Breaking the Biological Barriers to Plant Made Pharmaceuticals (PMPs)

mutebabiesBiotechnology

Dec 6, 2012 (4 years and 4 months ago)

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Breaking the Biological Barriers
to Plant Made Pharmaceuticals
(PMPs)

(Nov. 02, 2007)


Kyun Oh Lee, Ph.D.

Division of Applied Life Science & EB
-
NCRC

Gyeongsang National University

Increasing demands for recombinant
proteins after human genome project


Therapeutic proteins


Diagnostic Proteins


Hormones


Antibodies


Vaccines


Cosmetics Additives


Feed Additives

Completed
in 2003

Biopharmaceutical

Year of approval

Condition or disease treated

recombinant somatrem (human growth hormone) for injection

1985

human growth hormone (hGH) deficiency in children

recombinant interferon alfa
-
2b

1986

hairy cell leukemia

1988

genital warts


1988


Kaposi’s sarcoma


1991


hepatitis C

1992

hepatitis B

recombinant interferon alfa
-
2a

1986

hairy cell leukemia

1988

Kaposi’s sarcoma

Muromonab
-
CD3

1986

reversal of kidney transplant rejection

1993

reversal of heart and liver transplant rejection

recombinant hepatitis B vaccine

1986

hepatitis B prevention

recombinant somatropin for injection

1987

human growth hormone (hGH) deficiency in children

Alteplase

1987

acute myocardial infarction

1990

acute massive pulmonary embolism

Epoetin alfa (rEPO, Epogen)

1989

anemia of chronic renal failure

recombinant hepatitis B vaccine

1989

hepatitis B

interferon alfa
-
n3

1989

genital warts

adenosine deaminase

1990

severe immunodeficiency in infants

interferon gamma
-
1b

1990

chronic granulomatous disease

filgrastim (rG
-
CSF)

1991

neutropenia caused by chemotherapy

1994

bone marrow transplantation

1994

chronic, severe neutropenia

sargramostim (yeast
-
derived GM
-
CSF)

1991

bone marrow transplantation

Aldesleukin (interleukin
-
2)

1992

renal cell carcinoma

Protein
-
b
ased therap
eutics

Protein
-
b
ased therap
eutics

Biopharmaceutical

Year of approval

Condition or disease treated

Staumonab pendetide (OncoScint)

1992

colorectal and ovarian cancers

recombinant antihemophiliac factor (rAHF)

1992

hemophilia A

recombinant interferon beta
-
1b

1993

relapsing, remitting multiple sclerosis

dornase alpha (Pulmozyme)

1993

cystic fibrosis

Pegaspargase

1994

lymphoblastic leukemia

imiglucerase for injection (Cerezyme, recombinant
lucocerebrosidase)

1994

Gaucher’s disease

abciximab (ReoPro)

1994

prevention of blood clotting

Humulin 70/30 (biosynthesized human insulin)

1996

diabetes mellitus

Humatrope

1996

adult
-

or childhood
-
onset growth hormone deficiency

Serostim

1996

AIDS wasting associated with catabolism, weight loss, or cachexia

Saizen

1996

human growth hormone deficiency in children

Nutropin

1996

Turner’s syndrome

Infanrix (vaccine)

1997

diphtheria and tetanus toxoids absorbed

coagulation factor IX (recombinant)

1997

factor IX deficiencies (Christmas disease)

Novolin 70/30 (biosynthesized human insulin)

1997

diabetes mellitus

Velosulin human (semisynthesized purified human insulin)

1997

diabetes mellitus

Genotropin

1997

human growth hormone deficiency in adults

Oprelvekin (Neumega)

1997

prevention of thrombocytopenia

Rituximab (Rituxan)

1997

follicular B
-
cell non
-
Hodgkin’s lymphoma

Becaplermin (Regranex Gel)

1997

diabetic foot ulcers

daclizumab (Zenapax)

1997

acute renal allograft rejection

Nutropin AQ

1997

human growth hormone deficiency in adults

recombinant human insulin

1982

diabetes mellitus

2006 Top
-
selling Classes of Biological
Products

No.

Class of Products

2006 sales
(US$ bln.)

