Wild-type apolipoprotein L-I for use in the prevention of kidney diseases

Abstract

A wild-type human Apolipoprotein L-I is used in a method for the prevention and/or the treatment of renal disease.

Description

This application is a National Stage Application of PCT/EP2012/052731, filed 17 Feb. 2012, which claims benefit of Serial No. 11154776.6, filed 17 Feb. 2011 in the European Patent Office and which applications are incorporated herein by reference. To the extent appropriate, a claim of priority is made to each of the above disclosed applications.

FIELD OF THE INVENTION

The present invention is in the field of Molecular Biology and is related to a pharmaceutical (therapeutical or prophylactic) use of apolipoprotein L-I, especially for a treatment and/or a prevention of diseases affecting the kidney such as glomerulosclerosis.

BACKGROUND OF THE INVENTION AND STATE OF THE ART

Normal human serum (NHS) is able to kill T. b. brucei, but not T. b. rhodesiense and T. b. gambiense. The lytic factor was identified as being apolipoprotein L-I (apoL1) that forms pore in endosomal membranes of the parasite. This protein is associated with HDL particles that are efficiently taken up by the parasite through specific binding to a haptoglobin-hemoglobin surface receptor, due to the simultaneous presence of haptoglobin-related protein (Hpr) acting as a ligand in these particles.

The T. brucei subspecies rhodesiense and gambiense resist this lytic activity and can infect humans, causing sleeping sickness. T. b. rhodesiense resistance to lysis involves interaction of the Serum Resistance-Associated (SRA) protein with the C-terminal helix of apoL1.

SUMMARY OF THE INVENTION

The present invention discloses a therapeutic composition for patients suffering from a renal disease, possibly in relation with (caused by) a mutation in ApoL1 gene in this patient and preferably wherein this mutated ApoL1 protein is able to kill T. b. rhodesiense cells.

A first aspect of the present invention is related to the wild-type human Apolipoprotein L-I (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:7) for use in the treatment and/or the prevention of renal diseases.

Preferably, this wild-type human Apolipoprotein L-I is for use in the treatment and/or the prevention of renal diseases affecting a human patient, wherein this human patient harbours a mutated (variant) human Apolipoprotein L-I.

More preferably, this wild-type human Apolipoprotein L-I is for use in the treatment and/or the prevention of renal diseases affecting a human patient harbouring a mutated human Apolipoprotein L-I, wherein this mutated human Apolipoprotein L-I corresponds to the wild-type human Apolipoprotein sequence (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:7) modified by (which comprises) a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 (preferably 9) of its last C-terminal amino acids and/or wherein this mutated human Apolipoprotein L-I is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, and SEQ ID NO:9 and more preferably being selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5 and SEQ ID NO:8.

Alternatively or in addition, this wild-type human Apolipoprotein L-I is for use in the treatment and/or the prevention of renal diseases affecting a human patient harbouring a mutated human Apolipoprotein L-I, wherein this mutated human Apolipoprotein L-I is able to lyse Trypanosoma brucei rhodesiense cells.

Possibly, this wild-type human Apolipoprotein L-I for use in the treatment and/or the prevention of renal diseases affecting a human patient harbouring a mutated human Apolipoprotein L-I, wherein the mutated human Apolipoprotein L-I is not able to lyse Trypanosoma brucei gambiense cells, but preferably is able to lyse Trypanosoma brucei rhodesiense cells.

Advantageously, this wild-type human Apolipoprotein L-I is administered at a concentration comprised between (about) 100 μg/ml and (about) 20 mg/ml.

Possibly, this wild-type human Apolipoprotein L-I is a recombinant protein.

Alternatively (or in addition), this wild-type human Apolipoprotein L-I is present on HDL particles.

Preferably, these HDL particles comprise particles having a density comprised between 1.123 g/ml and 1.21 g/ml, more preferably these HDL particles consist (essentially) of HDL particles having a density comprised between 1.123 g/ml and 1.21 g/ml.

The present invention will be described in more details in the following detailed description of the invention as non limited illustration of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses a new pharmaceutical composition comprising an adequate pharmaceutical carrier, a wild-type Apolipoprotein L-I (in the form of an amino acid sequence, or a nucleotide sequence(s), a vector, a cell, a blood sample and/or particles including HDL particles) and/or fragments thereof that could be administrated to mammals, especially to humans to cure and/or prevent diseases affecting the kidney (possibly in the treatment and/or prevention of glomerulosclerosis including focal segmental glomerulosclerosis (FSGS) cause of idiopathic nephrotic syndrome, HIV associated nephropathy and hypertension associated end-stage kidney disease (ESKD) in these mammals, especially in humans.

The present invention is further related to a diagnostic method comprising the steps of:

• • analysing a blood sample (obtained from a human patient) for mutation (variant) in Apolipoprotein L-I through its capacity of lysis of a Trypanosoma brucei rhodesiense culture;
  • deducing whether this patient is carrying the mutation (variance) in Apolipoprotein L-I, possibly due to the specific lysis of Trypanosoma brucei rhodesiense cells.

