Surfing the First Wave: Recombinant DNA Drugs
September 2, 2004

I. Recombinant DNA Technology:

I. A (real quick) review: What is a gene?

1. Anatomy of a Eukaryotic, Protein-encoding Gene Figure:

• Exons: transcribed regions that encodes the polypeptide. Includes a 5' UTR, a 3'UTR, and a transcription termination signal.
• Introns: transcribed regions that will be removed from the mRNA before translation
• Transcription start site: binding site for RNA polymerase II (pol II), a complex of over 10 different proteins
• Regulatory sequences or "cis elements" that control whether the gene is 'on' or 'off':
  • Promoter: a regulatory sequence upstream of the transciption start site that binds transcription factors
  • Enhancer: a regulatory sequence that further influences the level of transcription

2. Pre-mRNA processing and alternative splicing

• Processing: a series of steps that mature pre-mRNAs into functional RNAs.
  • Cap: A 5' guanine cap is added to protect the RNA from degradation.
  • Splicing: removal of introns coupled with joining of exons is done by the spliceosome, a complex of snRNAs and over 145 different proteins!
  • Poly A+ tail: A long stretch of adenine (A) nucleotides added at the 3' end of the transcript needed to exit the nucleus as well as protect the mRNA from degradation.
• Alternative splicing generates many (subtle) forms of the same gene as a way to produce a large variety of proteins from a small number of genes. (Example: "One of the most dramatic examples is the DSCAM gene in Drosophila. This single gene contains some 108 exons of which 17 are retained in the final mRNA. Some exons are always included; others are selected from an array. Theoretically this system is able to produce 38,016 different proteins from one gene!)

3. The source of DNA for cloning: genomic or cDNA?

• Genomic clone: (DNA) the entire genome of an organism is isolated, digested with a restriction enzyme, ligated to millions of copies of a vector cut with the same enzyme, and transformed into the appropriate host cell. Each bacterial colony represents one piece of DNA from the original organism. It may or may not contain a whole gene, and it may contain introns and regulatory sequences inside or near the gene (or even non-coding DNA). Figure
• cDNA clone: (RNA) If a scientist is interested in studying the genes expressed (made into mRNA and then protein) in a cell during a particular condition, total RNA is isolated. RNAs with a poly A tail (mRNAs) are isolated, and a DNA copy of each mRNA (called cDNA) is made using the enzymes reverse transcriptase and DNA polymerase. Synthetic linkers are added to each of the cDNAs so they may be cloned into the restriction site of a vector. The cDNA contains ONLY the transcribed, processed part of the gene - the mRNA. There are NO introns, promoters, enhancers, etc - just the info that actually makes the final protein

 

II. Recombinant DNA Technology: the standard tools for cutting, pasting, and analyzing DNA

1. Plasmids - the "vehicle" for cloning Figure:

• Small, extrachromosomal pieces of bacterial DNA, often carry genes for antibiotic resistance
• Plasmids are the "shuttle vectors" to create, produce, and maintain recombinant DNAs in the lab
• These DNAs can be isolated from bacteria using a simple technique called a "plasmid prep"
• Other cloning vectors are used, depending on the cell type and the need to carry large DNA molecules:
  • Bacteriophages -bacterial viruses tht infect bacteria
  • YACs, or yeast artificial chromosomes, are used for yeast or animal cells
  • Ti plasmid of Agrobacterium tumefaciens, a bacteria that naturally infects plant cells .
  • Adenoviruses, retroviruses, and the SV40 virus are used to carry DNA into mammalian cells

2. Restriction enzymes - "cut and paste" DNA Figure

• Certain bacteria can specifically fight off (or "restrict") bacteriophages by producing restriction enzymes
• These bacterial enzymes cut DNA at very specific, often palindromic, sequences (each enzyme recognizes a specific sequence), leaving a specific "sticky end" or a blunt end
• Over 200 restriction enzymes are now available commercially
• If DNA from two different organisms have the same "sticky ends", they can find each other in a test tube and join together - creating recombinant DNA
• The two DNAs are chemically sealed together by DNA Ligase, which joins the sugar of one base to the phosphate of another.
• Animation

3. Host cells - to carry and replicate the cloned DNA Figure

• Bacteria = quick generation time, ease of transformation, and simple growth requirements
• Yeast cells = when it is necessary to study a cloned gene in a eukaryotic environment (ie if you are studying RNA splicing).
• Animal cells or plant cells (culture cells, whole tissues, or transgenic organisms) = when it is necessary to study the expression of a gene (or protein) in a multicellular environment where development and differentiation take place.
• During a process called transformation, cells are made competent (able to take up foreign DNA) by treating them with CaCl2 and a brief heat shock or by electroporation or microparticle bombardment, or "biolistics" where the DNA of interest is shot at high velocity into tissues using a particle gun.

