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EP-4735052-A1 - PROMOTERS

EP4735052A1EP 4735052 A1EP4735052 A1EP 4735052A1EP-4735052-A1

Abstract

The present invention relates to hybrid promoters comprising a super core promoter and a kidney-specific promoter. The present invention also relates to polynucleotides and vectors comprising said promoters and use of said promoters.

Inventors

  • MADILL, Martin
  • GRIFFITH, Alan

Assignees

  • Purespring Therapeutics Limited

Dates

Publication Date
20260506
Application Date
20240628

Claims (20)

  1. CLAIMS 1. A hybrid promoter comprising a super core promoter and a kidney-specific promoter.
  2. 2. The hybrid promoter according to claim 1, wherein the super core promoter is downstream of the kidney-specific promoter.
  3. 3. The hybrid promoter according to claim 1 or 2, wherein the super core promoter is selected from super core promoter 1 (SCP1), super core promoter 2 (SCP2), or super core promoter 3 (SCP3).
  4. 4. The hybrid promoter according to any preceding claim, wherein the super core promoter is SCP1.
  5. 5. The hybrid promoter according to any preceding claim, wherein the super core promoter comprises from 5’ to 3’: a TATA box, an initiator motif (Inr), a motif ten element (MTE), and a downstream promoter element (DPE).
  6. 6. The hybrid promoter according to claim 5, wherein: (i) the TATA box is located from about -31 to about -26 relative to the super core promoter +1 transcription start site; (ii) the Inr is located from about -2 to about +4 relative to the super core promoter +1 transcription start site; (iii) the MTE is located from about +18 to about +27 relative to the super core promoter +1 transcription start site; and/or (iv) the DPE is located from about +28 to about +33 relative to the super core promoter +1 transcription start site.
  7. 7. The hybrid promoter according to claim 5 or 6, wherein the TATA box is a CMV TATA box.
  8. 8. The hybrid promoter according to any of claims 5 to 7, wherein the TATA box comprises or consists of the nucleotide sequence TATAWAAR, preferably wherein the TATA box comprises or consists of the nucleotide sequence TATATAAG.
  9. 9. The hybrid promoter according to any of claims 5 to 8, wherein the Inr is a composite Inr derived from the AdML core promoter and the Drosophila melanogaster G element core promoters, or a CMV Inr.
  10. 10. The hybrid promoter according to any of claims 5 to 9, wherein the Inr comprises or consists of the nucleotide sequence TCAKTY or YYANWYY, preferably wherein the Inr comprises or consists of the nucleotide sequence TCAKTY, more preferably wherein the Inr comprises or consists of the nucleotide sequence TCAGTC.
  11. 11. The hybrid promoter according to any of claims 5 to 10, wherein the MTE is a Tollo MTE.
  12. 12. The hybrid promoter according to any of claims 5 to 11, wherein the MTE comprises or consists of the nucleotide sequence CSARCSSAAC, preferably wherein the MTE comprises or consists of the nucleotide sequence CGAGCCGAGC.
  13. 13. The hybrid promoter according to any of claims 5 to 12, wherein the DPE is a Drosophila G DPE or a CALM2 DPE.
  14. 14. The hybrid promoter according to any of claims 5 to 13, wherein the DPE comprises or consists of the nucleotide sequence RGWYVT, preferably wherein the DPE comprises or consists of the nucleotide sequence AGACGT.
  15. 15. The hybrid promoter according to any preceding claim, wherein the super core promoter comprises or consists of a nucleotide sequence having 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to one or more of SEQ ID NOs: 7-11.
  16. 16. The hybrid promoter according to any preceding claim, wherein the super core promoter comprises or consists of a nucleotide sequence having 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to one or more of SEQ ID NOs: 7-9.
  17. 17. The hybrid promoter according to any preceding claim, wherein the super core promoter comprises or consists of a nucleotide sequence having 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to SEQ ID NO: 7.
  18. 18. The hybrid promoter according to any preceding claim, wherein the super core promoter comprises or consists of the nucleotide sequence of SEQ ID NO: 7.
  19. 19. The hybrid promoter according to any preceding claim, wherein the kidney-specific promoter is a podocyte-specific promoter.
  20. 20. The hybrid promoter according to any preceding claim, wherein the kidney-specific promoter is a NPHS1 or a NPHS2 promoter.

