GENE THERAPY

Abstract

The present invention provides an adeno-associated virus (AAV) vector gene therapy comprising a vascular endothelial growth factor (VEGF)C transgene; and minimal nephrin promoter NPHS1 or podocin promoter NPHS2. The gene therapy vector can be used to target podocytes within the glomerulus of the kidney in order to treat or prevent kidney disease, such as diabetic kidney disease.

Claims

1. An adeno-associated virus (AAV) vector comprising: a vascular endothelial growth factor (VEGF)C transgene; and minimal nephrin promoter NPHS1 or podocin promoter NPHS2.

2. An AAV vector according to claim 1, wherein the AAV vector is AAV serotype 2/9, LK03 or 3B.

3. An AAV vector according to claim 1, wherein the VEGFC transgene comprises a polynucleotide encoding a VEGFC prepropeptide, or an intermediate form of VEGFC or a mature form of VEGFC.

4. An AAV vector according to claim 1, wherein the AAV vector additionally comprises a Woodchuck hepatitis post-transcriptional regulatory element (WPRE).

5. An AAV vector according to claim 1, wherein the VEGFC transgene is human and/or comprises a hemagglutinin (HA) tag.

6. An AAV vector according to claim 1, wherein the AAV vector additionally comprises a Kozak sequence between the promoter and the VEGFC transgene.

7. An AAV vector according to claim 1, wherein the AAV vector additionally comprises a polyadenylation signal such as bovine growth hormone (bGH) polyadenylation signal.

8. A method for treating or preventing kidney disease in a subject, comprising administering to the subject an effective amount of the AAV vector of claim 1, thereby treating or preventing the kidney disease in the subject.

9. The method according to claim 8, wherein the kidney disease is diabetic kidney disease.

10. The method according to claim 9, wherein the diabetic kidney disease is early stage diabetic kidney disease.

11. The method according to claim 8, wherein the subject is a human patient.

12. The method according to claim 11, wherein the human patient has type 1 diabetes or type 2 diabetes.

13. The method according to claim 8, wherein the AAV vector is administered systemically to the subject.

14. The method according to claim 8, comprising administering the AAV vector is by intravenous injection to the subject.

15. The method according to claim 8, comprising administering the AAV vector by injection into the subject's renal artery.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] FIG. 1 shows an example DNA sequence for the minimal human nephrin promoter (NPHS1).

[0034] FIG. 2 shows an example cDNA sequence for a human VEGFC transgene.

[0035] FIG. 3 shows an example DNA sequence for a WPRE sequence.

[0036] FIG. 4 shows an example DNA sequence for a bGH poly(A) signal sequence.

[0037] FIG. 5 shows the construct used for the production of AAV LK03 VEGFC. In this construct hpodocin was replaced by VEGFC.

[0038] FIG. 6 shows expression of human VEGFC-FLAG in a positive control (transfected HEK293) and in AAV LK03 VEGFC infected podocytes. No expression was observed in proximal tubule cells or in glomerular endothelial cells, indicating that VEGFC expression was specific to podocytes.

[0039] FIG. 7 shows the effect of VEGFC conditioned media obtained from HEK cells transfected with pCMV3-VEGFC expression plasmid or mock, and podocytes transfected with Nphsl.AAV-VEGFC or control virus. There was a strong trend for an increase in WGA binding to glomerular endothelial cells stimulated with conditioned media form HEK cells (see FIG. 7A) and a significant increase in WGA binding to glomerular endothelial cells stimulated with podocyte conditioned media (see FIG. 7B).

EXAMPLES

[0040] DKD, a disease that initiates in the glomerulus, lacks a glomerular-specific therapeutic strategy. Currently the mainstay of treatment is to target elevated blood pressure. Elevated glomerular filtration rate and microalbuminuria, early indicators of DKD, are both related to changes in glomerular endothelial ultrastructure before podocyte ultrastructural changes can be seen. Initiation of targeted treatment at this point would be most beneficial.