Selected Branded Products

1

Erythropoietins

11.94

Aranesp, Procrit Eprex, Epogen, Neo
-
Recormon, ESPO

2

Major cancer antibodies

10.62

Rituxan/MabThera, Herceptin, Avastin, Erbitux, Vectibix

3

Anti
-
TNF antibodies

10.28

Enbrel, Remicade, Humira

4

Insulin and insulin analogs

8.97

Humalog, Humulin, Lantus, Levemir, Novorapid, Actrapid,
Novolin

5

Rec. coagulation factors

4.71

Novoseven, Kogenate, Helixate, Refacto, Advate, Recombinate,
Benefix

6

Interferon beta

4.40

Avonex, Rebif, Betaferon / Betaseron

7

G
-
CSF

4.36

Neulasta, Neupogen, Neutrogin, GRAN

8

Human growth hormone

2.47

Genotropin, Norditropin, Humatrope, Nutropin, Saizen,
Serostim

9

Interferon alpha

2.26

Pegasys, Peg
-
Intron, Intron A

10

Enzyme Replacement

1.71

Cerezyme, Fabrazyme, Aldurazyme, Myozyme, Replagal,
Naglazyme, Elaprase

11

Antiviral antibody

1.1

Synagis

12

Follicel stimulating

hormones

1.04

Gonal
-
f, Puregon

Total:

$63.83 (58



)

Source: La Merie Business Intelligence, February 2007

56.8
27.5
24.8
24.4
17
14.4
11.4
6.9
6.9
6.7
0
10
20
30
40
50
60
Cancer MAbs
Enzyme Replacement
TNF Mabs
Insulins
Coagulation Factors
Interferons beta
G-CSFs
hGH
Interferons alpha
EPO
Growth Rate (%)
Growth Rates (2006 vs 2005) of Top
-
selling Classes of Biological Products

Current production system
for
protein
-
based

therap
eutics

Chinese Hamster Ovar
y
(CHO)

Cells


Expensive


Not economically viable for some
drugs


Not safe from some pathogen
infection


Need to develop new cost
efficient substituting systems

Plants: a solution to the shortfall in
manufacturing capacity for new drugs

Molecule

Problematic

Plant Solutions

Hemoglobin


Blood demand
increase; Donors
decrease


Serotypes
incompatibility


S
afe
ty concern
(HIV; HCV; HBV)


Large scale production


No concern about serotypes


No human pathogens

Antibody


Expensive


Production
capacity limited


Cost reduction


Large scale production

Enbrel

(
TNF blocker
)


Capacity shortage


Large scale production

Factor VIII

(
C
oagulation factor
)


Worldwide
shortage (40% of
hemophilia patients
have access to the
product)


Large scale production: accessibility to all
patients

Insulin


Too expensive for
non industrialized
countries


Cost reduction: accessibility for non
industrialized countries

Plant m
olecular
f
arming

(Biopharming)?


The use of modified plants to produce
therapeutically
active proteins or
industrial products


The end product of crop harvests is not food, feed or fi
bers, but plant
-
made pharmaceuticals

(PMPs)


Biopharming industry may be worth $100 billion by 2015




수매가격


50,000

⼴〠歧k

Plant Products

I.
Plant
-
derived pharmaceuticals (non
-
GE)


Over 120 pharmaceutical products currently in use are
derived from plants, mainly from tropical forest species

II.
Plant
-
made pharmaceuticals and industrial products (GE)

1.
Pharmaceuticals


Recombinant human proteins


Therapeutic proteins


Enzymes


Antibodies (plantibodies)


Vaccines

2.
Industrial products


Proteins


Enzymes


Modified starches


Oils


Waxes


Plastics

Why use plants?

1.
Plants are the cheapest, most abundant source of protein on the
planet:
low
cost


2.
Plant provide
low upstream production costs
:
low
cost

3.
Plants, as eukaryotes, can express and process most prokaryotic
and eukaryotic proteins:
stability

4.
Plant provide scalable production capacity and flexibility:
easy
scale up

5.
Plant seed permit stockpiling of inexpensive inventory:
good
storage

6.
Plants are f
ree
from

animal and human pathogens
:
safety

Plant Golgi


-
1,2
-
Mannosidase I

N
-
acetyl
-
glucosaminyl
-
transferase I (
GnT
-
I)

