Biotechnology and Genetic Engineering-PBIO 450 ... - Ohio University

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Chapter 10
-
Protein Therapeutics

Chapter 11
-
Nucleic Acids as Therapeutic Agents


Pharmaceutical proteins and enzymes


Monoclonal antibodies and recombinant antibodies


Nucleic acids (antisense RNA and oligonucleotides,
ribozymes, interfering RNAs or RNAi)


Gene therapy


Stem cells and therapeutic cloning

Table 10.1 Some recombinant proteins approved for
human use ($15 billion
-
2001)

Protein

Company

Disorder

Factor VIII

Baxter, Bayer

Hemophilia A

Factor IX

Genetics Institute

Hemophilia B

Tissue plasminogen
activator (TPA)

Genetech

Acute myocardial
infarction

Insulin

Eli Lilly, Novo Nordisk

Diabetes mellitus

Human growth
hormone

Eli Lilly,
Genetech
,
Upjohn, Novo Nordisk

GH deficiency in
children (dwarfism)

Erythropoietin

Amgen, Ortho Biotech

Anemia

DNase I

Genetech

Cystic fibrosis

Various interferons
(IFN)

Schering, Biogen, Chiron,

Genetech

Hepatitis B and C,
multiple sclerosis

Recombinant proteins
-
from
http://en.wikipedia.org/wiki/List_of_recombinant_proteins

-
10/1/08

Human recombinants that largely replaced animal or harvested from human types


Human growth hormone

(
rhGH
)
Humatrope

from
Lilly

and
Serostim

from
Serono

replaced
cadaver

harvested
human growth hormone


Human insulin

(
rhI
)
Humulin

from
Lilly

and
Novolin

from
Novo Nordisk

among others; largely replaced
bovine and porcine insulin for human therapy. Some prefer to continue using the animal
-
sourced
preparations, as there is some evidence that synthetic insulin varieties are more likely to induce
hypoglycemia unawareness
. Remaining manufacturers of highly
-
purified animal
-
sourced insulin include the
U.K.'s
Wockhardt

Ltd. (headquartered in India), Poland's
Polfa

Tarchomin

S.A., Argentina's
Laboratorios

Beta
S.A., and China's
Wanbang

Biopharma

Co.


Follicle
-
stimulating hormone

FSH replaced
Serono
's

Pergonal

which was previously isolated from post
-
menopausal female urine


Factor VIII

Kogenate

from
Bayer

replaced
blood

harvested factor VIII

Human recombinants with recombination as only source


Erythropoietin

(EPO)
Epogen

from
Amgen


Granulocyte colony
-
stimulating factor

(G
-
CSF)
filgrastim

sold as
Neupogen

from
Amgen
;
pegfilgrastim

sold as
Neulasta


alpha
-
galactosidase

A

Fabrazyme

by
Genzyme


alpha
-
L
-
iduronidase

(
rhIDU
;
laronidase
)
Aldurazyme

by
BioMarin

Pharmaceutical

and
Genzyme


N
-
acetylgalactosamine
-
4
-
sulfatase

(
rhASB
;
galsulfase
)
Naglazyme

(TM) by
BioMarin

Pharmaceutical


DNAse

Pulmozyme

by
Genentech


Tissue
plasminogen

activator

(TPA)
Activase

by
Genentech


Glucocerebrosidase

Ceredase

by
Genzyme


Interferon

(IF) Interferon
-
beta
-
1a as
Avonex

from
Biogen

Idec;
Rebif

from
Serono
; Interferon beta
-
1b as
Betaseron

from
Schering


Insulin
-
like growth factor 1

(IGF
-
1)

Animal recombinants


Bovine
somatotropin

(
bST
)


Porcine
somatotropin

(
pST
)


Bovine
Chymosin


Expression systems are based on the insertion of a gene into a host cell for its translation and expression into protein.

Host cells include :


Bacteria
-

e.g.
Escherichia coli (
E.coli
), Bacillus
subtilis

(
B.
subtilis
)


Yeast


Cultured insect cells


Cultured mammalian cells

The choice of cell type used depends upon the protein to be expressed.

All require DNA to be cloned into the an appropriate vector.

Advantages of bacterial cells

simple physiology

short generation times, as bacteria grow and multiply rapidly

large yields of product
-

up to 10 % of mass (low cost)

With
B.
subtilis

and some others, it is possible to induce secretion of a gene product into the surrounding medium. This method is in use in t
he

pharmaceutical industry in the production of hormones such as insulin and human growth hormone.

Disadvantages of bacterial cells

The expressed proteins often do not fold properly and so are biologically inactive.