[0041] The aim of this research is to combine a successful strategy to protect from microvascular complications in DKD, with a safe and successful gene delivery approach so that VEGFC gene expression can be delivered to podocytes early in disease.

[0042] Hypothesis: Targeted podocyte adeno-associated viral VEGFC gene therapy will protect from endothelial dysfunction and prevent DKD

[0043] Objective 1: Create and validate AAV gene therapy tool for podocyte-specific VEGFC transduction.

[0044] Objective 2: Demonstrate that this protects from the development of experimental DKD.

[0045] Objective 3: Proof in principle: targeting podocyte VEGFC gene expression in human glomerular tissue.

[0046] Podocyte targeted gene therapy: We have developed a targeted gene delivery system in human and mouse podocytes using adeno-associated virus (AAV) (see PCT/GB2020/050097). Using a podocyte-specific promoter (nephrin), AAV serotype 2/9 successfully infected podocytes in vivo, inducing podocin expression. In animals where podocin was knocked down using the Cre-Loxp system (NPHS2fl/fl), resulting in proteinuria, AAV treatment successfully recovered podocin expression and ameliorated proteinuria. In addition, we have shown efficient and specific transduction of GFP by AAV LK03 (with better efficiency than AAV2/9) in human podocytes using the same promoter. Combining this technology, we aim to drive podocyte VEGFC gene transduction in mice and then show proof of principle in human glomerular tissue.

Objective 1: Create and Validate AAV Gene Therapy Tool for Podocyte-Specific VEGFC Transduction

[0047] This project will use AAV2/9 for mouse work and AAVLK03 for human work. AAV3B will also be used for human work and will be used for large animal studies. We have demonstrated using AAV, in both cell culture and mouse models, that a minimal nephrin promoter (1.2 Kb) successfully induces transduction in podocytes, despite the restricted packaging size of AAV (4.7 Kb). Both the human and mouse minimal nephrin promoter was effective in driving transduction in mouse tissue, therefore the human nephrin promoter will be used throughout this project. This demonstrates that we can effectively drive gene transduction in podocytes in vivo.

[0048] Human rVEGFC has previously been shown to have an effect in vivo in mouse kidneys, when delivered by osmotic mini-pump, and human VEGFC was transgenically overexpressed in the skin of mice with functional effects. Therefore, human VEGFC will be used in the same construct for mouse and human work.

[0049] AAV vectors are considered the leading platform for gene delivery in humans. They are 26 nm diameter capsids with a single stranded DNA genome. They are non-pathogenic with low immunogenicity and have been proven successful in many clinical trials, the first; Glybera, an AAV1 encoding lipoprotein lipase, followed by others including systemic application (AAV8 and AAV9). Targeted transduction to the podocytes should remove the impact of liver tropism, following systemic application.

[0050] Human full length VEGFC (Jha et al.) with an N-terminal HA tag (1260 bp, SinoBiological) or N-terminal MyC tag will be ligated into our AAV2/9, AAVLK03 and AAVL3 vectors containing a human minimal nephrin promoter (NPHS2).

[0051] Murine podocytes, glomerular endothelial cells and proximal tubule epithelial cells will be infected with AAV2/9 VEGFC or empty vector. Suitable titres will be determined. Infection will be quantified by RNA extraction and QPCR for viral particles. Transduction will be confirmed by immunofluorescence staining of HA, MyC and/or VEGFC expression quantified by ELISA on cell lysates.

[0052] Human podocytes and glomerular endothelial cells and proximal tubule epithelial cells will be infected with AAV VEGFC (LK03 or 3B) or empty vector. Infection and transduction will be confirmed as mouse cell lines above.

[0053] The expectation is that all cell types will be infected, but expression of VEGFC will only occur in podocytes.

Objective 2: Demonstrate that this Protects from the Development of Experimental DKD

[0054] Two mouse models of type 1 diabetes with diabetic nephropathy are available: STZ DBA2/J and OVE26 FVB. The latter provides a more severe model of diabetic nephropathy, more closely resembling human pathophysiology (albuminuria by 8 wk, hyper filtration at 3 months and reduced GFR at 9 months) and increased blood pressure at 8 months (systolic and diastolic). Podocyte loss is observable at 12 weeks.