UDP
-
GlcNAc

Transporter


-
Manno獩dase 䥉




䙵c
-
呲an獦era獥

GnT
-


Xyl
-
呲an獦erase

N
-
a捥tyl
-
Glu捯獡minidase

Comp汥x 呹pe

F

F

F

X

X

F

Golgi complex


1,2
-
mannosidase I

GnT1

UDP
-
GlcNAc

Transporter


-
manno獩dase 䥉

GnT
-
II

UDP
-
Gal

Transporter

GalT

SAT

CMP
-
NeuAc

synthetase

Complex Type glycan




수매가격


50,000

/㐰g

Comparison of pharmaceutical expression
systems


Expression
system

Advantages

Disadvantages

Applications

Cost per
gram

Bacteria

Established regulatory track; well
-
understood genetics; cheap and easy to
grow

Proteins not usually secreted;
contain endotoxins; no
posttranslational modifications

Insulin (
E. coli
; Eli Lilly);
growth hormone (Genentech);
growth factor; interferon

$50

100

Yeast

Recognized as

safe;


long

history of use; fast; inexpensive;
posttranslational modifications

Overglycosylation can ruin
bioactivity; safety; potency;
clearance; contains
immunogens/antigens

Beer fermentation;
recombinant vaccines;
hepatitis B viral vaccine;
human insulin

Insect
cells

Posttranslational modifications;
properly folded proteins; fairly high
expression levels

Minimal regulatory track; slow
growth; expensive media;
baculovirus infection (extra
step); mammalian virus can infect
cells

Relatively new medium;
Novavax produces virus
-
like
particles

Mammalian

cells

Usually fold proteins properly; correct
posttranslation modifications; good
regulatory track record; only choice
for largest proteins

Expensive media; slow growth;
may contain allergens/
contaminants; complicated
purification

Tissue plasminogen activator;
factor VIII (glycoprotein);
monoclonal antibodies
(Hercepin)

$500

5,000

Transgenic
animals

Complex protein processing; very high
expression levels; easy scale up; low
-
cost production

Little regulatory experience;
potential for viral contamination;
long time scales; isolation/GMPs
on the farm

Lipase (sheep, rabbits; PPL
Therapeutics); growth
hormone (goats; Genzyme);
factor VIII (cattle)

$20

50

Transgenic
plants

Shorter development cycles; easy seed
storage/scaling; good expression levels;
no plant viruses known to infect
humans

Potential for new contaminants
(soil fungi, bacteria, pesticides);
posttranslational modifications;
contains possible allergens

Cholera vaccine (tobacco;
Chlorogen, Inc.); gastric lipase
(corn; Meristem); hepatitis B
(potatoes; Boyce Thompson)

$10

20

Note. Data from BioPharm International (2003).


Impact on the environment;

-

Food supply contamination; accidental or
intentional gene flow
,
cross
-
pollination
,

-

Outcrossing

(
non
-
target species near field
sites e.g. butterflies, bees, etc)


Allergenicity, antigenicity, immunogenicity


Inappropriate use of plant by
-
products


Occupational exposure


Expression levels


Reproducibility


Differences in glycosylation patterns


Biological containment


Regulatory issues

Risks and concerns

State of the art


~
25 companies world wide are developing plants
for molecular farming


16 plant based products are under clinical trials


commercial products are launching

PMPs in clinical trial phase II

Plant

Active substance

Use

Country, company

Tobacco

Monoclonal antibody

Dental caries prophylaxis

USA, Planet Biotechnology

Maize

Enzyme, gastric lipase

Cystic fibrosis, pancreatitis

France, Meristem Therapeutics

Tobacco

Antibody, cancer vaccine

Non
-
Hodgkin lymphoma

USA, Large Scale Biology

Duckweed

Alpha interferon

Hepatitis C

USA, Biolex

Potato

Antigen

Hepatitis B

USA, Arizona State University

Arabidopsis

Protein, intrinsic factor

Diagnostic test, Absorption of vitamin 12

Denmark, Cobento Biotech

Tobacco

Antibody

Colds caused by rhinoviruses

USA, Planet Biotechnology

other PMPs in clinical trial phase I

Plant

Product/ active substance

Use

Country, company

Lettuce

Vaccine

Hepatitis B

Polish Science Academy

Potato

Vaccine

Norwalk virus

USA, Arizona State University

Spinach

Vaccine

Rabies

USA, Thomas Jefferson University, Philadelphia

Maize

Protein, lactoferrin

Dry eye syndrome

France, Meristem Therapeutics

Potato

Vaccine

Travel diarrhoea

USA, Arizona State University

Mais

Vaccine

Travel diarrhoea

USA, Arizona State University

Mais

Vaccine

Travel diarrhoea

USA, ProdiGene

Duckweed

Alpha interferon

Hepatitis B and C

USA, Biolex

n.s.

