Transcript Biotechnology and Genetic Engineering-PBIO 450

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 Genetech
activator (TPA)
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, Hepatitis B and C,
multiple sclerosis
Genetech
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 postmenopausal 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
Cloning and expression of a foreign protein in a suitable host
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 the
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 to
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 problem 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 produced 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
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, HoffmannLaRoche
Prevents kidney
transplant rejection
Chimeric
Centocor
Prevents blood clots
Chimeric
Genetech, HoffmannLaRoche
Non-Hodgkin
lymphoma
Humanized
(Herceptin)
Genetech
HER2-positive breast
cancers
Humanized
Am Home Prod, Celltech, Certain leukemias
Schering, Millen. Pharm.
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
antisense oligonucleotide
poly A addition signal
mRNA-antisense
RNA complex
Fig. 11.8 Ribozymes: A. Hammerhead B. Hairpin
Fig. 11.13 RNA interference (RNAi)
dsRNA
sense
antisense
Binding of dsRNA-specific nuclease
Nuclease-ssRNA complex
Hybridizes to mRNA
cleavage
mRNA is cleaved!
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.
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, sugarrich 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)