Roscoe O. Brady, M.D.

Chief, Developmental and Metabolic

Neurology Branch
National Institute of Neurological
Disorders and Stroke
National Institutes of Health
Bethesda, Maryland
 CURRENT AND FUTURE STRATEGIES FOR THE

TREATMENT OF METABOLIC STORAGE DISORDERS

1. BONE MARROW TRANSPLANTATION

2. ENZYME REPLACEMENT THERAPY

3. SUBSTRATE REDUCTION THERAPY

4. MOLECULAR CHAPERONE THERAPY

5. GENE THERAPY
Hereditary Lipid Storage Disorders

Sphingolipidoses
 SPHINGOSINE

18 3 2 1
CH3 -(CH2)12-CH=CH-CH-CH-CH2OH

OH NH2

Carbon atoms 1 and 2 arise from the amino acid serine

Carbon atoms 3 to 18 arise from palmitic acid

 CERAMIDE

Sphingosine

CH3 -(CH2)12-CH=CH-CH-CH-CH2OH

OH NH

CH3 - (CH2)22 - C = 0

Long Chain Fatty Acid

GAUCHER DISEASE
 Gaucher
Disease
Type 1
(Non-
Neuronopathic) Anemia

Easy bruising
due to low
blood platelets Bone damage

Enlarged
liver Huge spleen
ACCUMULATING LIPID IN GAUCHER DISEASE

GLUCOCEREBROSIDE

SPHINGOSINE GLUCOSE

FATTY ACID
ENZYMATIC DEFECT IN GAUCHER DISEASE
DEFICIENCY OF GLUCOCEREBROSIDASE

SPHINGOSINE GLUCOSE

FATTY ACID

R. O. Brady et al. Biochem Biophys Res Commun 1965; 18: 221

WHAT IS THE ORIGIN OF THE ACCUMULATING LIPID?
MAJOR LIPID OF WHITE BLOOD CELLS

CERAMIDELACTOSIDE

SPHINGOSINE GLUCOSE GALACTOSE

FATTY ACID
 MAJOR LIPID OF RED BLOOD CELLS

GLOBOSIDE

SPHINGOSINE—GLUCOSE—GALACTOSE—GALACTOSE—N-ACETYGALACTOSAMINE

FATTY ACID

• 20-40 times more glucocerebroside arises from senescent

white blood cells than red blood cells

TREATMENT OF PATIENTS WITH

LYSOSOMAL STORAGE DISORDERS

1. Bone Marrow Transplantation

 BONE MARROW TRANSPLANTATION (BMT)

If a suitable match is available, BMT can cure a

patient with type 1 Gaucher disease

Risks
Graft-versus host disease
Continuous immunosuppression probably necessary

Implication
? Gene therapy using transduced bone marrow stem cells
 TREATMENT STRATEGIES

2. ENZYME REPLACEMENT THERAPY

R. O. BRADY N Engl J Med 1966; 275: 312

 GAUCHER DISEASE

The required enzyme glucocerebrosidase is currently

produced recombinantly in Chinese hamster ovary cells.

It is necessary to modify the glycoform of this enzyme

in order to target it to macrophages, the principal lipid-

storing cells in the body of patients.

Amino Acid Chain
GLUCOCEREBROSIDASE IS TREATED WITH

3 EXOGLYCOSIDASES
Amino Acid Chain
Delivery of mannose-terminal glucocerebrosidase

to lipid-storing macrophages (Kupffer cells in the

liver) is increased 50-fold over that of unmodified

glucocerebrosidase
RESULTS OF ENZYME REPLACEMENT THERAPY
IN GAUCHER PATIENTS USING MACROPHAGE-
TARGETED GLUCOCEREBROSIDASE

Spleen size decreases

Liver size decreases
Hemoglobin increases
Blood platelets increase
Skeleton improves
 SPLEEN
MRI OF
ABDOMEN

ERT 7 months ERT

MORE THAN 4,300 PATIENTS WITH GAUCHER

DISEASE ARE NOW RECEIVING ENZYME

REPLACEMENT THERAPY
 TYPE 2 GAUCHER DISEASE

Acute Neuronopathic Gaucher Disease

Neuronophagia in the brain of a patient with Type 2 Gaucher disease
WHAT IS THE SOURCE OF GLUCOCEREBROSIDE

IN THE BRAIN?
 Ganglioside GDIa

SPHINGOSINE -GLUCOSE -GALACTOSE- N-ACETYGALACTOSAMINE- GALACTOSE

FATTY ACID N-ACETYLNEURAMINIC ACID N-ACETYLNEURAMINIC ACID

 ENZYME REPLACEMENT THERAPY IN

PATIENTS WITH TYPE 2 GAUCHER DISEASE

No benefit of intravenous glucocerebrosidase on brain

WOULD DIRECT INTRACEREBRAL INJECTION

OF GLUCOCEREBROSIDASE BE EFFECTIVE?