Monoclonal antibody

Lessening the side
-
effects of
chemotherapy

USA, Planet Biotechnology

From: TAB report 104, 2005; Armin Sp
ö
k, Molecular farming on the rise, 2006

PMPs in or nearing clinical trials

Examples of current plant
-
made
pharmaceutical research

Alfalfa

Plasma proteins

Arabidopsis

Human intrinsic factor (vitamin B12 uptake)

Corn

Anti
-
HIV and anti
-
Herpes simplex antibodies, Microbicides for pulmonary
infection

MABs for cancer, arthritis, and other autoimmune diseases like Crohn's disease

Vaccines for Hepatitis B transmission and Traveler's disease

TGEV for animal health, Aprotinin for blood loss and heart surgery

Vaccines and antibodies for animal disease prevention, Antibodies

Lemma

Human plasminogen for peripheral arterial occlusion, Alpha interferon

Moss

Factor IX for treatment of haemophilia B

Rice

Alternatives to antibiotics in poultry diets

Lysozyme for gastrointestinal health, topical infections and inflammations

Safflower

Pharmaceuticals and oil
-
body
-
based products for oral and dermal delivery

Spinach

Protective antigen for vaccine against
Baccillus anthracis

Tobacco

TGF
-
b glucocerebrosidase for Gaucher's disease

Alpha galactosidase for enzyme replacement therapy

IgGs for the prevention of dental decay, prevention of common cold, and
neutralization of chemotherapeutic drug toxicity

GAD 7 cytokines for type 1 Diabetes, IL
-
10 for inflammatory bowel disease

Glycoprotein B of human cytomegalovirus (hCMV)

Tobacco, Corn

Gastric lipase for cystic fibrosis

Lactoferrin for gastro
-
intestinal infection and dry eye syndrome

Tomato, Potato,
Potato tuber

Edible vaccines against
E.coli,
Norwalk virus, Hepatitis B, Antimicrobial peptides

InfoSource is produced by Ag
-
West Biotech Inc 2003.

USA, 108,
59%
Canada, 51,
28%
Eu, 24, 13%
Worldwide release application for PMP plants

Maize
43%
Tobacco
19%
Oilseed rape
9%
Soya
7%
Rice
5%
Others
17%
The types of plant used for PMPs


Erythropoietein


Human Growth Factor


β
-
Interferon


Malaria epitopes


Streptococcus Surface
Antigen IgA (Dental Caries)


Carcinoembryonic Antigen


Colon Cancer Antigens


Interleukin 10 (Chrohn’s
Disease; Inflammatory
Bowel Syndrome)


Glucocerebrosidase
(Gaucher’s Disease)


Interleukin 4


Urokinase (Breaks Blood
Clots)


Human Serum Albumin


Rabies Antigens


Hepatitis B Surface
Antigen


Rotavirus VP6


Labile Eneterotoxin
(Botulism)


Zonna pellucida ZB3 Protein
(Contraceptive)


Gastric Lipase (Cystic Fibrosis)


Creatine Kinase


Protein C (Anticoagulant)


Neutropenia (Granulocyte
Macrophage Colony Stimulating
Factor)


Epidermal Growth Factor


α and β Hemoglobin


Angiotensin Converting Enzyme
(Hypertension)


Insulin Like Growth Factor
(Diabetes)


Tissue Necrosis Factor (Rheumatoid
Arthritis)


HIV
-
1 Peptide


Lactoferrin


Substance P (Neuropeptide)


Animal Pharmaceuticals (Vaccines)


Feline Parvovirus (Panleukopenia)


Canine Parvovirus


Bovine Foot and Mouth


Measles


Aprotinin (Protease
Inhibitor)


α
-
Galactosidase (Fabry’s
Disease)


Interferon α 2a and 2b


Personalized Non
-
Hodgkin’s
Lymphoma Vaccines


Papilloma Virus Vaccines


Lysosomal Acid Lipase
(Woman’s Disease;
Atherosclerotic Plaques)


