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Gene Therapy for Hemophilia B

DR. DEMET SAG

THE UNIVERSITY OF NORTH CAROLINA, CHAPEL HILL, NC USA

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TOPICSPART I: Sine Wave Alternative Current Gene Delivery• Cardiovascular and Coagulation Biology• Waterfall Coagulation Cascade • Hemophilia• Mice models• Electrotransfer: Gene Delivery and Gene Therapy PART II: Using VKOR to improve FIX specific activity• Anticoagulant Therapy and new approaches• VKOR, GCCX and Vitamin K Cycle• Post translation: Carboxylation reaction Galactolysation

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Cardiovascular and Coagulation System

Cardiovascular System – Blood (plasma+cells) + Vessels + Heart

• Carrier + transporter + Pump

Coagulation– Blood, plasma, endothelial cells, membrane,

coagulation factors, transmembrane proteins, PARs etc.

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Hemostasis

• Hemostasis: The balance between clotting and bleeding

• Components of Hemostasis:– Vasculature– Coagulation proteins– Platelets

Clot Formation

Clot Dissolution

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Coagulation Cascade – Then & Now

• “Waterfall” theory developed in the 1960’s– Believed that clotting occurs through a series of reactions

in which serine protease zymogens are converted into active enzymes in a step-wise process

• For many years, intrinsic pathway was believed to be the more important clotting mechanism; didn’t take into account the significance of the extrinsic pathway (TF/FVII pathway)

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“Contact Activation” Tissue Factor + VII

XIIIa

XIII

ThrombinXL-

FibrinPolymer

Fibrinogen Fibrin Monomer

IXXI

XIaIXa

XaVa

XIIaProthrombin

TF-VIIa

(Prothrombinase)

PL

PL, Ca2+(Tenase)

VIIIaPL, Ca2+

X

Intrinsic Pathway

PrekallikreinHMW

Kininogen

Extrinsic Pathway

Common Pathway

“TF Pathway”

Coagulation Cascade

Anticoagulation proteins: Protein C, Protein S, Antithrombin III, TFPI

Ca2+

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Waterfall Coagulation Cascade Model

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Coagulation Cascade• THROMBUS/CLOT: Vascular damage, abnormal

activation of blood coagulation and/or depressed fibrinolytic activity initiates the coagulation cascade results in the generation of thrombin at the site of injury, that may lead to stroke, heart attacks.

• BLEEDING: In contrast, a defect or deficiency in the coagulation process and/or accelerated fibrinolysis is associated with a bleeding tendency.

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Blood Coagulation• Under normal circumstances, the coagulation system is

balanced in favor of anti-coagulation• Thrombin is the key affector enzyme of the clotting

cascade• Antagonists of vitamin K inhibit a Vitamin-K-dependent

post-translational modifications of several coagulation proteins, which is required for these proteins to attain a phosphol-lipid binding conformation

• Gene therapy may soon be a therapeutic option for inherited deficiencies of factors VIII and IX.

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ARTERY AND VEIN

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When coagulation cascade meets PARs (Proteinase Activated Receptors)

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The relative importance of the coagulation factors in vivo has been elucidated by knock-out mice technology.

• Lacking mice TF die as embryos.• Deficient in factor VII develop normally in utero but die shortly after birth

due to severe bleeding.• The difference between deficiency of TF and deficiency of factor VII

suggests a role for tissue factor during embryogenesis beyond fibrin formation.

• Deficiencies of factor V and prothrombin are both associated with fatal hemorrhage and partial embryonic lethality.

• Deficiencies in FIX and FVIII develop normally during fetal life, but show hemophilia after birth.

• Fibrinogen deficiency in mice associated with normal fetal development and a moderate to severe bleeding phenotype, which shows that thrombin generation is more important than fibrin deposition.

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Hemophilia• rare inherited bleeding disorders with prevalence of about one in 10 000.

• The genes for both factors are on X chromosome, – which why only males affected rarely females, – mostly females are carriers of the disease.

• Almost half of the disease causing mutations are novel• Many boys are born families with no previous history of the disease.

• concentration of either factor IX or factor VIII of the normal plasma – less than 1% severe– 1-5% moderate and mild forms – 5-20% of normal.

• The normal plasma concentrations of these components seem therefore to be higher than required for a physiological response, which is noteworthy considering the very low concentrations of factor VIII in particular.

