Chapter 10Therapeutic Agents

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Chapter 10-Therapeutic Agents

• Pharmaceutical proteins and enzymes
• Monoclonal antibodies
• Nucleic acids (antisense RNA and olignucleotides,
  ribozymes, interfering RNAs or RNAi)
• Gene therapy
• Stem cells and Therapeutic Cloning

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

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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

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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.
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Table 10.3 Some therapeutic monoclonal antibodies
approved for human use
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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.

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Fig. 10.27 Inhibition of translation of specific
RNA by antisense nucleic acid molecules
Promoter antisense cDNA poly A addition signal
-antisense RNA complex
mRNA
antisense oligonucleotide
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Fig. 10.30 Ribozymes A. Hammerhead B. Hairpin
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Fig. 10.34 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.

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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)

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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.
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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

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Vectors/methods used to deliver genes in Human
Gene Therapy

• Retroviruses
• Adenoviruses
• Adeno-associated viruses
• Herpes simplex virus
• Liposomes
• Naked DNA

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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)

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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).
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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.
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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 230 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.

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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. Parkinsons disease, spinal cord
  injury)

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