Personalized Vaccines for
Follicular Lymphoma


α Trichosanthin (HIV)


Diagnostics for Ovarian
Cancer


Diagnostics for Ecclampsia


Biomarkers for Alzheimers
Disease

Status of Biotech Pharmaceutical
Production

Commercial Pharma/Industrial

Crop Products

Product


Trade Name


Pharma Crop

Source of
Genes


Commercial
Purpose

¹


Company
Producing


Company Selling

²

(product number)

Aprotinin

AproliZean

Corn

Cow


Research and
manufacturing


ProdiGene


Not available

Aprotinin

Apronexin



Tobacco ³

Cow


Research and
manufacturing


Large

Scale

Bi
ology


Sigma Chemical Company
(A6103)

Avidin

Recombinant
avidin


Corn


Chicken


Research and
diagnostic reagent


ProdiGene


Sigma Chemical

Company
(A8706)


B
-
glucuronidase
(GUS)

Not available

Corn


Bacteria


Research and
diagnostic reagent


ProdiGene


Not available

Trypsin

TrypZean


Corn


Cow


Research
and

manufacturing


ProdiGene


Sigma Chemical Company
(T3568 and

T3449)


Lactoferrin

Not available


Rice


Human


Research


Ventria
Bioscience


Sigma Chemical

Company
(L4040)


Lysozyme

Not available


Rice


Human


Research


Ventria
Bioscience


Sigma Chemical Company
(L1667)


Notes

(1)
Information on specific commercial uses of pharmaceutical and industrial

crop products is not available to the public. Some have

potential applications for human or veterinary medicine but none has been approved by the Food and Drug Administration, which

regulates drugs.

(2)
Most of these products have been commercialized through Sigma Chemical Company, which specializes in products for research
and diagnostic purposes. AproliZean and B
-
glucuronidase have been commercialized according to ProdiGene documents, but their
current availability is unknown.

(3)
Unlike pharmaceutical corn, tobacco itself is not genetically engineered. Rather, a tobacco mosaic virus is engineered to con
tai
n
a gene for aprotinin. The engineered virus then produces aprotinin in tobacco plants infected with the virus.

Representative diseases potentially
treatable with PMPs


Alzheimer’s disease


Arthritis


Cancers


Chronic obstructive pulmonary disease


Crohn’s disease


Cystic fibrosis


Diabetes


Heart disease


Hepatitis C


HIV


Kidney disease


Multiple sclerosis


Obesity


Spinal cord injuries

Source: Phillips 2003

Golgi complex


1,2
-
mannosidase I

GnT1

UDP
-
GlcNAc

Transporter


-
manno獩dase 䥉

GnT
-


UP
-
Gal

呲an獰orter

Ga汔

SAT

CMP
-
NeuAc

獹nt桥ta獥

Comp汥 呹pe g汹捡n

Host development

Recombinant DNA

technology

Transformation &

regeneration

Production of PMPs

Purification

Cell culture and harvest

Cultivation

The process of m
olecular
f
arming

Strategies for efficient Molecular
farming system: synthetic biology


Synthetic Biology: the design and construction of new biological parts, devices,
and systems, and the re
-
design of existing, natural biological systems for useful
purposes


For high yield protein production:


Fast growing, high yield, stress and pathogen resistant host development


Strong tissue specific promoter


Gene silencing inhibition


Codon usage optimization


Using translation enhancer


Efficient vector system development



Efficient purification:


Use of tag or fusion protein for efficient purification


Secretion of protein using signal sequence


Development of
efficient protein purification technique



Stable & safe protein:


Glycosylation control and humanization


ER stress (Unfolded Protein Response) control


Use of chaperone and redox proteins


Use of plant secretory and vacuolar systems

Plant expression systems for molecular
farming

1.
Vegetative systems


2.
Seed
-
based systems


Seed have evolved to naturally store large amounts of
lipid, protein and carbohydrate


Seed desiccation allows intact storage for years


Allow for an inventory of an intermediate between
production and processing


Uncouples harvesting from processing

Target product selection


To

use

plants

effectively

we

should

choose
:


Products

with

very

large

volume

applications

(>
100
kg,

eg
.