II. Biotech's First Wave: Recombinant DNA Drugs

Homework Note: The notes below are in OUTLINE form and require you to do a bit of homework (~2 hours of time before class). For each drug listed below, go to the link provided and read about the drug to determine:
(1) The Protein Product: what human protein is formulated as the drug?
(2) Indication: why is the drug prescribed and what it does it do in the body?
(3) Other notes (Alternate forms of this drug, competing products on the market, "the dark side" scandals, etc)

It may be more difficult to get the info for some drugs than others - but get as much info as you can before class. The Full Notes will be posted on September 2.

Information about each drug is taken from the referenced website (linked)

I. Amgen: (Nasdaq: AMGN) Corporate History: Founded in Thousand Oaks, California in 1980 as AMGen (Applied Molecular Genetics) by George Rathmann et al. The world's largest independent biotechnology company. These first 5 drugs are among the best-selling biotechnology drugs in the world!
rDNA products on the market:

1: Epogen: (recombinant erythropoetin) [FDA approval 1989] A recombinant erythropoetin that stimulates production of RBCs. Erythropoietin is a protein produced in functioning kidneys; circulates through the bloodstream to bone marrow; stimulates production of RBCs. Epo has truly revolutionized treatment of anemia associated with dialysis, chronic renal failure, chemo, and HIV infection. Amgen has licensed Ortho Biotech, Inc., to market and distribute Epo under the trademark Procrit, for non-dialysis uses only. The Dark Side of Epo: Epo Doping - See the Good For article! Sales of ~$2.4 B annually

2. Aranesp: (recombinant erythropoetin) [FDA Approval September 2001] (Darbepoetin): A modified version of Epo "developed to simplify anemia management" via the addition of 2 additional N-linked carbohydrate chains. This sugar addition creates a longer lasting effect in the body (three fold greater that Epo), requires fewer injections that Epo. Used to treat anemia associated with dialysis, chronic renal failure & chemotherapy. The Dark Side of Darbepoetin: 2/24/02: Salt Lake City: New Drug Darbepoetin Behind Olympic Scandal . Sales of $2.8 B annually

Aranesp®: innovative science

3: Neupogen: (recombinant granulocyte colony-stimulating factor G-CSF) [FDA approvals 1991- 1998] G-CSFs are glycoproteins that act on hematopoietic cells - stimulating proliferation and growth of neutrophils (WBCs). Decreases neutropenia (low WBC count) and febrile neutropenia (low WBC count with fever/ infection) associated with chemotherapy, bone marrow transplantation, and acute myelogenous leukemia (AML). Must be given as a daily dose during each cycle of chemotherapy. Sales of $1.3 B annually

4: Neulasta (recombinant granulocyte colony-stimulating factor G-CSF) [FDA Approval Feb 2002] A recombinant GCS-F conjugated to a polyethylene glycol (PEG) molecule. The addition of PEG enlarges the molecule, extending the half-life. This means that only a single dose per chemotherapy cycle is needed. Sales of $1.1 B annually

5: Infergen: (recombinant type-I interferon alpha) [FDA approval 1997] Used to treat chronic hepatitis C viral (HCV) infection, particularly non-respondong or relapsing HCV. Infergen is a synthetic 'consensus' interferon based on the most common amino acid sequences in 12 natural interferons. Interferons are immunity-boosting proteins in WBCs - they signal antiviral, antiproliferative, and 'immunomodulatory' activities in response to viral infection, cancer, or autoimmune diseases". In June 2001, InterMune acquired Infergen from Amgen. Sales of Infergen were less than $10 million in the last two years."

II. Biogen Idec (Nasdaq: BGEN - now BIIB) Corporate History: Biogen was co-founded in 1978 by Nobel Prize winners Phillip Sharp of MIT and Walter Gilbert of Harvard, in Cambridge MA. In November 2003, Biogen and IDEC Pharmaceuticals merged (NASDAQ: BIIB) .
rDNA products on the Market

1. Avonex, (recombinant interferon beta-1a) [FDA Approval May 1996] For the treatment of relapsing forms of multiple sclerosis (MS). Slows the progression of MS by regulating the body's immune response against myelin. Given as an IM injection (ouch!) once per week. Annual sales exceeded $200 M / year in 2000. How Avonex is made ; Sales of ~1 B annually

2. Intron A (recombinant interferon alpha-2b) [FDA Approval 1986], A genetically engineered anticancer / antiviral interferon approved for over 20 indications, including chronic hepatitis B and C, malignant melanoma, hairy cell leukemia, Kaposi's sarcoma, and prostate cancer. "Intron A is the leading product in the $2 billion global alpha interferon market." Licensed to Schering-Plough Corporation, Intron A is one of S-P's best-selling products with total worldwide annual sales exceeding $440 million in each of the last three years. Good news: 8/21/2002: Intron A being tested as a therapy to treat west nile virus