[0055] Pilot Study Using STZ and OVE26 in Parallel:

[0056] AAV-VEGFC or vehicle will be tail vein injected at 6 or 12 weeks post-STZ or 6 and 12 weeks old (OVE26). N=2 in group. Urine samples will be taken very two weeks. Cheek vein blood will be sampled every two weeks. Urine albumin creatinine ratios (uACR) and eGFR will be calculated. Glomerular VEGFC expression will be correlated with podocyte viability in each model. This will help to define the (latest) time of intervention in each model and experimental end point.

[0057] STZ DBA2/J mice—male N=9 each condition [0058] (i) Sham+vehicle [0059] (ii) STZ+vehicle [0060] (iii) STZ+AAV VEGFC

[0061] Readout: uACR, eGFR, glomerular permeability assay, histological features including by EM.

[0062] OVE26 FVB mice—male and female. N=9 each condition. [0063] (i) Littermate control non-diabetic+vehicle [0064] (ii) Diabetic+vehicle [0065] (iii) Diabetic+AAV VEGFC

[0066] Readout: uACR, eGFR, glomerular permeability assay, histological features including by EM.

[0067] This aim will confirm that VEGFC transduction prevents early GEnC changes and albuminuria in DKD and that this treatment is effective long term.

Objective 3: Proof in Principle: Targeting Podocyte VEGFC Gene Expression in Human Tissue

[0068] Glomeruli will be isolated from human donor kidneys unsuitable for transplant. We already have the infrastructure set up to receive these kidneys regularly on existing projects. Human kidney organoids from pluripotent stem cells, infected with AAV, have previously shown expression by day. Glomeruli will be cultured in suspension for 1 day before AAVLK03 VEGFC, AAV3B VEGFC or empty vector is added to the culture media. Five days later glomeruli will lysed be fixed in tissue-tek and sectioned as we have done previously for human glomeruli cultured in suspension.

[0069] Infection: Glomerular lysates will be mRNA extracted and viral particles quantified by QPCR.

[0070] VEGFC expression: Confocal immunofluorescence colocalization studies will be carried out to demonstrate transduction of VEGFC by podocytes and not endothelial or mesangial cells. VEGFC expression will also be quantified in lysed glomeruli for human VEGFC ELISA.

[0071] Human glomerular viability: We have shown that human glomeruli can be cultured up to 10 days in suspension and remain physiologically responsive and that human glomeruli are viable in culture up to 7 days. At end point (6 days of culture), viability will be confirmed on fresh glomeruli.

[0072] These experiments will be carried out on a minimum of three separate populations of isolated human glomeruli (i.e., 3 kidneys).

[0073] If successful, this aim will demonstrate effective transduction of VEGFC in human glomeruli using a clinically safe vector.

Example 2

Cloning Human VEGFC into AAV Vector

[0074] Human VEGFC-FLAG was cloned into an AAV LK03 vector, expressing under the minimal nephrin promoter (hNPHS1) using AflII and SbfI restriction sites (see FIG. 5). The clone sequence was verified, then grown and purified.

VEGFC-FLAG Cloning into AAV Vector

[0075] VEGFC insert was amplified from pCMV3-ORF-FLAG from Sinobiologicals (HG10542-CF) as template using primer sequence GATCcttaagGCGATCGCCATGCACTTGCTGG containing AflII restriction as forward and GATCcctgcaggTTAAACCTTATCGTCGTCATCCTT containing the SbfI restriction site as reverse. NEB Q5 HF 2X Master Mix (M042S) was used for amplification following manufacturer instructions and 60 C as anneal temperature for primers. Single product at correct size (band at 1200 bp) was confirmed by gel electrophoresis before using the Qiagen PCR Purification kit (28104) to clean up PCR reaction. VEGFC amplicon and AAV vector pAV.Hnphs1.hpodHA.WPRE.bGH were double digested with AflII and SbfI at 37 C for 2 hours. Restriction digest for AAV vector was ran on 1% Agarose gel for 1.5 hours at 100V to allow for separation of linearized double digested vector from digest products. VEGFC PCR digest was once again cleaned up using the Qiagen PCR Purification kit. Digested AAV vector was cut out of gel (6,500 bp band) and purified with Qiagen Gel purification kit (28115). Once clean up and purification was complete, ligation was set up using a 1:1 ratio of vector to insert, using 100 ng of vector. Promega T4 Ligase (M180) was used for ligation following manufacturer instructions. Once ligation was complete, ligation products were transformed using use NEB 5-alpha competent E. coli (high efficiency) (C2987) cells following manufacturer instructions. Transformation was plated on LB agar plates with 100 ug/ml of Ampicillin and put at 37 C overnight. Colonies were screened for VEGFC insert and sequence verified.

