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Therapeutic Targets in Alport Syndrome: Disclosure of Relevant Insights from the Big Picture Financial Relationships

USCAP requires that all planners (Education Committee) in a position to influence or control the content of CME disclose any relevant financial Jeffrey H. Miner, Ph.D. relationship WITH COMMERCIAL INTERESTS which they or their Professor of Medicine spouse/partner have, or have had, within the past 12 months, which relates to Division of Nephrology the content of this educational activity and creates a conflict of interest. Washington University School of Medicine St. Louis, Missouri

The The

Endothelial Cell

Glomerular

Basement Membrane The Glomerular Filtration Barrier

Urine

Blood

Epithelial Basement Membrane

The Glomerular Basement Membrane (GBM)

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Green ≈ LM‐521 The Major Glomerular Basement Red ≈ LM‐511 Membrane Proteins • ‐521 – α5β2γ1 heterotrimer • α3α4α5(IV) • Nidogens‐1,2 • Agrin (heparan sulfate proteoglycan)

The Major Glomerular Basement The Major Glomerular Basement Membrane Proteins Membrane Proteins • Laminin‐521 Pierson Syndrome – α5β2γ1 heterotrimer • Collagen α3α4α5(IV) Alport Syndrome • Nidogens‐1,2 • Agrin (heparan sulfate proteoglycan)

Human mutations in 4 of these 9 proteins cause disease LM‐521 α3α4α5

Alport Syndrome Split, Thickened, “Basket Weave” GBM

• A hereditary glomerular disease usually accompanied by hearing and characteristic lens and retina defects; incidence of ~1 in 10,000 • Often diagnosed in children based on the presence of hematuria and stereotypical defects in the glomerular basement membrane (GBM) seen in biopsy by TEM • Almost invariably progresses to ESRD by late adolescence or thereafter; progression is signaled by the onset of • Treatment with ACE inhibitors delays proteinuria and ESRD, but is not a cure

Normal Alport

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Loss of Collagen 345(IV) and Alport Syndrome Increased α1/1/2(IV) in Alport GBM • Caused by mutations in glomerular basement membrane type IV collagen genes – (COL4A3, COL4A4, or COL4A5) • The COL4A5 gene is X‐linked, so most patients are males – COL4A5+/‐ females can manifest various aspects of the syndrome, from hematuria to ESRD • 15% of cases are autosomal recessive homozygous COL4A3 or COL4A4 mutations – Heterozygous COL4A3 or COL4A4 carriers can manifest “Thin Basement Membrane Nephropathy”

Type IV Collagen Chains, Genes, and Trimers Alport Syndrome: Three Genes & A Spectrum of Manifestations • α1COL4A1 α1α1α2 • α2COL4A2

• α3COL4A3 • α4COL4A4 α3α4α5 GBM • α5COL4A5 α5α5α6 • α6COL4A6

Miner, Kidney Int, 2014

Stereotypical GBM Abnormalities There Are Great Animal Models for Alport Syndrome in Alport Mice • Both mouse and dog models recapitulate most aspects of the human • Genetic background influences the rate of Normal progression to ESRD in mice – 129’s progress quickly (ESRD at 80 to 90 days) – C57BL/6J’s progress slowly (ESRD at 8 to 9 months) – 129/B6 hybrids progress moderately (ESRD at ~4 months) • Identification of modifier loci could provide Alport opportunities for therapeutics

Splitting, Thickening, Basket‐weave Miner et al., 1996

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3D‐EM defines GBM defects GBM Abnormalities in “3‐D”

Control Mouse GBM Alport Mouse GBM

A combination of compositional and likely biophysical changes impact the overlying Randles MJ, Lennon R et al. Sci Rep 2016

Podocyte‐GBM invasion Alport Syndrome

• GBM structural defects eventually lead to: – infiltration of immune cells – increased expression of extracellular matrix proteins and proteases – glomerular scarring – obstruction of glomerular Invasive phenotype of podocytes in Alport syndrome may be – reduced renal flow/GFR induced by GBM compositional or structural changes

Randles MJ, Lennon R et al. Sci Rep 2016

There are Great Animal Models for Alport Syndrome Alport Mice: A Good Model for CKD • Both mouse and dog models recapitulate most • Amenable to testing hypotheses about aspects of the human kidney disease disease pathogenesis and potential • Genetic background influences the rate of therapeutics progression to ESRD in mice – Generating double mutant mice – 129’s progress quickly (ESRD at 80 to 90 days) – C57BL/6J’s progress slowly (ESRD at 8 to 9 months) – Treating mice with candidate therapeutics – 129/B6 hybrids progress moderately (ESRD at ~4 • In general, reducing inflammatory cell months) infiltration or interstitial fibrosis (genetically or • Identification of modifier loci could provide pharmacologically) has had only modest opportunities for logical, targeted therapeutics impact on slowing progression to ESRD