3. Engerix-B/Recombivax (recombinant hepatitis B vaccine) [FDA aproval 8/99, 1/2000 for teens] First human vaccine produced using genetic engineering. Engerix-B is the #1 selling hepatitis B vaccine worldwide. According to the CDC, the hepatitis B vaccine is recognized as the first anti-cancer vaccine because it can prevent primary liver cancer Hepatitis B is one of the world's most serious and widespread chronic diseases. The global hepatitis B vaccine market exceeds $1 billion dollars annually. It is expected to continue to grow as more countries adopt World Health Organization recommendations for the vaccination of newborns, teenagers, healthcare workers and other at-risk populations. Sold under license worldwide by SmithKline Beecham and Merck.

4. Amevive: (Alefacept) [FDA approval 1/31/03] a recombinant fusion protein between the Fc portion of human IgG1 and the extracellular domain of the "leukocyte function-associated antigen-3" (LFA-3). Amevive treats moderate-to-severe chronic plaque psoriasis by suppressing overactive T lymphocytes. Because Amevive reduces T-cell counts (important for fighting off infections, etc), T-cell levels are monitored closely after each of 12 weekly injections. Mechanism of action

1. Betaseron (recombinant Interferon beta-1b). [FDA approval 7/93] Berlex Laboratories, Inc., is the developer and marketer of Betaseron, and Chiron is the manufacturer. Betaseron significantly delays the progression of secondary MS. Mechanism of action; Sales of ~$118 M annually

2. Proleukin (recombinant Interleukin-2 - IL-2) [FDA approval 1992] Proleukin was approved by the FDA in 1992 for use in treating advanced stages of kidney cancer (metastatic renal cell carcinoma) and in 1998 for skin cancer (metastatic melanoma). IL-2 has a variety of activities but the major one is to stimulate T cell and natural killer (NK) cell proliferation, increasing and activating these immune system cells to find and destroy cancer cells. Recently Proleukin has been considered for use as an immune system supplement for AIDS patients. Mechanism of action; Sales of ~$92 M annually

3. Regranex (recombinant platelet-derived growth factor.) [FDA approval 12/97] PDGF is a cytokine (growth factors that promote cell-cell communication) that stimulates cellular processes (skin cell and blood vessel production) involved in developing protective tissue and skin after a wound or ulcer (a process called granulation). In 1997, Chiron and its partner Johnson & Johnson (& now Ortho-McNeil) obtained clearance to market Regranex Gel for treatment of diabetic foot ulcers. Mechanism of action ; Sales of $48 M annually

OUCH! Chiron and flu vaccine Added 12/04

Flu's in the News

• 7 October 2004: Shortage Was Predicted By Denise Grady
  • From WebMD: 12 Tips to Prevent Cold and Flu the "Natural" Way
• 6 October 2004: U.S. Will Miss Half Its Supply of Flu Vaccine

IV. Genentech (NYSE: DNA) Corporate History: Founded 25 years ago in 1976 by Robert Swanson and Herb Boyer. Located in South San Francisco CA. Genentech was the first company to produce a recombinant DNA product for the market - Humulin (liscensed to Eli Lilly), and 18 of ~70 biotechnology-based products on the market today were developed by Genentech. Genentech has reserves of $1.3 billion in cash and investments revenues exceeding $1 billion.
rDNA products on the Market:

First three are thrombolytics ('clot buster enzymes') that break up deadly blood clots during acute myocardial infarction (heart attacks, or AMI), strokes, and in patients with central lines. All are tissue-plasminogen activators (t-PA) which, in the presence of the clotting protein fibrin activate plasminogen to plasmin and cause fibrinolysis (breaking up of a blood clot). The clot is then dissolved, restoring blood flow to the heart, sometimes in seconds. Combined annual sales for all thrombolytics of ~$200 M annually.

1. Activase (recombinant TPA, tissue-plasminogen activator). [FDA approval 4/95] Approved for treatment of AMI, cardiac ischemia, acute massive pulmonary embolism, and management of stroke. Binds to fibrin and converts plasminogen to plasmin

1a. CathFlo Activase: [FDA approval Sept 2000] "A form of Activase that can be directly injected into a the catheter of a person with a "central line" or a central venous access device (CVAD) to restore function to central venous access ('central line')
devices that have become occluded due to a blood clot."

2. TNKase (Tenecteplase, recombinant TPA), [FDA approval June 2000] A single-bolus tPA, approved for the treatment of AMI. "TNKase is the first 'clot-buster' that can be administered over five seconds in a single dose, offering the fastest administration of a thrombolytic to date in the treatment of heart attack. TNKase is a bioengineered variant of Activase. It is constructed with amino acid substitutions at three sites (the letters T, N and K represent the three amino acids (Threonine, Asparagine, Lysine) changed from the natural t-PA protein." Investigate the molecule.

