Gene therapy application for cancer: virotherapy

Concept of gene therapy and virotherapy
1. What is gene therapy?
  1. A technique of correcting defective genes responsible for disease by introducing genetic material into the patient to treat or cure a disease
2. Why gene therapy?
  1. Many diseases caused by genetic events (e.g. cancer)
    1. Single gene mutations or abnormal expression
    2. Include; cystic fibrosis, X-linked severe combined immuno deficinecy (SCID), haemophilia, thalassaemia, CML, infectious diseases; AIDS
      1. Neurological; Huntington's, Parkinson's, Alzheimer's(?)
        Blindness; Leber congenital amaurosis (LCA)
        Rare metabolic syndromes; lipoprotein lipase deficiency (LPLD)
        Only disease with an approved virotherapy - Glybera (EU approved, 2012)
        Virotherapy with adeno-related virus under clinical trials with promising results
  2. No current drugs available to treat many diseases
  3. Drug-resistance and drug-desensitisation of many cancers
3. How?
  1. Insert a normal gene in a non-specific location within the genome to substitute for a non-functional one
  2. Insert a normal gene in a specific place to replace a non-functional one
  3. Intorduce new genetic material without inserting it into the patient genome
  4. How can we deliver the genes?
    1. Cells
    2. Viruses
    3. DNA
    4. siRNA
How gene therapy and oncolytic viruses can be used in the treatment of cancer
1. Vectors
  1. Most common viral vectors; adeno-associated viruses (adenovirus) and vaccinia (replicates and transcribes genes outside of host nucleus)
  2. Using replicating viruses (replication-competent)
    1. Tissue selectivity through;
      1. Mutational complementation (cancer cells feature a mutation that enhances virus uptake/activity)
        Only mutated cells are infected/affected by gene
      2. Tissue/tumour specific promter
        Viral gene can only be activated in specific tissue/tumour type
      3. Specific receptor targetting
  3. Using replication-defective viruses
    1. Deliver genetic material but lack genes to replicate
2. Onyx-015 (mutated adenovirus with E1B55K and E1B19K mutations)
  1. Can only replicate in cells lacking p53 and that are immune to apoptosis
    1. Mutational selectivity (complementation)
  2. The first oncolytic adenovirus in clinical trials - for; head/neck, glioblastomas, ovarian, bladder and colorectal cancers
    1. Not efficacious as a single agent - improved activity with cytotoxic drugs
  3. Can carry the genetic material to code for thymidine kinase (TK)
    1. HSV TK is a suicide gene which when inserted into a cancer converts the harmless prodrug ganciclovir into a cytotoxic metabolite
  4. Can carry the genetic material to code for cytosine deaminase (CD)
    1. CD is another suicide gene that converts 5-fluorocytosine (5-FC) into 5-FU within the tumour cell
3. H101 (first approved replicating viral therapy in China) - for treating head an neck cancers
  1. Like onyx-015 - can only replicate in cells lacking p53 (i.e. cancer cells)
How to manipulate viruses to target cancer cells
1. Viral biology, life cycle and engineering
2. Examples
  1. Adenovirus is commonly used for virotherapy
    1. Easy to modify - small genome and life cycle well known
    2. Does not integrate - no risk of cancer
    3. Can enhance anti-tumour immune response
    4. Safe in patients - but only efficacious in combination with drugs
    5. Replication-competent - this can be exploited to make it tumour-selective
      1. Life cycle and proteins well known
      2. Naturally the virus can only replicate in epithelial cells that are replicating
        However it can hijack the host cell to force it into S-phase
        Viral proteins; E1a (binds and inhibits Rb), E1B55K (inhibits p53)
        Therefore deletion of E1a (adenovirus mutant Ad-CR2) requires a cell that has lost Rb function for replication
