 Neuroreport 6, 49; Owens et al., 1991 J. Neurochem. 56, 1030). However, if one is to use local endogenous brain cells as L-DOPA factories for the treatment of PD in man it is likely that high levels of L-DOPA will be required to effect a treatment. These high levels must be efficiently converted to dopamine as the necessary neurotransmitter and primary therapeutic agent. It is likely therefore that it will be necessary not only to supply tyrosine hydroxylase but also DOPA decarboxylase (DD), the enzyme that converts L-DOPA to dopamine. This means that in a gene therapy strategy the genes for both of these enzymes will be required.
Amongst gene transfer systems retroviral vectors hold substantial promise for gene therapy. These systems can transfer genes efficiently and new vectors are emerging that are particularly useful for gene delivery to brain cells (Naldini et al., 1996 Science 272, 263). However, it is clear from the literature that retroviral vectors achieve the highest titres and most potent gene expression properties if they are kept genetically simple (PCT/GB96/01230; Bowtell et al., 1988 J.Virol. 62, 2464; Correll et al., 1994 Blood 84, 1812; Emerman and Temin 1984 Cell 39, 459; Ghattas et al., 1991 Mol.Cell.Biol. 11, 5848; Hantzopoulos et al., 1989 PNAS 86, 3519; Hatzoglou et al., 1991 J.Biol.Chem 266, 8416; Hatzoglou et al., 1988 J.Biol.Chem 263,17798; Li et al., 1992 Hum.Gen.Ther. 3, 381; McLachlin et al., 1993 Virol. 195, 1; Overell et al., 1988 Mol.Cell Biol. 8, 1803; Scharfman et al., 1991 PNAS 88, 4626; Vile et al., 1994 Gene Ther 1, 307; Xu et al., 1989 Virol. 171, 331; Yee et al., 1987 PNAS 84, 5197). This means only using a single transcription unit within the vector genome and orchestrating appropriate gene expression from sequences within the 5′ LTR. If there is a need to express two enzymes, such as TH and DD, from a single retroviral vector the only solution would be to use an internal ribosome entry site (IRES) to initiate translation of the second coding sequence in a polycistronic message (Adam et al 1991 J.Virol. 65, 4985). However, the efficiency of an IRES is often low and tissue dependent making this strategy undesirable when one is seeking to maximise the efficiency of metabolic conversion of tyrosine through to dopamine. The present invention addresses these problems.
The present invention provides in one aspect a polynucleotide sequence for use in gene therapy, which polynucleotide sequence comprises two or more therapeutic genes operably linked to a promoter, and encodes a fusion protein product of the therapeutic genes. The invention thus provides a way of expressing two therapeutic genes from a single “chimeric gene”.
In another aspect, the invention provides a vector carrying the polynucleotide sequence as described. The vector may be for example an expression vector such as a plasmid, or it may be a retroviral vector particle comprising an RNA genome containing the polynucleotide sequence as described herein.
In yet further aspects, the invention provides a DNA construct encoding the RNA genome for the retroviral vector particle; and a retroviral vector production system comprising a set of nucleic acid sequences encoding the components of the retroviral vector particle.
The invention further provides the use of retroviral vectors carrying the chimeric gene described herein, in gene therapy and in the preparation of a medicament for gene therapy; and a method of performing gene therapy on a target cell, which method comprises infecting and transducing the target cell using a retroviral vector particle as described herein. The invention further provides transduced target cells resulting from these methods and uses. The invention thus provides a gene delivery system for use in medicine.
In addition, the invention provides a polynucleotide sequence encoding a fusion protein comprising tyrosine hydroxylase and DOPA decarboxylase in either TH-DD or DD-TH order, linked by a flexible linker.
The therapeutic genes are chosen according to the effect sought to be achieved. The fusion protein has or is capable of having the desired activity of the therapeutic gene products. The product encoded by one or more of the therapeutic genes may be an enzyme. The fusion protein may thus display the activity of one or more enzymes. Where the therapeutic genes encode two different enzymes, the resulting fusion protein is a bifunctional enzyme. In the specific example described herein, the fusion protein comprises the enzymes tyrosine hydroxylase and DOPA dehydroxylase having enzyme activities as described above.
Preferably the therapeutic genes are linked by a sequence encoding a flexible linker. A suitable linker may comprise amino acid repeats such as glycine-serine repeats. The purpose of the linker is to allow the correct formation and/or functioning of the therapeutic gene products. It must be sufficiently flexible and sufficiently long to achieve that purpose. Where the therapeutic genes encode two different enzymes, the linker needs to be chosen to allow the functioning of both of the enzymes. The coding sequence of the flexible linker may be chosen such that it encourages translational pausing and therefore independent folding of the protein products of the therapeutic genes.
A person skilled in the art will be able to design suitable linkers in accordance with the invention. Some specific examples of suitable linkers are given below; it will be evident that the invention is not limited to these particular linkers.
1. (Gly-Gly-Gly-Gly-Ser)
3
(SEQ ID NO: 21) as described in Somia et al., 1993 PNAS 90, 7889.
2. (Gly-Gly-Gly-Gly-Ser)
5
(SEQ ID NO: 22), a novel linker.
3. (Asn-Phe-Ile-Arg-Gly-Arg-Glu-Asp-Leu-Leu-Glu-Lys-Ile-Ile-Arg-Gln-Lys-Gly-Ser-Ser-Asn) (SEQ ID NO: 23) from HSF-1 of yeast, see Wiederrecht et al., 1988 Cell 54, 841.
4. (Asn-Leu-Ser-Ser-Asp-Ser-Ser-Leu-Ser-Ser-Pro-Ser-Ala-Leu-Asn-Ser-Pro-Gly-Ile-Glu-Gly-Leu-Ser) (SEQ ID NO: 24) from POU-specific OCT-1, see Dekker et al., 1993 Nature 362, 852 and Sturm et al., 1988 Genes and Dev. 2, 1582.
5. (Gln-Gly-Ala-Thr-Phe-Ala-Leu-Arg-Gly-Asp-Asn-Pro-Gln-Gly) (SEQ ID NO: 25) from RGD-containing Laminin peptide, see Aumailly et al., 1

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