VEGFC-FLAG Expression in Kidney Cell Lines by Immunofluorescence

[0076] Temperature sensitive SV40 T-Antigen transformed glomerular endothelial cells (GEnC), podocytes (LY), and proximal tubule epithelial cells (PTEC) were seeding on cover slips in 6-well plate and allowed to reach 80% confluency. Cells were then infected with 25 ul of purified AAV LK03 VEGFC virus and and thermoswitched from 33° C. to 37° C. which results in degradation of SV40 T-Antigen, allowing cells to differentiate. GEnCs and PTECs were differentiated for 5 days while LYs for 10 days. As positive control, 293 HEK cells were transfected with pCMV3-ORF-FLAG plasmid which expressed VEGFC under the CMV promoter, resulting in high expression levels. Cells were then washed with PBS, fixed with 4% PFA and stained with anti-FLAG M2 from Sigma (F3165) followed by anti-mouse 594 from Sigma (SAB4600092) to determine expression levels in each cell type. As positive control, 293 HEK cells were transfected with pCMV3-ORF-FLAG plasmid which expressed VEGFC under the CMV promoter, resulting in high expression levels. Cells were then imaged on epi-fluorescence microscope.

Results

[0077] The results show expression of human VEGFC-FLAG in a positive control (transfected HEK293) and in AAV LK03 VEGFC infected podocytes (see FIG. 6). No expression was observed in proximal tubule cells or in glomerular endothelial cells, indicating that VEGFC expression was specific to podocytes. The AAV LK03 VEGFC vector can therefore be successfully made and specifically targeted to podocytes. In view of earlier work by the inventors showing that VEGFC promotes the survival of glomerular endothelial cells and reduces albumin permeability (Foster et al 2008) and that VEGFC can enhance the glycocalyx layer on the surface of the endothelial cells, which controls endothelial albumin permeability (Foster et al, 2013), together these data show that the gene therapy vector of the present invention can be used to target podocytes within the glomerulus of the kidney in order to treat or prevent kidney disease, such as diabetic kidney disease.

Example 3

Conditioned Media from AAV-VEGFC Infected Podocytes Increases Cell Surface Lectin Binding to Glomerular Endothelial Glycocalyx

[0078] HEK cells were transfected with pCMV3-VEGFC expression plasmid or mock and podocytes were infected with Nphs1.AAV-VEGFC or control virus. Conditioned media was removed, concentrated using spin columns and resuspended in glomerular endothelial media.

[0079] The conditioned media was added to glomerular endothelial cells for 1 h, cells were fixed and immune fluorescent staining carried out using green labelled wheat agglutin lectin (WGA). This binds to the sugar residues on the surface of the endothelial cells, the endothelial glycocalyx. Cell surface fluorescent intensity was quantified. There was a strong trend for an increase in WGA binding to glomerular endothelial cells stimulated with conditioned media form HEK cells (see FIG. 7A) and a significant increase in WGA binding to glomerular endothelial cells stimulated with podocyte conditioned media (see FIG. 7B, unpaired t-test, p<0.05). This suggests that podocytes infected with Nphs1.AAV-VEGFC secrete VEGFC, which acts on the glomerular endothelial cells to enhance the glycocalyx layer.

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