Lactoferrin,

lysozyme)


Products

which

are

already

well
-
known
,

even

generics

(eg
.

insulin,

growth

hormone



SemBioSys
;

α
-
interferon



BioLex)


Products

which

have

proven

difficult

to

make

in

other

systems

(eg
.

sec

lgAs



Planet

Biotech

)



Products

that

do

not

make

sense

currently


Products

that

are

required

in

small

volumes

(eg
.

Erythropoietin
-
Amgen,

approximately

8

~

10

kg

per

year

=

$
3

billion)


Products

that

require

glycosylation

for

function

(certain

lgGs

require

sugars

for

effecter

function)


Products

requiring

unusual

post
-
translational

modifications

(eg
.

amidation,

sulfonation

etc)

Suitable host system for protein production


WT

Vector

8

10

Overexpressed

5 weeks

7 weeks

WT

Vector

8

10

Overexpressed

Lumen

Cytoplasm

Endoplasmic

Reticulum

Golgi Complex

N
-
Acetylglucosamine

Galactose

Sialic acid

Mannose

Fucose

Glucose

Dolichylphosphate

Protein

Polypeptide chain

Modifications of
N
-
linked oligosaccharides
& protein
N
-
glycosylation

N
-
glycosylation in ER; Glc
3
Man
9
GlcNAc
2

glycans to Man
8
GlcNAc
2

glycans

: Folded peptide chain

: Glucose

: GlcNAc

: Mannose

: Phosphoric acid

Endoplasmic

Reticulum

Dolichol

Ribosome

mRNA

Oligosaccharyl

Transferase

Calnexin

Folding


-
Gl畣us楤慳e I



-
Gl畣us楤慳e II

E删

-
䵡湮os楤ise

Golgi complex

Glc
3
Man
9
GlcNAc
2

Man
8
GlcNAc
2

Different
N
-
glycosylation in Golgi
-
complexes

Yeast Golgi


-
1,3
-
Mannosyl

Transferase

(
MNN1
)


-
1,6
-
Ma湮潳nl

T牡湳ne牡se (
OCH1
)

Mannan Type


-
1,2
-
Mannosyl

Transferase

(
MNN2,5?
)

Mannose
-
6
-

Phosphate

Synthesis

(
MNN4,6
and others)

n

Endoplasmic

Reticulum

Dolichol

Ribosome

mRNA

Oligosaccharyl

Transferase

Calnexin

Folding


-
䝬畣潳i摡de I



-
Glucosidase II

ER

-
Ma湮潳ndase

Glc
3
Man
9
GlcNAc
2

Man
8
GlcNAc
2

: Sialic acid

: Galactose

: Folded peptide chain

: Glucose

: GlcNAc

: Mannose

: Phosphoric acid

Mammalian Golgi


-
1,2
-
Mannosidase I

GnT
-
I

UDP
-
GlcNAc

Transporter


-
Ma湮潳楤ase II

GnT
-
II

UDP
-
Gal

Transporter

GalT

SAT

CMP
-
NeuAc

synthetase

Complex Type

Plant Golgi


-
1,2
-
Mannosidase I

N
-
acetyl
-
glucosaminyl
-
transferase I (
GnT
-
I)

UDP
-
GlcNAc

Transporter


-
Ma湮潳楤ase II




䙵F
-
T牡湳ne牡se

䝮G
-


Xyl
-
T牡湳ne牡se

N
-
acetyl
-
䝬畣潳ami湩摡de

Complex Type

F

F

F

X

X

F

Glucocerebrosidase production using
Arabidopsis and rice

Gaucher disease:
most common lysosomal
storage disorder

Glucocerebrosidase deficiency

(on chromosome 1, at 1q21)

Clinical Features

Found by
Phillipe Gaucher

in 1882

Neuraminidase

GC

β
-
Galactosidase

β
-
N
-
acetylglucosaminidase

GC

Glucocerebrosidase

N
-
acetylglucosamine

Mannose

Galactose

N
-
acetylneuraminic acid

Fucose

GC

GC

GC

(60,43 kDa)

(67,00 kDa)

Developed by

Roscoe O. Brady

in 1973

1.
Ceredase
®
:

a

placentally
-
derived

form,

1991

2.
Cerezyme
®
:

a

recombinant

form,

1994

3.
Approved

for

type

1

Gaucher

disease

4.
Targeting

to

the

appropriate

cells

is

difficult


5.
Does

not

provide

a

long

life

cure

6.
Too

expensive
.