The
synthesised

protein is often toxic to bacteria preventing the cell cultures from reaching high densities. A solution to this problem is t
o
incorporate an inducible promoter, which may be turned on to transcribe the inserted gene after the culture has been grown

Lack of enzymes responsible for post
-
translational modifications (effect on function of proteins),
eg

if the protein to be expressed is a
glycoprotein, there is not apparatus in the bacterium to 'stick on' the necessary sugar residues.

Advantages of yeast cells

Yeast is a simple eukaryote and performs many of the post
-
translational modifications required for human proteins

Can be induced to secrete certain proteins into the growth medium for harvesting
-

e.g. Hepatitis B virus (HBV) vaccine.

Disadvantages of yeast cells

Presence of active proteases that degrade foreign (expressed) proteins, thereby reducing their yield (a solution to this prob
lem

is the
construction of yeast strains from which the protease genes have been deleted).

Insect Cells
-
Expression of foreign proteins in insect cells through incorporation of their genes into
baculovirus

vectors

Advantages of insect cells

High level of expression

Correct folding

Post
-
translational modifications similar to those in mammalian cells

Cost, though more than for culturing bacteria and yeast, less than for mammalian cells e.g. potential vaccine for AIDS virus
pro
duced by
expression of one of the HIV
glycoproteins

with this system

Disadvantages of insect cells

More difficult to work with

Expensive

Slow generation time

Not suitable for proteins with repetitive sequences

Cloning and expression of a foreign protein in a suitable host

Use of an appropriate expression vector and host

Example: A simple
E. coli

expression vector
utilizing the
lac

promoter
. (a) The
expression vector plasmid contains a
fragment of the
E. coli

chromosome
containing the
lac

promoter and the
neighboring
lacZ

gene. In the presence of
the lactose analog IPTG, RNA polymerase
normally transcribes the
lacZ

gene,
producing
lacZ

mRNA, which is translated
into the encoded protein,
b
-
galactosidase
.
(b) The
lacZ

gene can be cut out of the
expression vector with restriction enzymes
and replaced by the Granulocyte
-
Colony
Stimulating Factor
G
-
CSF
cDNA
. When the
resulting plasmid is transformed into
E. coli

cells, addition of IPTG and subsequent
transcription from the
lac

promoter
produces G
-
CSF mRNA, which is translated
into G
-
CSF protein.

Table 10.3 Some therapeutic monoclonal antibodies
approved for human use

Type of antibody

Company

Therapeutic use

Mouse, Humanized

Ortho Biotech, Protein
Design, Hoffmann
-
LaRoche

Prevents kidney
transplant rejection

Chimeric

Centocor

Prevents blood clots

Chimeric

Genetech, Hoffmann
-
LaRoche

Non
-
Hodgkin
lymphoma

Humanized
(Herceptin)

Genetech

HER2
-
positive breast
cancers

Humanized

Am Home Prod, Celltech,
Schering, Millen. Pharm.

Certain leukemias

Humanized

Genetech

Asthma

Antibody Structure


Antibodies are immune system
-
related proteins
called immunoglobulins. Each antibody consists of
four polypeptides


two heavy chains and two light
chains joined to form a "Y" shaped molecule.




The amino acid sequence in the tips of the "Y"
varies greatly among different antibodies. This
variable region, composed of 110
-
130 amino
acids, give the antibody its specificity for binding
antigen. The variable region includes the ends of
the light and heavy chains. Treating the antibody
with a protease can cleave this region, producing
Fab or fragment antigen binding that include the
variable ends of an antibody.



The constant region determines the mechanism
used to destroy antigen. Antibodies are divided
into five major classes, IgM, IgG, IgA, IgD, and
IgE, based on their constant region structure and
immune function.

Fig. 10.40 Making
antibodies even
more effective
therapeutic agents:
two ways

Fig. 11.1 Inhibition of translation of specific RNA
by antisense nucleic acid molecules

Promoter


antisense cDNA


poly A addition signal

antisense oligonucleotide

mRNA

-
antisense

RNA complex

Fig. 11.8 Ribozymes: A. Hammerhead B. Hairpin

Fig. 11.13 RNA interference (RNAi)

A cellular nuclease binds to the dsRNA cleaving it into ssRNAs of 21
-
23 nucleotides each.

The nuclease
-
RNA oligonucleotide complex binds and cleaves specific mRNA.


dsRNA

Binding of dsRNA
-
specific nuclease

cleavage

mRNA is cleaved!