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A Notable Pre‐Clinical Success

Mesangial Sclerosis Massive 44% increase in the age (Kimmelstiel‐Wilson Nodules GBM Thickening at which 50% of Alport mice reach ESRD Hyperfiltration, Microalbuminuria, Progressive and Chronic Kidney Disease Is itself injurious to the nephron?

A Notable Pre‐Clinical Success Generation and Characterization of Alb‐/‐ Mice

• Normal lifespan • Alb-/- mice: – No albumin – Reduced total serum protein concentration – Hypertriglyceridemia – Normal – Normal kidneys • Alb+/- mice: – Reduced albumin concentration (1/2 of normal) – Hypertriglyceridemia

The Absence of Albumin Improves Kidney Pathology in Alport Mice The Absence of Albumin Reduces Proximal Tubular Injury in Alport Mice

KIM‐1 and WGA

Col4a3-/-;Alb+/- Col4a3-/-;Alb-/- Col4a3+/-;Alb+/- Col4a3-/-;Alb+/- Col4a3-/-;Alb-/- Col4a3+/-;Alb+/-

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Alport Mice (B6) Survive Longer in the Conclusions Absence of Albumin

• Filtered albumin is injurious to the nephron in Alport syndrome and perhaps in other proteinuric diseases • Defining pathways activated by filtered albumin (in podocytes) and resorbed albumin (in tubular cells) could reveal novel targets for therapy.

A Notable Clinical Success The Nephron

Glomerulus

Tubule

Arteriole

Collecting Duct

Vein

“earlier therapy in younger patients significantly delayed dialysis by 13 years compared to later or no therapy in older siblings.”

The Glomerulus Split, Thickened, “Basket Weave” GBM

Endothelial Cell Podocyte

Glomerular Basement Membrane “Fix it!” (Karl Tryggvason)

Normal Alport

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Potential Targets for Therapy The Major Glomerular Basement in Alport Syndrome Membrane Proteins

• The deranged matrix deposition/matrix degradation in glomeruli associated with GBM splitting and glomerular scarring • The inflammatory response in glomeruli and elsewhere in the kidney

• The defective GBM itself Could these – gene therapy approach approaches even – cell therapy approach change the GBM’s composition? – collagen replacement approach LM‐521 (α5β2γ1) α3α4α5

Structure of collagen IV protomers and Type IV Collagen Chains, Genes, and Trimers their assembly into networks

• α1COL4A1 α1α1α2 • α2COL4A2

• α3COL4A3 • α4COL4A4 α3α4α5 GBM • α5COL4A5 α5α5α6 • α6COL4A6

Hudson, B. G. J Am Soc Nephrol 2004;15:2514-2527

Copyright ©2004 American Society of Nephrology

Restoration of the Collagen IV Network in the Can the Defective GBM Be Repaired? GBM (Nephrin‐rtTA; Dox at 3 weeks) • Express a Doxycycline‐inducible Col4a3 transgene in the podocytes of Col4a3‐/‐ mice 14440_40x, Col+/+ Col4a3 14440_40x, Col+/+ Col4a4 14440_40x, Col+/+ Merged Control

14441_40x, Col‐/‐ Col4a3 14441_40x, Col‐/‐ Col4a4 14441_40x, Col‐/‐ Merged Alport Mouse “Rescued” Mouse P21 P21 P21 “Rescue” The Real Test: Restore the missing Collagen IV Lin et al., JASN 2014 after the GBM is mature and functioning. The Mature GBM Exhibits Plasticity

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Restoration of the Collagen IV Network The Glomerular Slit Diaphragm‐Actin Cytoskeleton Connection Slows Progression to ESRD

Miner and Abrahamson In The Kidney, 2012 Lin et al., JASN 2014

Glomerular Therapeutic Targets Glomerular Therapeutic Targets

• Restore the missing Collagen IV network • Restore the missing Collagen IV network – a.k.a., “Fix the Defect” – a.k.a., “Fix the Defect” • Define aberrant signaling within podocytes and • Define aberrant signaling within podocytes and inhibit it normalize it – “neo”‐collagen IV interactions – “neo” collagen IV interactions with podocyte receptors – impaired laminin interactions – impaired laminin interactions with integrin α3β1 – pathogenic mechanical strain – pathogenic mechanical strain