        Therefore deletion of E1B55K (adenovirus mutant Onyx-015) requires a cell that has lost p53 activity for replication
        Host cells can detect the presence of the virus and intiate apoptosis
        Adenovirus can also hijack this by viral protein E1B19K (inhibits pro-apoptotic proteins Bax-bak)
        Therefore to replicate adenovirus mutants lacking E1B19K (Ad-19K) require cells that can self-evade apoptosis
3. Some viruses are inherently selective for tumours; e.g. Reovirus
  1. Others can be made selective by deletion of certain genes; HSV1, vaccinia, adenovirus
Novel therapeutic developments
1. Examples
  1. (1990) Using retroviral therapy to insert a gene for adenosine deaminase (ADA) into patient T-cells with ADA-SCID
    1. Number of T cells normalised and so did cell and humoral immune responses
      1. ADA expression in immune cells persisted despite the discontinuing of the treatment - following 2 years of treatment
        Proved that gene therapy could be achieved safely
  2. (2010) Human PE65 gene therapy for Leber congenital amaurosis (LCA)
    1. Early visual improvements and no unwanted effects after 1 year
    2. Using adeno-associated virus vector
2. Of the >1,800 gene therapy trials currently going one a majority are still only at Phase I (~60%)
  1. Most of these are aimed at a therapy for treatment of cancer (~65%)
3. H101 and onyx-015
  1. Showed promising signs whien used in combination with cytotoxic drugs - however very little activity alone (14% necrotic response)
    1. Needs further work - investigations into the host immune response againstthe virus
4. How to improve efficacy of viral therapy
  1. In onyx-015
    1. Include immunomodulating proteins?
      1. E3 proteins - inhibit MHC class I molecules and cytotoxic T cell killing; inhibit fas mediated apoptosis
  2. Include tumour specific promoters with prodrug converting enzymes, protein inhibitors, androgen receptor inhibtors etc.
5. Other viruses
  1. Vaccinia - JX-594 (phase I trial)
    1. Advantages
      1. Can be administred sytemically
      2. Fast and potent lysis
      3. Large genome (+++ cloning capacity)
      4. Infects many cell types - prefers cancer cells
    2. Disadvantages
      1. Large genome - still some unknown functions
      2. Uptake mechanisms uknown
  2. HSV-1 - Oncovex-GMCSF (currently phase III)
    1. Delivers genetic material coding for GMCSF - promotes an immune response
      1. In trial against unresectable late stage melanoma
Colon, prostate and pancreatic cancer
1. Current treatment is with cytotoxic compounds (and suregery and radiotherapy)
  1. Capecitabine (5-FU prodrug)
  2. 5-FU (thymydilate synthase inhibtor - no nucleotide synthesis)
  3. Mitoxantrone (Inhibitor of topoisomerase II - inhibits DNA synthesis/transcription)
  4. Cisplatin/carboplatin (platinum-based - causes direct DNA damage [crosslinking] - apoptosis)
  5. Gemcitabine (nucleoside analogue - replaces cytosine)
  6. Docetaxel (taxane - disrupts microtubules - mitotic spindle, intracellular transport, cell shape and interaction with ECM)
2. Commonly become drug-resistant - then metastasise
  1. We need new therapies!
    1. Oncolytic viruses - adenovirus (+ chemo drugs + cytotoxic genes), vaccinia etc
Gene therapy approaches to kill cancer cells
1. Prodrug activation therapy: thymidine kinase (TK), cytosine deaminase (CD), nitroreductase (NR)
2. Tumour supressor replacement: p53, PTEN
3. Inhibiton of oncogenes: shRNA, siRNA, ribozyme
4. Immunomodulation: cytokines, granulocyte macrophage colony stimulatinig factor (GMCSF)
5. Stromal targetting - antiangiogenesis
6. Cell therapies and vaccines
7. Oncolytic biotherapy - the vector as therapy: adenovirus, vaccinia, HSV
8. Delivery of suicide genes

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Gene therapy application for cancer: virotherapy

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