Enzyme replacement therapy of Gaucher
disease

CR

FNII

CRDs

TM

CT

CR: Cysteine rich domain

Mediates Ca
2+

independent binding to sulphated N
-
linked
sugars


FNII: Domain containing fibronectin type II repeats




CRDs: Carbohydrate recognition domains

Mediates Ca 2+ dependent binding to carbohydrate

Sugar specificity (monosacchride level):

L
-
Fuc=D
-
Man>D
-
GlcNac>>>D
-
Gal


CRD 4,5 and 7
are required for high affinity binding for
complex sugars with terminal mannose residues




TM and CT: Transmembrane domain and cytoplasmic tail

Contain signals that mediate endocytosis and phagocytosis

4

5

7

1

3

2

6

8

Mannose
-
specific

glycoprotein

uptake

of

macrophages

via

mannose
-
specific

receptor

Genetic

manipulation

of

plants

for

mannose
-
terminated

glycan

production

Endoplasmic

Reticulum

Dolichol

Ribosome

mRNA

Oligosaccharyl

Transferase

Calnexin

Folding


-
䝬畣潳i摡de I



-
䝬畣潳i摡de II

E删

-
Ma湮潳ndase

: Sialic acid

: 䝡lact潳o

: 䙯F摥搠灥灴i摥dc桡楮

: 䝬畣潳e

: 䝬cNAc

: Ma湮潳n

: P桯獰桯物c acid

Glc
3
Man
9
GlcNAc
2

Man
8
GlcNAc
2

Mammalian Golgi


-
1,2
-
Mannosidase I

GnTI

UDP
-
GlcNAc

Transporter


-
Ma湮潳楤ase II

GnT
-
II

UDP
-
Gal

Transporter

GalT

SAT

CMP
-
NeuAc

synthetase

Genetically manipulated

plant Golgi


-
1,2
-
Mannosidase I

Δ
gnt1

Mannose

Type

X

Plant Golgi


-
1,2
-
Mannosidase I

N
-
acetylglucosaminyl
-
transferase I (
GnTI)

UDP
-
GlcNAc

Transporter


-
Ma湮潳楤ase II




䙵F
-
T牡湳ne牡se

GnT
-
II

Xyl
-
Transferase

F

F

F

X

F

Human

proteins

β
(1,4)

β
(1,4)

β
(1,4)

β
(1,4)

β
(1,2)

β
(1,2)

α
(1,3)

α
(1,6)

α
(1,6)

α
(2,6)

α
(2,3)

β
(1,3)

Plant

proteins

α
(1,4)

α
(1,4)

β
(1,4)

β
(1,4)

β
(1,2)

β
(1,2)

α
(1,3)

α
(1,3)

α
(1,6)

β
(1,2)

β
(1,3)

GlcNAc

Gal

NeuAC

Man

Xyl

Fuc

‘Lewis a’ epitope

Different
N
-
linked glycans structures

β
(1,3)

α
(1,4)

α
(1,4)

β
(1,3)

Human

proteins

β
(1,4)

β
(1,4)

β
(1,4)

β
(1,4)

β
(1,2)

β
(1,2)

α
(1,3)

α
(1,6)

α
(1,6)

α
(2,6)

α
(2,3)

β
(1,3)

Plant

proteins

α
(1,4)

α
(1,4)

β
(1,4)

β
(1,4)

β
(1,2)

β
(1,2)

α
(1,3)

α
(1,3)

α
(1,6)

β
(1,2)

β
(1,3)

GlcNAc

Gal

NeuAC

Man

Xyl

Fuc

β
(1,4)

β
(1,4)

β
(1,4)

β
(1,4)

β
(1,2)

β
(1,2)

α
(1,3)

α
(1,6)

α
(1,6)

α
(2,6)

α
(2,3)

Plant

proteins

Proteins from
β
1,2
-
xylt
,
α
1,3
-
fuct
,
(
β
1,3
-
galt
,
α
1,4
-
fuct
)
,

β
1,4
-
GalT
,

α
1,6
-
FucT
plant

Plant

proteins

β
(1,4)

β
(1,4)

β
(1,2)

β
(1,2)

α
(1,3)

α
(1,6)