Alternatively (or in addition) the present invention is related to this diagnostic method (further) comprising the step of performing the sequencing of the ApoL1 gene from the (blood) sample and possibly deducing whether this patient carries a mutation (variance) in ApoL1 gene.

Preferably in this diagnostic method, the two genes (chromosomal copies) of ApoL1 are sequenced and the possible deduction step discriminates between patient expressing a mutated (variant) ApoL1 at heterozygous levels (in addition to a wild-type ApoL1) and patient expressing two mutated copies of ApoL1 (possibly these two copies of ApoL1 have the same mutation and the patient is homozygous), and no wild-type ApoL1, these patients being the most affected by the renal disease.

Alternatively (but less preferably), a related diagnostic method comprises the step of:

• • analysing a blood sample (obtained from a human patient) for mutation (variance) in Apolipoprotein L-I through its absence of binding to Serum Resistance-Associated (SRA) protein (of Trypanosoma brucei rhodesiense);
  • deducing whether the patient is carrying the mutation (variance) in Apolipoprotein L-I. This method can be combined with the above-described preferred diagnostic methods.

Preferably, in these diagnostic methods, the (blood) sample is obtained from a patient suffering for a renal disease and/or who is suspected to develop a renal disease.

Preferably, in these diagnostic methods, the blood sample is obtained from a patient being at an early stage of a renal disease.

Preferably, in these diagnostic methods, the mutation (variance) in Apolipoprotein L-I corresponds to wild-type human Apolipoprotein L-I (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:7) modified by (which comprises) a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 (preferably 9) of its last C-terminal amino acids and/or this mutated Apo L-I is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, and SEQ ID NO:9, being more preferably selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5 and SEQ ID NO:8.

A related aspect of the present invention is a diagnostic kit comprising means to detect whether a patient carries a mutated (variant of) ApoL1, these means being selected from the group consisting of immobilized SRA, Trypanosoma brucei rhodesiense cells or extracts thereof, nucleotidic probes specifically recognizing the mutations (variance) in ApoL1, antibodies specifically recognizing the mutations (variance) in ApoL1 and tools for the sequencing of ApoL1 gene or messenger RNA.

Possibly, this diagnostic kit comprises:

• • nucleotide probes able to identify the mutated (variants) Apolipoprotein L-I or
  • antibodies specifically recognizing mutated (variant) Apolipoprotein L-I and/or
  • Trypanosoma brucei rhodesiense culture andmeans to identify whether a subject is expressing the mutated (variant) Apolipoprotein L-I, wherein this mutated (variants) human Apolipoprotein L-I corresponds to the wild-type human Apolipoprotein sequence (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:7) modified by (which comprises) a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 (preferably 9) of its last C-terminal amino acids and/or wherein this mutated (variant) human Apolipoprotein L-I is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, and SEQ ID NO:9 and more preferably being selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5 and SEQ ID NO:8.

Another aspect of the present inventions relates to a pharmaceutical composition (including a vaccine) comprising an adequate pharmaceutical carrier (or diluent and possibly one or more adequate adjuvant(s)) and a sufficient amount of an element selected from the group consisting of the (wild-type) Apolipoprotein L-I (amino acid sequence; SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7)), the corresponding polynucleotide or vector, or the cell expressing (an active fragment of) this (wild-type) Apolipoprotein L-I (possibly in the form of a pharmaceutically-acceptable lysate or lyophilisate); preferably, this pharmaceutical composition is used in (for) a treatment and/or a prevention of a disease affecting the kidney of a human patient.

Preferably, the pharmaceutical composition is a solution comprising the (wild-type) Apolipoprotein L-I (SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7) at concentrations between (about) 100 ng/ml and (about) 20 mg/ml, more preferably at concentrations between (about) 10 μg/ml and (about) 10 mg/ml, still more preferably at concentrations between (about) 100 μg/ml and (about) 1 mg/ml.

The inventors used preferably the (pharmaceutical composition comprising the) wild-type ApoL1 at concentrations between (about) 10 μg/ml and (about) 20 mg/ml and still more preferably at (about) 2 mg/ml and (about) 10 mg/ml.

Advantageously, the wild-type ApoL1 is recombinant.

Preferably, the wild-type (recombinant) ApoL1 is not denaturated, such as by mixture with (about 8 M) urea.

A related aspect of the invention is a pharmaceutical composition comprising a blood sample (preferably a serum) or extract thereof comprising the (wild-type) Apolipoprotein L-I.

Preferably this blood sample or extract thereof is in the form of (HDL) particles, more preferably, being (essentially; i.e. at more than 50%, preferably more than 60%, more preferably more than 90%) in the form of HDL particles having a density comprised between 1.12 g/ml and 1.21 g/ml.