Description

PROMOTERS FIELD OF THE INVENTION The present invention relates to promoters for kidney-specific transgene expression. The present invention also relates to polynucleotides and vectors comprising said promoters and use of said promoters. BACKGROUND TO THE INVENTION There are many genetic diseases which affect kidney function by attacking the glomerulus. The glomerulus filters approximately 180 litres of plasma each day, and the healthy glomerular filtration barrier has an astonishing ability to retain about 99.9% of large proteins including albumin over our lifetimes without clogging. The glomerular filtration barrier (GFB) comprises 3 main layers: the glomerular endothelial cell, the glomerular basement membrane (GBM) and the podocyte. The podocyte has also been implicated as a key cell in the progression of glomerular disease. Podocytes are mesodermally derived cells that are highly specialized and found only in the renal glomerulus. They exhibit unique characteristics such as foot processes and slit diaphragms, which are critical for glomerular filtration. Podocyte-associated genetic glomerular diseases include Nephrotic Syndrome, Frasier syndrome and Denys–Drash syndrome, Schimke immuno-osseous dysplasia, and Epstein and Fechtner syndrome. (see Chiang, C.K. and Inagi, R., 2010. Nature Reviews Nephrology, 6(9), p.539). Accordingly, kidney cells, in particular podocytes, represent a potential target for gene therapy approaches. In order to maximise kidney gene therapy potential, promoter sequences which can drive transgene expression in kidney cells, in particular podocytes, are required. Wong, M. et al., 2000. American Journal of Physiology Renal Physiology, 279(6), pp.F1027-32 describes a 1.25-kb DNA fragment from the human nephrin promoter and 5′-flanking region that is capable of directing podocyte-specific expression. This promoter has been used to achieve kidney-specific expression of GFP using an AAV2/9 vector, but showed reduced transfection efficiency compared to a CMV promoter (see Picconi et al., 2014. Molecular Therapy – Methods & Clinical Development; 1, p.14014). Thus, there is a need in the art for further promoters which can drive higher expression of a protein-coding sequence in kidney cells, in particular podocytes. SUMMARY OF THE INVENTION The present inventors have developed a promoter which can drive higher expression of a protein-coding sequence in kidney cells, in particular podocytes. In one aspect, the present invention provides a hybrid promoter comprising a super core promoter and a cell-specific or tissue-specific promoter. A super core promoter may comprise from 5’ to 3’: a TATA box, an initiator motif (Inr), a motif ten element (MTE), and a downstream promoter element (DPE). Suitably (i) the TATA box is located from about -31 to about -26 relative to the super core promoter +1 transcription start site; (ii) the Inr is located from about -2 to about +4 relative to the super core promoter +1 transcription start site; (iii) the MTE is located from about +18 to about +27 relative to the super core promoter +1 transcription start site; and/or (iv) the DPE is located from about +28 to about +33 relative to the super core promoter +1 transcription start site. Suitably, the TATA box comprises or consists of the nucleotide sequence TATAWAAR. In some embodiments, the TATA box is a CMV TATA box. In some embodiments, the TATA box comprises or consists of the nucleotide sequence TATATAAG. Suitably, the initiator motif (Inr) comprises or consists of the nucleotide sequence TCAKTY or YYANWYY. In some embodiments, the Inr is a composite Inr derived from the AdML core promoter and the Drosophila melanogaster G element core promoters, or a CMV Inr. In some embodiments, the Inr comprises or consists of the nucleotide sequence TCAKTY. In some embodiments, the Inr comprises or consists of the nucleotide sequence TCAGTC. Suitably, the motif ten element (MTE) comprises or consists of the nucleotide sequence CSARCSSAAC. In some embodiments, the MTE is a Tollo MTE. In some embodiments, the MTE comprises or consists of the nucleotide sequence CGAGCCGAGC. Suitably, the downstream promoter element (DPE) comprises or consists of the nucleotide sequence RGWYVT. In some embodiments, the DPE is a Drosophila G DPE or a CALM2 DPE. In some embodiments, the DPE comprises or consists of the nucleotide sequence AGACGT. Any super core promoter may be used. Suitably, the super core promoter is selected from super core promoter 1 (SCP1), super core promoter 2 (SCP2), or super core promoter 3 (SCP3). In some embodiments, the super core promoter is SCP1. In some embodiments, the super core promoter comprises or consists of a nucleotide sequence having 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to one or more of SEQ ID NOs: 7-11. In some embodiments, the super core promoter compr