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Procoagulant and Fibrinolytic Factors

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Location of human F.IX mutations on the secondary structure of F.IX

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Gla Domain

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In vivo: Genetics of mouse models

FIXKO, loss of mutation, null– Deletion of FIX between exon 4-7

Lin HF, Maeda N, Smithies O, Straight DL, Stafford DW. A coagulation factor IX-deficient

mouse model for human hemophilia B. Blood. 1997;90:3962-3966. R333Q, missense mutation

– 2/3 of human population has missense mutation that don’t develop immune response as much as the ones have big deletions

Jin DY, Zhang TP, Gui T, Stafford DW, Monahan PE. Creation of a mouse expressing defective human factor IX. Blood. 2004;104:1733-1739.

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The muscle tissue: is an attractive target organ for gene therapy

• The tissue is abundant, makes up 40% of the body weight of an adult

• The skeletal muscle is accessible to most of the deliversy systems in currently used for gene therapy

• There is no significant cell replacement in muscle tissue, hence, the introduced genes are not rapidly lost following mitosis, and transgene expression kept longer.

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• Membrane permeablization, small molecules EDTA, histamine etc

• Strength of the electrophoresis and duration, high voltage (800 cm/volt) vs. low voltage (80volt/cm) duration of 20 ms.

• Receptor mediated • Energetic metabolism (role of ATP and ADP) to alow the

DNA crossing the plasma through the membrane and its migration to the nucleus

• Inflammation may be induced by Electrotransfer Plasmid DNA injection to various tissues with electrotransfer after gene delivery improves the gene expression.

Mechanism of Electrotransfer:

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Gene deliveryapplied in:

1. vaccination, 2. oncology, 3. gene therapy

• Muscle disorders, secreted protein expression in liver, brain, cornea besides muscle expanding.

• Cancer, blood disorders, muscle disorders, rheumatoid arthritis, muscle ischemia.

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Considerations for Electrotransfer (part 1)• Important Factors:

1. cell permeabilization, 2. safety, 3. size of plasmid, 4. optimizing plasmid biodistribution,5. design of encoded protein, and 6. the structure of plasmid.

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Considerations for Electrotransfer (part 2)

Kinetic expression of the transgene dependson many factors including:

1. cellular localization of the protein,

2. physiological activity,

3. regulation.

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Experiments• Field Strength• Pulse Length • DNA dose• Time Course• Tissue Damage • Safety • Gene Expression in alive mice and on tissue

– In adult mice different tissues, liver, skin, tumor- In newborn mice for safety and expression

- Comparing Alternative Current Sine Wave (ACSW) vs. Direct Current Square Wave (DCSW)

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Field Strength

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Pulse Length

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Gene Expression in alive mice with Luciferase

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The Field Strength and Length in vivo

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SAFETY and Tissue Damage

• H&E staining for the examination of muscle damage after electro-gene transfer (6pulses) using a syringe electrode

• The samples were collected and sectioned 5 days after gene transfer.

• The pictures were taken from muscle sections (10 μm thick) after H&E staining (100X).

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H&E staining for the examination of muscle damage after electro-gene transfer (6pulses) using a syringe electrode.

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DNA dose

For luciferase expression muscle tissues were examined after ACSW gene transfer of luciferase plasmid to muscle with 20 V/cm, 6 pulses and 600 ms duration using a syringe electrode.

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Time course

The second electro-transfer was performed at day 80 after the first gene transfer and gene expression was detected at one and three weeks after 19 the second transfer.

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Gene expression in adult mice

• in different tissues of adult mice, • 4 mice/group, • were transferred with

– 10 μg luciferase plasmid for skin (6 pulses, 30 V/cm, 400 ms duration) and

– tumor (6 pulses, 60 V/cm, 400 ms duration) or

– 20 μg for the middle lobe of the liver (6 pulses, 30 V/cm, 400 ms duration).

• Approximately 1 cm2 of injected skin was cut for luciferase expression assay.

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Gene expression in different tissues of adult mice

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Gene expression in the skin of newborn mice

Using a syringe electrode: • 5 μg of luciferase plasmid in 10 μl saline was

injected into the skin on the back of the baby mouse

• within 24 h of birth,• 3 per group, • followed by electroporation using ACSW (6

pulses, 20 V/cm, 600 ms, 0.4 cm gap).• Approximately 0.8 cm2 of injected skin was cut

for luciferase expression assay.