Preferably, this blood sample (serum or extract, including in the form of (HDL) particles) is for use in the treatment or the prevention of diseases affecting the kidney of a patient (more preferably a human).

Preferably, this patient has a mutated ApoL1 protein, and more preferably the patient does not express wild-type ApoL1, i.e. for instance the patient carries a mutation on both (chromosomal) copies of ApoL1 (possibly the patient is homozygous and expresses two identically mutated ApoL1 proteins).

More preferably, the mutated ApoL1 protein (present in a blood sample of the patient) is able to kill (in vitro) Trypanosoma brucei rhodesiense cells or Trypanosoma mutants having a similar phenotype.

Alternatively, or in addition, the patient expresses a mutated ApoL1 protein corresponding to wild-type human Apolipoprotein L-I (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:7) modified by (which comprises) a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 (preferably 9) of its last C-terminal amino acids and/or this mutated Apo L-I is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, and SEQ ID NO:9, being more preferably selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5 and SEQ ID NO:8 (and preferably the patient does not express the wild-type ApoL1).

Another aspect of the present invention is a therapeutic method to prevent and/or cure renal disease, comprising the steps of:

• • selecting a (human) patient having or suspected to have a renal disease;
  • analysing a blood sample (obtained from this patient) for the presence of mutations in ApoL1 protein (preferably carrying the mutations on the two ApoL1 genes of this patient and/or analysing the absence of wild-type ApoL1);
  • treating this patient having or suspected to have a renal disease and preferably having a mutation in ApoL1 protein by wild-type ApoL1 protein and/or by a nucleotide sequence encoding this wild-type ApoL1 protein.

The inventors found various naturally-occurring deletions and mutations in the C-terminal domain of apoL1 (the mutated ApoL1 of the present invention) and studied their effect on the trypanolytic potential of these mutated proteins against T. b. brucei and T. b. rhodesiense.

The inventors then screened patients that carry mutated (variants of) ApoL1 (patients that express the ApoL1 of the invention, being heterozygotes or, more preferably, patients expressing mutated ApoL1 and not wild-type ApoL1, possibly this patient is homozygous and/or express the mutated ApoL1 of the present invention).

The inventors then draw correlations between the trypanolytic potential of the mutated form (variant) of ApoL1 and the development of renal syndromes.

In addition, rodent expressing the mutated ApoL1 of the present invention were investigated for their renal pathologies and for the development of corresponding treatments.

The inventors ran in parallel cell models of kidney disease and provoked kidney disease by the addition of the mutated ApoL1 they fermented.

The inventors then added wild-type ApoL1 protein to these transgenic animals and to these cells and observed an overall tendency towards a reduction and/or delay of the renal symptoms.

Finally, the inventors administered ApoL1 to human patients suffering for a renal disease and carrying a mutation according to the invention and observed a tendency towards a reduction of the symptoms and/or to the progression of the disease.

Claims

1. A method for the treatment of a renal disease affecting a human patient harboring a mutated human Apolipoprotein L-I comprising the step of administering to said human patient a wild-type human Apolipoprotein L-I, wherein said wild-type human Apolipoprotein L-I comprises a sequence selected from a group consisting of (SEQ ID NO:1, SEQ ID NO:4 and SEQ ID NO:7).

2. The method of claim 1, wherein said mutated human Apolipoprotein L-I corresponds to the wild-type human Apolipoprotein sequence (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:7) modified by a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 of the last C-terminal amino acids and/or wherein said mutated human Apolipoprotein L-I is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, and SEQ ID NO:9.

3. The method of claim 1, wherein said mutated human Apolipoprotein L-I lyses Trypanosoma brucei rhodesiense cells.

4. The method of claim 3, wherein the mutated human Apolipoprotein L-I does not lyse Trypanosoma brucei gambiense cells.

5. The method of claim 1, wherein said human patient does not express wild-type human Apolipoprotein L-I.

6. The method of claim 1, wherein the renal disease is glomerulosclerosis.

7. The method of claim 1, wherein the wild-type human Apolipoprotein L-I is in a concentration between 100 μg/ml and 20 mg/ml.

8. The method of claim 1, wherein the wild-type human Apolipoprotein L-I is a recombinant protein.

9. The method of claim 1, wherein the wild-type human Apolipoprotein L-I is on HDL particles.

10. The method of claim 9, wherein the HDL particles consist essentially of particles having a density between 1.123 g/ml and 1.21 g/ml.

11. A method for the treatment of a renal disease affecting a human patient harboring a mutated human Apolipoprotein L-I comprising the step of administering to said human patient a nucleotide sequence encoding wild-type human Apolipoprotein L-I protein, wherein said protein sequence is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:7.

12. The method of claim 11, wherein the nucleotide sequence is in the form of a vector.

13. The method of claim 12, wherein the nucleotide sequence is a plasmid.