(CONVECTION-ENHANCED DELIVERY)
 Safety and Distribution of Mannose-terminal
Glucocerebrosidase Injected into the Brain of Normal Rats

G.C. Zirzow et al. Neurochemical Res 1999; 24: 301

NEURONAL UPTAKE OF INTRA-CEREBRALLY

ADMINISTERED GLUCOCEREBROSIDASE
Safety Study of Intracerebrally Injected Glucocerebrosidase

in Non-human Primates

R. Lonser et al, Annals of Neurology 2005; 57: 543

 TREATMENT STRATEGIES

3. SUBSTRATE REDUCTION THERAPY

 REDUCE THE FORMATION OF
GLUCOCEREBROSIDE

SPHINGOSINE + UDP–GLUCOSE SPHINGOSINE GLUCOSE + UDP

FATTY ACID FATTY

ACID
Glucocerebroside

UDP-GLUCOSE = URIDINE DIPHOSPHATE GLUCOSE

= SITE OF INHIBITION OF GLUCOSYLTRANSFERASE

Small Molecule Inhibitor of Glucocerebroside Formation

MIGLUSTAT
 SUBSTRATE DEPLETION

Miglustat OGT 918 (Zavesca) has been approved for

the treatment of patients with type 1 Gaucher disease
for whom enzyme replacement therapy is not
appropriate.

(Cox T, Lachmann R, Hollak C, et al. Lancet 2000; 355: 1481)

Patient with
Type 3a Chronic
Neuronopathic Slow horizontal
Gaucher Disease eye movement
 SUBSTRATE DEPLETION

• Ongoing NIH Investigation of OGT 918 in

Patients with Type 3 (Chronic Neuronopathic)
Gaucher Disease Who Also Receive Enzyme
Replacement Therapy to Control the Systemic
Manifestations of the Disease
 TREATMENT STRATEGIES

4. MOLECULAR CHAPERONE THERAPY

GM1-GANGLIOSIDOSIS
GM1
Gangliosidosis
Phenotypes
Infantile

Juvenile Chronic Adult Adult

 Accumulation of Ganglioside GM1

SPHINGOSINE-GLUCOSE-GALACTOSE-N-ACETYGALACTOSAMINE-GALACTOSE

FATTY ACID N-ACETYLNEURAMINIC ACID

 Enzymatic Defect in GM1-Gangliosidosis

SPHINGOSINE-GLUCOSE-GALACTOSE-N-ACETYGALACTOSAMINE-GALACTOSE

FATTY ACID N-ACETYLNEURAMINIC ACID

-galactosidase deficiency
Okada and O’Brien 1968
CHEMICAL CHAPERONE THERAPY FOR BRAIN
PATHOLOGY IN GM1-GANGLIOSIDOSIS
CREATED A MOUSE MODEL WITH THE
JUVENILE PHENOTYPE OF GM1-
GANGLIOSIDOSIS BY CHANGING
ARGININE AT POSITION 201 OF
-GALACTOSIDASE TO CYSTEINE
(R201C)
 Chemical Chaperone

N-0ctyl-4-epi--valienamine (NOEV)

J. Matsuda et al. Proc Natl Acad Sci USA 2003; 100: 15912
EFFECT OF N-OCTYL-4--VALIENAMINE (NOEV)
ON -GALACTOSIDASE ACTIVITY IN CULTURED
MURINE FIBROBLASTS
Additions
None 0.2 mM NOEV
Fold
(nmols/h/mg protein)
Wild type 68 79
1.2

Juvenile GM1 23 116

5.1
REDUCTION OF GM1 IN THE BRAIN OF MICE
WITH THE JUVENILE PHENOTYPE OF GM1-
GANGLIOSIDOSIS WITH THE NOEV

CHAPERONE IN THE DRINKING WATER

Ganglioside GM1
? Chaperone Therapy for Gaucher
Disease

N-Octyl--valienamine up-regulates
activity of

F213I mutant -glucosidase in cultured

cells:
a potential chemical chaperone therapy
for
Gaucher disease.
FABRY DISEASE
PRINCIPAL ACCUMULATING LIPID IN FABRY DISEASE

CERAMIDETRIHEXOSIDE

SPHINGOSINE – GLUCOSE – GALACTOSE - GALACTOSE

FATTY ACID
 ENZYMATIC DEFECT IN FABRY DISEASE
CERAMIDETRIHEXOSIDASE
(Alpha-Galactosidase A)

SPHINGOSINE – GLUCOSE – GALACTOSE - GALACTOSE

FATTY ACID

R. O. Brady et al New Engl J Med 1967

ENZYME REPLACEMENT THERAPY IN FABRY

PATIENTS HAS PRODUCED DISTINCT BENEFIT,

BUT NOT ALL OF THE MANIFESTATIONS ARE

COMPLETELY RESOLVED
MOLECULAR CHAPERONE THERAPY FOR
FABRY DISEASE
TREATMENT OF METABOLIC STORAGE DISORDERS

5. GENE THERAPY

1. GAUCHER DISEASE

Retroviral transduction of patients’ autologous bone-

marrow stem and progenitor cells -- 2 patients no clinical

benefit

2. FABRY DISEASE

Intravenous injection of adeno-associated virus with

human -galactosidase A gene into -galactosidase A

knock-out mice -- spectacular results - ?neoplasms

 CURRENT AND FUTURE STRATEGIES FOR THE

TREATMENT OF METABOLIC STORAGE DISORDERS

1. BONE MARROW TRANSPLANTATION

2. ENZYME REPLACEMENT THERAPY

3. SUBSTRATE REDUCTION THERAPY

4. MOLECULAR CHAPERONE THERAPY

5. GENE THERAPY ?