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Gene expression in the skin of newborn mice

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ACSW transfer of hFIX plasmid DNA into two diseased models of

hemophilia B mice• FIX Antigen Levels by ELISA and FIX Activity

• ACSW transfer of hFIX DNA into the hamstring muscles of both rear legs of the mice (4 mice per group) using a syringe electrode.

• The hFIX protein levels in R333Q mice were subtracted from the background (time 0).

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hFIX antigen levels (ELISA) with ACSW

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FIX clotting activity

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The pulse waves for DC square-wave and AC sine-wave with 50 ms length.

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SUMMARY• Field Strength: 10 to 30V/cm• Pulse Length: 600 ms• DNA dose: 1 to 5ug• Time Course: at least 80 days• Tissue Damage: ACSW has 70% less than DCSW• Safety: confirmed with creatine kinase levels,

indicative for muscle damage • Gene Expression: 10-20 fold more with ACSW

– In adult mice different tissues, liver, skin, tumor- In newborn mice for safety and expression

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Acknowledgement

• Dr. Paul Monahan, The Gene Therapy Center• Dr. Leaf Huang, Dept of Pharmacology• Dr. Feng Liu, Dept of Pharmacology• Dr. Darrel Stafford, Dept of Biology UNC• Dr. Richard Samulski, Director of Gene Therapy

Center

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PART IIINCLUDING VKOR TO IMPROVE FIX

SPECIFIC ACTIVITY THROUGH BETTER CARBOXYLATION

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Monitoring Warfarin Therapy is a Balancing Act!

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Vitamin K Cycle

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VKOR STRUCTURE

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Molecular basis for anticoagulant therapy

• The enzymes and the substrates are vitamin-K dependent proteins that interact with the phospholipid domain via their amino terminal domains, which contain g-carboxyloglutamic acid residues.

• This post-translationally modified glutamic acid residues present only in the vitamin K-dependent proteins.

• The residues are involved in calcium binding, important for the correct folding of the g-carboxyglutamic acid domain.

• Inhibition of the g-carboxylation reaction by antagonists of vitamin K results in defective calcium binding of the g-carboxyglutamic domains and the loss of ability to interact with the phospholipid membrane.  

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Glutamic Acid to Gamma Carboxyl Glutamic Acid(GGCX)

• Posttranslational modification of glutamate (Glu) to gamma carboxyl glutamic acid (Gla) is required for the activity of a few proteins, most of which are related to coagulation.

• These modified proteins are often called vitamin K–dependent proteins because they require reduced vitamin K for their Gla modification.

• The enzyme that catalyzes the modification of Glu to Gla is the gamma-glutamyl carboxylase (GGCX), an 87.542-kDa1

integral membrane protein with 5 transmembrane domains.2

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The carboxylation reaction

• For the carboxylation reaction, a propeptide on the substrate binds to an exosite on the vitamin K–dependent gamma-carboxylase, holding the substrate in place while multiple glutamic acids are modified to Gla.

• The carboxylation also requires CO2, O2, and reduced

vitamin K.

• For each Glu modified, at least 1 molecule of vitamin K epoxide is formed.4

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Vitamin K Cycle

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New Anticoagulants

Limitations of traditional anticoagulants, – both with heparin and – warfarin,

have prompted the development of new agents

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Stanford Lab (UNC, Chapel Hill USA)• Vitamin K recycles into its active, reduced form through

the activity of vitamin K epoxide reductase (VKOR);

• Warfarin targets this step and inhibits the regeneration of reduced vitamin K.

• The investigators utilized the clinical observation from rats, humans, and mice with warfarin resistance that the genetic regions in each species were homologous to that in humans with combined vitamin K-dependent protein deficiency that mapped to Chromosome 16.

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Stanford Lab Approach (Part 2)

• After eliminating proteins with known function, they refined their search to 13 genes because biochemical evidence suggested that the protein was transmembrane.

• Utilized a cell line that expresses high amounts of VKOR activity,

• disrupted expression of each of the 13 candidate genes with siRNA

• narrow their search to one gene encoding a protein with a molecular mass of 18,200.