Proteins from
β
1,2
-
xylt
,

α
1,3
-
fuct,
(
β
1,3
-
galt
,

α
1,4
-
fuct

)

plant

α
(1,3)

α
(1,6)

Plant

proteins

β
(1,4)

β
(1,4)

α
(1,3)

α
(1,6)

Proteins from
cgl
(
gnt1
)
plant

Genetic

manipulation

of

plants

for

humanized

glycan

production

1.
β
1,2
-
xylt
/
α
1,3
-
fuct
-
1
: (completed)


2.
β
1,2
-
xylt
/
α
1,3
-
fuct
-
2
: (completed)


3.
α
1,3
-
fuct
-
1
/
α
1,3
-
fuct
-
2
: (completed)


4.
β
1,2
-
xylt
/
α
1,3
-
fuct
-
1
/
α
1,3
-
fuct
-
2
: (completed)


5.
β
1,2
-
xylt
/
α
1,3
-
fuct
-
1
/
α
1,3
-
fuct
-
2
/
α
1,4
-
fuct
: (completed)


6.
β
1,2
-
xylt
/
α
1,3
-
fuct
-
1
/
α
1,3
-
fuct
-
2
/
β
1,3
-
galt
/
α
1,4
-
fuct
:


(under construction)

Progress

of

genetic

manipulation

in

Arabidopsis

Codon usage optimization for high
glucocerebrosidase expression in plants

Strong seed specific promoter and
signal
sequence for efficient glycosylation



Pea vicilin promoter (7S storage
protein)


Rice 1Cys
-
Prx promoter (aleurone
layer specific)


Rice amylase 3D (germination
specific)


Arabidopsis cruciferin3, 12S
seed storage protein, contains an
efficient vacuolar
-
targeting
signal in the N
-
terminal region


Rice a
-
globulin, 2S albumin
-
type
seed storage protein, contains an
efficient vacuolar
-
targeting
signal in the N
-
terminal region


Secretion signal sequences

The

plant

secretory

and

vacuolar

systems
.

Indicated

are

endoplasmic

reticulum

(ER),

Golgi,

cell

wall

(CW),

protein

storage

vacuole

(PSV)

and

lytic

vacuole

(LV)
.

Plant

Molecular

Biology

38
:

127

144
,

1998
.

Lytic vacuole

Protein storage vacuole

Apoplasmic space

Unfolded protein response (UPR) in yeast

Ire1p

Hac1p

Unfolded proteins,
ER stress

UPR

nucleus

cytosol

HAC1
u

HAC1
i

Induction of ER chaperones, glycosylation/modification,

and secretion pathway genes

ER stress,
Unfolded proteins

S. cerevisiae

Ire1p

HAC1

mRNA
splicing

Transcriptional upregulation of ER chaperones,
glycosylation/modification,

and secretion pathway genes

Plants

Mammals

ATF6

Splicing of ATF6
cytosolic domain

PEK
-
1

elF
-
2
α

phosphorylation

Attenuation of
protein synthesis

Ire1
α

Ire1
β

Other Ire1


family members?

Xbp
-
1

mRNA
splicing

Up
-
regulation of
Xbp
-
1

mRNA

ATF6???

Ire1
α

Ire1
β

Xbp
-
1

mRNA???


An additional ER kinase
(PERK/PEK)


Alternative transcriptional
factors (ATF6, Xbp1)


ATF6 is a rER
transmembrane protein
with a luminal sensing
domain, and a C
-
terminal
cytosolic transcription
transactivation domain
subject to release by
proteolysis by S1P/S2P
proteases.

Transcriptional upregulation of ER
chaperones by
Xbp1 (Hac1) and ATF6

Strategies for efficient Molecular
farming system: synthetic biology


Synthetic Biology: the design and construction of new biological parts, devices,
and systems, and the re
-
design of existing, natural biological systems for useful
purposes


For high yield protein production:


Fast growing, high yield, stress and pathogen resistant host development


Strong tissue specific promoter


Gene silencing inhibition


Codon usage optimization


Using translation enhancer


Efficient vector system development



Efficient purification:


Use of tag or fusion protein for efficient purification


Secretion of protein using signal sequence


Development of
efficient protein purification technique



Stable & safe protein:


Glycosylation control and humanization


ER stress (Unfolded Protein Response) control


Use of chaperone and redox proteins


Use of plant secretory and vacuolar systems