The Major Glomerular Basement Laminin β2 Knockout Mice Membrane Proteins Nephrotic syndrome and neuromuscular defects A model for Pierson Syndrome

Lamb2 -/-

Lamb2 +/-

LM‐521 (α5β2γ1) α3α4α5 Lethal at ~1 month of age Heavy albuminuria at 3 weeks

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Human LAMB2 Mutations Cause Pierson Syndrome Foot Process Effacement in Lamb2‐/‐ Mice (3 weeks of age)

Mostly Null/ Nonsense Mutations Hypothesis: Laminin β2 in the GBM is a signal that induces podocytes to form foot processes and slit diaphragms.

Lamb2+/‐ Lamb2‐/‐

Proteinuria is Followed by Foot Process Typical Podocyte Architecture, with Proteinuria at 2 days of age Effacement in Lamb2‐/‐ Mice Control Lamb2‐/‐

High Proteinuria and Widespread Foot Process Effacement at 3 weeks

Proteinuria is Followed by Foot Process Effacement in Lamb2‐/‐ Mice

Protein Therapy: Inject the missing LM‐521 i.v. to deliver it to the GBM before widespread foot process effacement.

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Treatment with hLAM‐521 from day 12 Localization of Injected hLM‐521 by Inhibited Proteinuria up to day P18 Super‐Resolution Microscopy Uninjected Lamb2‐/‐ Injected Lamb2‐/‐ ITGB1 0.050 n=2 LAMA5 * n=3 0.040 n=4 controls creatinine 0.030 to

(g/mg) mutants 0.020 hLAM‐treated mutants albumin Ratios

0.010 n=4 n=7 n=6 n=7 n=6 n=2 0.000

Urinary P12 P18 P20

Podocyte Injury in Lamb2-/- Kidney at P18 was Ameliorated by hLAM-521 Treatment

25.0 n=4 20.0 controls

podocytes: 15.0

mutants

desmin (+)/WT1(+) 10.0

& injured n=3

hLAM‐treated

of 5.0 (+) n=4 mutants %. 0.0 WT1 P18

“Pierson Syndrome” LAMB2 Null and Missense Mutations Laminin Polymerization

Matejas et al. Hum. Mutat. 2010 59

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Introduce S80R into the Mouse Lamb2 Gene by CRISPR/Cas9‐Mediated Genome Editing What about the LAMB2‐S80R mutation?

Required Cas9 Nuclease Guide RNA (gRNA)

Optional DNA oligo or dsDNA for HDR

Ocular abnormalities from infancy Proteinuria from age 6 Nephrotic range proteinuria by age 11 Include a 200 nt oligo to change Diffuse mesangial sclerosis on biopsy AGT (Ser) to CGT (Arg) at S80

CRISPR‐engineered LAMB2‐S80R Allele: No Phenotype LAMB2‐S80R in Alport mice: LAMB2‐S80R Acts as a Modifier Allele Lamb2 WT Lamb2 S80R Lamb2 KO Lamb2 7weeks

Lamb2+/S80R Nidogen

Lamb2: WT +/SR Lamb2: WT +/SR SR/SR Col4a3: ‐/‐‐/‐ Col4a3: +/‐‐/‐‐/‐ P7 P21 Merged

Early and Severe GBM and Podocyte Hypothesis: Strengthening Laminin/Collagen IV Defects in Lamb2+/S80R Alport Mice Interactions Will Slow Splitting/Thickening

Normal

Alport

Post‐natal d7: Lamb2+/S80R; Col4a3‐/‐ Miner et al., 1996

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Conclusions

Miner Lab Contributors Collaborators • Modifying the composition of the GBM in Pierson Hani Suleiman, MD, PhD Andrey Shaw, MD (Genentech) syndrome via the bloodstream can slow the Steven Funk, PhD Yamato Kikkawa, PhD (Tokyo) onset of proteinuria. Meei‐Hua Lin, PhD Rachel Lennon (Manchester, UK) • Altering or enhancing the interactions of the George Jarad, MD Michael Randles laminin and collagen IV networks with each Gloriosa Go Jennifer Richardson FUNDING other or with linking proteins in Alport syndrome NIDDK could reduce GBM splitting and thickening and American Heart Association slow the onset of proteinuria and CKD. Alport Syndrome Foundation

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