3. Pulmozyme (recombinant human deoxyribonuclease I (rhDNase) [FDA approval 1994] Inhalation Solution - For management of cystic fibrosis in children 3 months - 5 years old.

Mechanism of action: In cystic fibrosis (CF) patients, lungs and airways become clogged with thick secretions that contain very high concentrations of extracellular DNA released by degenerating leukocytes (neutrophils). Pulmozyme selectively cleaves / hydrolyzes the DNA in sputum and airways of CF patients.
Sales of ~$120 M annually

4. Nutropin (somatropin rDNA origin) [FDA approval Nov 93] human growth hormone - or somatropin, a protein identical to pituitary-derived GH. Indicated for treatment of GHD in children and in adults, growth failure associated with chronic renal insufficiency (CRI) prior to kidney transplantation, and short stature associated with Turner syndrome (45, XO). Sales of $320 M annually for all Genentech growth hormone products

4a: Neutropin AQ: [FDA approval 4/97] a liquid formulation of somatropin
4b: Neutropin AQ pen: [FDA approval 4/02]same stuff, single use throw-away 'pen' injection
4c: Nutropin Depot: [FDA approval 12/99] a slow-release, injectable suspension form of Nutropin formulated using rhGH embedded in slow-release biodegradable polylactide-coglycolide (PLG) microspheres.

5. Protropin (somatrem rDNA origin) [FDA approval October 1985] human growth hormone nearly identical to human GH- Genentech's first product! For long-term treatment of growth hormone deficiency (GHD) in children.

V. Eli Lilly: (NYSE: LLY) Corporate History: Founded in 1876 by Colonel Eli Lilly in beautiful Indianapolis, Indiana. The goal was to create "a company that manufactured pharmaceutical products of the highest possible quality, dispensed at the suggestion of physicians rather than by eloquent sideshow hucksters, and based on the best science of the day." (Lilly History) Today, Lilly employs more than 31,000 people around the world, and markets its products in over 150 countries
rDNA products on the Market: ( rDNA origin )

1. Humulin (insulin rDNA origin) [FDA approval 1982] Human insulin made in bacterial cells. Brought to market in 1982. Used by over 4 million people worldwide every day. Diabetes care products, composed primarily of Humalog and Humulin hasworldwide revenues of $2.5 B annually. Comes in different versions:

1a: Humulin pens (single use injectables), and derivatives (Humalog, etc)
1b: Humalog: Fast acting insulin that better mimics the body's response to insulin
1c: Humalog Mix 75/25 ...and lots more!

Take a look at this cool chart comparing Time Activity Profiles:

2. Humatrope (somatropin rDNA origin), [FDA approval 8/96]a synthetic human growth hormone (hGH) For Somatropin Deficiency Syndrome (SDS) in adults and GHD in children. HGH is produced in large quantities by the pituitary gland, and adults with SDS may have hypopituitarism, a pituitary tumor or other pituitary disorder, or they may have been treated for growth hormone deficiency as children. Annual revenues of ~$330 million.

July 25, 2003: FDA Approves Humatrope for Short Stature: Eli Lilly recently announced that the FDA had granted approval for the use of Humatrope (human growth hormone) in children who are healthy, but short in stature (only the shortest 2-3% of children qualify). Discuss some of the realities of this treatment, and whether the benefits of this treatment justify its use in children.
* Drug delivery * Cost to patients * Drug side effects * Will it be covered by insurance

3. Xigris (drotrecogin alfa) [FDA approval 11/01]is a recombinant version of Activated protein C. "Xigris is the first drug approved for treatment of severe sepsis, an overwhelming systemic response to infection. Sepsis is a leading cause of death in the United States, claiming 215,000 lives each year - more than breast, colon/rectal, pancreatic, and prostate cancer combined. Sepsis can rapidly lead to the formation of blood clots, organ dysfunction and death by suppressing the activation of Protein C, a regulatory molecule that has multiple properties (inhibits blood coagulation, increases fibrinolysis, and controls inflammation). Xigris (activated protein C) prevents or reduces inflammation and blood clotting in people who develop sepsis and increases the odds of survival by over 38%. The estimated costs associated with treatment of sepsis are $17 Billion annually in the US. Worldwide sales of $1.4 B annually. "

4. Forteo [FDA Approval Nov 26, 2002] a recombinant form of parathyroid hormone for the treatment of osteoporosis in women and men. The drug is the first approved osteoporosis treatment that stimulates new bone formation, increases the number and action of osteoblasts, and increases bone mineral density (BMD) and bone strength. About 10 million people in the US suffer from osteoporosis. US consumer group Public Citizen is warning the public against Forteo due to fears that bone cancer (osteosarcoma) might be stimulated by a drug that stimulates osteoblast formation.