• Expression of this gene conferred VKOR activity on an insect cell line that does not normally express it, and the activity was inhibited by warfarin in vitro

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Oldenburg Lab (Germany)• by using a positional cloning approach

• in patients that were identified on a genetic basis to have reduced vitamin K-dependent clotting factors or warfarin resistance.

• Their characterization revealed that the protein is widely expressed in a number of tissues, including liver.

• Mutations in the gene encoding VKORC1 were confirmed in patients:1. deficient in vitamin K-dependent clotting factors and 2. with warfarin resistance.

• The investigators found the same point mutation in the unrelated families with combined deficiency of vitamin K-dependent clotting factors 2, and four distinct mutations in those with warfarin resistance.

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VKOR and GGCX activity for the various cell lines Ref: Cloning and expression of the cDNA for human gamma-glutamyl

carboxylase SM Wu, WF Cheung, D Frazier, and DW Stafford

Pro-peptide of Gla FIX change to FII for better binding

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Comparison of VKOR, GGCX, and FX (A6) mRNA expression levels determined by real-time Q-PCR

  Cell line  

  VKOR mRNA expression level ratio  

  GGCX mRNA expression level ratio  

  FX (A6) mRNA expression level ratio  

  HEK293-FX (A6)     1     1     1  

  HEK293-FX (A6)-VKOR     10.69     1.63     1.24  

  HEK293-FX (A6)-GGCX     1.51     86.14     1.32  

  HEK293-FX (A6)-VKOR-GGCX  

  11.97  

   

Pro-peptide of gla FIX change to FII for better binding

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Separation of -carboxylated and uncarboxylated FX by hydroxyapatite chromatography

Pro-peptide of gla FIX change to FII for better binding

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FIX Expression Controls

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FIX on treated leg

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THE QUANTITATIVE ANALYSIS

COMPARING THREE CONSTRUCTS• FIX ALONE, • FIX-VKOR SINGLE GENE CO-INTRODUCTION AND • COMBINED DOUBLE GENE INTRODUCTION

ANALYSIS • ELISA, FOR PROTEIN EXPRESSION• FIX ACTIVITY• SPECIFIC ACTIVITY

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FIX Antigen ExpressionELISA

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D2 D5 D7 D10 D13

DAY

NG/M

L

FIX FIX-VKOR COMBINED

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Functional Analysis FIX

FIX ACTIVITY

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D2 D5 D7 D10 D13

DAY

(%)

FIX FIX-VKOR CO0MBINED

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THE SPECIFIC ACTIVITY of FIX

SPECIFIC ACTIVITY

0.002.004.006.008.00

10.0012.0014.0016.00

D2 D5 D7 D10 D13

DAY

IU/M

L

FIX FIX-VKOR COMBINED

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Sequential Double Immunofluorescence for Combined Plasmid

DAPIDAPI

ALEXA 488ALEXA 488

ALEXA 488ALEXA 488

ALEXA488-ALEXA488-ALEXA594ALEXA594

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Acknowledgement• Dr. Paul Monahan, The Gene Therapy Center

• Dr. Darrel Stafford, UNC Chapel Hill, Dept Of Biology• Dr. Feng Liu, Pharmacology Dr. Jude Samulski, UNC

Chapel Hill, The Gene Therapy Center• Dr. Leaf Huang, Pharmacology

• Dr. Jue Wang, China• Dr. Lisa M Shollenberger, University of Pittsburg

• Feilong Niu, China• Xing Yuan, Duke University• Shyh-Dar Li, Pharmacology

• Mike Thompson, Pharmacology

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FIX SINGLE PLASMID GENE DELIVERY ELISA FIX

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FIX ACTIVITY FOR FIX

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D2 D5 D7 D10 D13

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ACTI

VITY

(%)

SPECIFIC ACTIVITY FOR FIX

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FIX VKOR SINGLE PLASMIDCO-GENE DELIVERY

FIX-VKOR ELISA

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FIX ACTIVITY FOR FIX-VKOR

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ACTI

VITY

(%)

SPECIFIC ACTIVITY FOR FIX-VKOR

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IU/M

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FIX-VKOR DOUBLE GENE PLASMID(COMBINED) GENE DELIVERY

COMBINED ELISA

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FIXACTIVITY FOR COMBINED

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VITY

(%)

SPECIFIC ACTIVITY FOR COMBINED

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