Glucose-inducible recombinant viral vector

Details Glucose-inducible recombinant viral vector Glucose-inducible recombinant viral vector

1997-12-17

2001-10-30

McKelvey, Terry (Department: 1636)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

C435S320100, C435S366000, C435S370000

Reexamination Certificate

active

06309878

ABSTRACT:

The present invention relates to recombinant vectors of viral origin, to the preparation of these vectors, to pharmaceutical compositions containing them and to their therapeutic use, in particular in gene therapy, and more especially for the treatment and/or prevention of pathologies associated with a hyperglycaemia.
Gene therapy consists in correcting a deficiency or an abnormality (mutation, aberrant expression, and the like) by introducing genetic information into the cell or organ affected. Traditionally, this genetic information may be introduced either in vitro into a cell extracted from the organ, the modified cell then being reintroduced into the body or directly in vivo into the appropriate tissue. Different techniques have been described for the introduction of this genetic information, among them various techniques of transfection involving complexes of DNA and DEAE-dextran (Pagano et al., J. Virol. 1 (1967) 891), of DNA and nuclear proteins (Kaneda et al., Science 243 (1989) 375) and of DNA and lipids (Felgner et al., PNAS 84 (1987) 7413), the use of liposomes (Fraley et al., J. Biol. Chem. 255 (1980) 10431), and the like.
More recently, the use of viruses as vectors for gene transfer has been seen to be a promising alternative to these physical transfection techniques. In this connection, different viruses have been tested for their capacity to infect certain cell populations. In particular, retroviruses (RSV, HMS, MMS, and the like), the HSV virus, adeno-associated viruses and adeno viruses [lacuna].
The present invention relates more especially to the development of new viral vectors which are, in particular, of special interest for the treatment of pathologies associated with hyperglycaemia such as diabetes.
Sugar diabetes is a syndrome, usually chronic, whose most characteristic symptom is a hyperglycaemia causing, in the absence of an elevation of the renal glucose threshold, a glycosuria. Its main cause is an absolute or relative deficiency of insulin, giving rise to abnormalities not only of carbohydrate metabolism but also of those of proteins and fats.
Diabetes is generally treated by administration of insulin, a treatment which proves very restricting for the patient.
The objective of the present invention is specifically to provide a novel viral vector enabling this disorganization of glucose metabolism to be compensated in the body via the restoration in a controlled manner of an appropriate in vivo insulin concentration.
Glucose is known to be a factor regulating gene transcription in the majority of living organisms. It has been demonstrated, in particular, that glucose is capable of stimulating the transcription of genes coding for glycolytic and lipogenic enzymes in hepatocytes and adipocytes, such as, for example, the gene for type L pyruvate kinase, a tissue-specific glycolytic enzyme which plays a decisive part in the regulation of glycolysis and gluconeogenesis in the liver. The expression of the L-PK protein in the liver is under nutritional and hormonal control. It is induced by a glucose-rich diet and, on the contrary, inhibited by deprivation and in diabetics. Thus, it has been demonstrated that the expression of its gene is positively regulated by the glucose/insulin system and negatively by glucagon via its second messenger cyclic AMP (A. Kahn; J. Bio. Chem. 264, (1989), 11584-11590).
Recently, the Applicant has characterized different binding sites (elements L1 to L4) for transcription factors, and especially a glucose/insulin response element, or “GIRE”, in the region upstream of the promoter of the gene for this L-PK enzyme (A. Kahn et al., J. Mol. Biol. 209, (1989), 205-219). Thus he identified, in the 3′ to 5′ direction starting from the TATA box, the L1 element, a binding site for hepatocyte nuclear factor 1 (HNF1), the L2 element, a binding site for nuclear factor 1 (NF1), the L3 element, a binding site for hepatocyte nuclear factor 4 (HNF4) and the L4 element, a binding site for the major late transcriptional factor (MLTF)/USF (see FIG.
1
), respectively. The specific element for glucose/insulin response has been precisely localized in the gene coding for L-PK, in the form of a perfect palindrome between nucleotides −168 and −144 from the cap site, that is to say in the L4 element (see FIG.
2
). This GIRE has been shown to be capable of conferring a transcriptional response to glucose on a minimal L-PK promoter consisting of the TATA box and the L1 element.
Unexpectedly, the Applicant demonstrated that it was possible to upgrade the capacity of this GIRE element to respond to glucose in a viral context in order to modulate the expression of a protein of interest in gene therapy.
More specifically, the present invention relates to a defective recombinant virus comprising at least one heterologous gene under the control of an expression signal which is inducible by glucose or one of its analogues.
Glucose analogue is understood to cover any compound displaying structural homologies with glucose and which is capable of inducing the activation of the expression signal. As analogues capable of being used according to the present invention, fructose, galactose, sucrose, lactose and all sugars capable of being hydrolysed to these substances may be mentioned in particular.
For the purposes of the present invention, an expression signal which is inducible with glucose covers any expression signal sequence whose activation, which conditions the subsequent transcription of the associated heterologous gene, is essentially induced in the presence of glucose or one its analogues.
More especially, the virus in question is a defective recombinant virus in which the expression of at least one heterologous gene is placed under the control of an expression signal derived wholly or partially from the promoter of the gene coding for type L pyruvate kinase, L-PK. More specifically, this expression signal is derived wholly or partially from the fragment consisting of the 183 bp located at the 5′ end of the coding frame of the gene coding for L-PK.
Preferably, the expression signal according is to the invention comprises all or part of the sequence SEQ ID NO: 1.
According to a preferred embodiment, this expression signal comprises at least all or part of the L4 element of this promoter.
The L4 element is preferably represented by all or part of the sequence SEQ ID NO: 2, of a sequence hybridizing with all or part of the latter or of a sequence which is derived therefrom, and capable of interacting with the factor MLTF/USF.
For the purposes of the present invention, derivative is understood to mean any sequence obtained by one or more modifications and which retains at least one of the biological properties of the original sequence, and in particular its capacity to interact with its specific binding factor or factors, in the present case consisting of the factor MLTF/USF. Modification is understood to mean any mutation, substitution, deletion, addition or modification of a genetic and/or chemical nature. These modifications may be carried out by the techniques known to a person skilled in the art.
In a variant of the invention, the L4 element is repeated in the form of 4 successive oligomers in the expression signal.
According to a preferred embodiment of the invention, the expression signal comprises, besides the L4 element, all or part of the L3 element of the promoter of the gene coding for L-PK.
It has, in effect, been mounted that this L3 element contributes to an improved efficacy of the L4 element (A. Kahn et al., Nucleic Acids Research 20, (1992), No. 8 p. 1871). The L3 element is preferably represented wholly or partially by the sequence SEQ ID NO: 3, a sequence hydridizing with all or part of the latter or of a sequence which is derived therefrom, capable of interacting with the factor HNF4.
According to a preferred embodiment of the invention, the expression signal comprises the L4 element of the promoter of the gene coding for L-PK, fused upstream of the L3 element of this same promoter. It is then

Affiliated with

Also associated with

Rating

Glucose-inducible recombinant viral vector does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Glucose-inducible recombinant viral vector, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Glucose-inducible recombinant viral vector will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2589940