Chapter 10-Protein Therapeutics Chapter 11-Nucleic Acids as Therapeutic Agents

1. Chapter 10-Protein TherapeuticsChapter 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

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

3. 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 PolfaTarchomin S.A., Argentina's Laboratorios Beta S.A., and China's WanbangBiopharma Co. • Follicle-stimulating hormone FSH replaced Serono'sPergonal which was previously isolated from post-menopausal female urine • Factor VIIIKogenate 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 AFabrazyme by Genzyme • alpha-L-iduronidase (rhIDU; laronidase) Aldurazyme by BioMarin Pharmaceutical and Genzyme • N-acetylgalactosamine-4-sulfatase (rhASB; galsulfase) Naglazyme (TM) by BioMarin Pharmaceutical • DNAsePulmozyme by Genentech • Tissue plasminogen activator (TPA) Activase by Genentech • GlucocerebrosidaseCeredase 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

4. 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

5. 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.

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

7. 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.

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

9. Fig. 11.1 Inhibition of translation of specific RNA by antisense nucleic acid molecules Promoterantisense cDNApoly A addition signal -antisense RNA complex mRNA antisense oligonucleotide

10. Fig. 11.8 Ribozymes: A. Hammerhead B. Hairpin

11. 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.

12. 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)

13. 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.

14. 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

15. Vectors/methods used to deliver genes in Human Gene Therapy • Retroviruses • Adenoviruses • Adeno-associated viruses • Herpes simplex virus • Liposomes • Naked DNA

17. 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)

18. Seehttp://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 Severe Combined ImmunoDeficiency (SCID) 6-yr-old Ashanthi DeSilva-SCID sufferer treated with gene therapy-coloring at home in N Olmstead, OH (March 1993).

19. 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.

20. 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-linkedgenetic 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.

21. 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)