Lecture 11.2: Biopharmaceuticals

Question 1

What is a Biopharmaceutical (Bio-P)?

Answer 1

• A biological macromolecule or cellular component,
  such as blood product, used as a pharmaceutical
• A pharmaceutical derived from a biological source and
  especially one produced by biotechnology

Question 2

What biopharmaceuticals have been harvested from cadavers? (1)

Answer 2

Human Growth Hormone

Question 3

What are 1st Generation Bio-Ps? Example?

Answer 3

• Simple copies of human proteins
• Put the appropriate part of the human genome into an
  expression system, harvest, purify and use
• Insulin

Question 4

What are 2nd Generation Bio-Ps? Examples?

Answer 4

• Have undergone some form of ‘engineering’
• Either a change to the genome prior to transfection, or
  a change to the purified product
• Faster and longer acting insulin (aa sequence altered)
• Erythropoietin (CHO residue added to prolong half-life)

Question 5

Types of Potential Modifications that can be made to Bio-Ps? (3)

Answer 5

• Alteration of nucleotide sequence
• Creation of fusion proteins
• Humanising the protein

Question 6

What is the structure of Etanercept? What is it used for?

Answer 6

• Fusion Protein
• Combination of TNF receptor combined with IgG
  antibody
• Used in RA

Question 7

Why is Humanising protein’s important?

Answer 7

• Makes it less immunogenic

Question 8

What is Herceptin? What is it used to treat?

Answer 8

• A humanized mouse Mab directed to the HER2
  receptor (human epidermal growth factor receptor)
• Used in the treatment of metastatic breast cancer
  when the tumour over-expresses HER2 (proto-
  oncogene)

Question 9

Herceptin MOA

Answer 9

Blocks HER2 by promoting its internalisation, blocks tyrosine kinase activity of HER2

Question 10

What are Oligonucleotides?

Answer 10

An alternative way of modifying genetic material – without really changing a gene

Question 11

Antisense Oligonucleotides MOA

Answer 11

• 15-25 base pairs long
• Can enter cells via endocytosis & diffusion
• Complementary to a gene you wish to influence
• Form a triplex with DNA or a duplex with mRNA
• Act by either causing RNA cleavage OR RNA blockage

Question 12

What is Eteplirsen?

Answer 12

• Antisense Oligonucleotide
• Targets the dystrophin gene in Duchenne Muscular
  Dystrophy
• Removes a faulty exon, resulting in a functional
  protein

Question 13

What is Short interfering RNA (siRNA)?

Answer 13

• Short lengths of RNA induce an enzyme complex (RNA
  induced silencing complex, RISC)
• Results in selective degradation of the corresponding
  mRNA
• Very effective in gene silencing

Question 14

What is NOAEL?

Answer 14

No Observable Adverse Effect Level

Question 15

How are gene therapies transported into desired site?

Answer 15

Via vectors, often viruses

Question 16

A Gene Delivery System must have..? (4)

Answer 16

• Capacity: to deliver large amounts of material
• Efficacy: to enter & be utilised by cells
• Longevity
• Safety: especially important when using viral vectors

Question 17

Gene Delivery Strategies: In-vivo

Answer 17

Administration of the vector to the patient

Question 18

Gene Delivery Strategies: Ex-vivo

Answer 18

Cells removed from patient, altered, then re-administered

Question 19

Vectors must be…? (4)

Answer 19

• Safe
• Efficient (insert into target cells)
• Selective (only expressing desired protein and no
  other viral proteins)
• Persistent (potentially an issue in cells with high
  turnover)

Question 20

Retroviruses as Viral Vectors: Pros

Answer 20

• Excellent if introduced into stem cells
• All daughter cells express the desired protein
• Principally used for ex-vivo infections

Question 21

Retroviruses as Viral Vectors: Cons

Answer 21

• BUT, retroviral integrase inserts the therapeutic gene
  randomly -> damage?
• May need to alter the viral envelope so that it infects
  the cell of interest
• Don’t really cross the nuclear membrane – this breaks
  during cell division, so retroviruses only useful for
  targeting dividing cells

Question 22

Adenoviruses as Viral Vectors: Pros

Answer 22

• High expression rates, don’t insert into the host
  genome = limited risk of disturbing other genes
• Good for in-vivo transfection

Question 23

Adenoviruses as Viral Vectors: Cons

Answer 23

Viral genome needs to be engineered to reduce the likelihood of causing widespread infection

Question 24

Other Viral Vectors (2)

Answer 24

• Disabled HIV
• Herpes (long-lived, does not associate with host DNA,
  lon-lived in neural tissue)

Question 25

What other structures can be used as vectors for gene therapies? (3)

Answer 25

• Liposomes
• Microspheres (spheres loaded with plasmid DNA)
• Plasmids

Question 26

How is an inserted gene controlled?

Answer 26

• Co-administration of an inducible promoter e.g. target
  gene linked to a promoter activated by doxycycline
• Linking target gene to a tissue specific promoter would
  further enhance activity

Question 27

What is CRISPR?

Answer 27

• Clustered Regularly Interspaced Palindromic Repeats
• Ability to target nucleases (especially Cas9) to edit
  genes of interest
• Has the potential to repair faulty genes