Nuclease
-
ssRNA complex

Hybridizes to mRNA

sense

antisense

Table 10.5 Human gene therapy

(# clinical trials 1990
-
1999)


AIDS (19)


Amyotrophic lateral
sclerosis


Cancer (280)


Cardiovasc. dis. (20)


Cystic fibrosis (24)


Familial
hypercholesterolemia


Gaucher disease (3)


Hemophilia A (2)


Hemophilia B (2)


Hunters disease


Multiple sclerosis


Muscular dystrophy


Rheumatoid arthritis


Severe combined
immunodeficiency (3)

Consider somatic vs germline gene therapy; the later is currently banned.

Note that gene therapy is limited to somatic cells and disorders that are

caused by a single gene.

Two types of gene therapy


Ex vivo

-
cells are removed from the body, the
gene of interest is inserted into them, the cells
are cultured to increase cell numbers, and
they are returned to the body by infusion or
transplantation (time consuming and
expensive)


In vivo

-
a gene is introduced directly into
specific cells within the body (quick and
inexpensive), but targeting certain cells (e.g.,
bone marrow stem cells) is difficult

Vectors/methods used to deliver genes in
Human Gene Therapy


Retroviruses


Adenoviruses


Adeno
-
associated viruses


Herpes simplex virus


Liposomes


Naked DNA


Human gene therapy

(# clinical trials 1990
-
1999)


AIDS (19)


Amyotrophic lateral
sclerosis


Cancer (280)
-
p53


Cardiovasc. dis. (20)


Cystic fibrosis (24)


Familial
hypercholesterolemia


Gaucher disease (3)


Hemophilia A (2)


Hemophilia B (2)


Hunters disease


Multiple sclerosis


Muscular dystrophy


Rheumatoid arthritis


Severe combined
immunodeficiency (3)

Severe Combined ImmunoDeficiency (SCID)


See

http://www.scid.net/about.htm

How is ADA deficiency treated?

There are no real cures for ADA deficiency, but doctors have
tried to restore ADA levels and improve immune system
function with a variety of treatments:


Bone marrow transplantation from a biological match (for
example, a sibling) to provide healthy immune cells


Transfusions of red blood cells (containing high levels of ADA)
from a healthy donor


Enzyme replacement therapy, involving repeated injections
of the ADA enzyme


Gene therapy
-

to insert synthetic DNA containing a normal
ADA gene into immune cells


6
-
yr
-
old Ashanthi DeSilva
-
SCID sufferer
treated with gene therapy
-
coloring at
home in N Olmstead, OH (March 1993).

Cystic fibrosis transmembrane conductance regulator protein (CFTR)

CFTR involved with chloride
ion transport out of cells; if
defective
Cl
-

builds up inside
cells and draws water inside
resulting in a sticky, sugar
-
rich extracellular mucus.

Is gene therapy safe?


What do you think?


Jesse Gelsinger story

Jesse Gelsinger

(
June 18
,
1981

-

September 17
,
1999
) was the first person publicly identified as having died in a clinical trial for
gene therapy
. He was 18 years old. Gelsinger suffered from
ornithine transcarbamylase deficiency
, an
X
-
linked

genetic
disease

of the
liver
, whose victims are unable to metabolize
ammonia

-

a byproduct of
protein

breakdown. The disease is
usually fatal at birth, but Gelsinger had not inherited the disease; in his case it was the result of a genetic mutation and
as
such was not as severe
-

some of his cells were normal which enabled him to survive on a restricted diet and special
medications.

Gelsinger joined a clinical trial run by the
University of Pennsylvania

that aimed to correct the mutation. On Monday, September
13 1999, Gelsinger was injected with
adenoviruses

carrying a corrected gene in the hope that it would manufacture the
needed enzyme. He died four days later, apparently having suffered a massive immune response triggered by the use of the
viral vector used to transport the gene into his cells. This led to multiple organ failure and brain death. Gelsinger died on

Friday, September 17th at 2:30 PM.

A
Food and Drug Administration

(FDA) investigation concluded that the scientists involved in the trial, including the lead
researcher Dr. James M. Wilson (U Penn), broke several rules of conduct:

Inclusion of Gelsinger as a substitute for another volunteer who dropped out, despite having high ammonia levels that should
have led to his exclusion from the trial

Failure by the university to report that two patients had experienced serious side effects from the gene therapy

Failure to mention the deaths of monkeys given a similar treatment in the informed consent documentation.

The University of Pennsylvania later issued a rebuttal
[1]
, but paid the parents an undisclosed amount in settlement. The Gelsinger
case was a severe setback for scientists working in the field.


Stem Cells


Stem cells are the progenitors of many
different cell types, depending upon which
type of stem cell is used (e.g., bone marrow
stem cells, neural stem cells, embryonic stem
cells)


Stem cell therapy
-
the goal is to repair
damaged tissue (e.g. Parkinson’s disease,
spinal cord injury)