Newer cancer therapies gene therapy

gene therapy Direct genetic modification of cells in patients

‘Trojan horses’ that sneak the gene into the cell 3 challenges in gene therapy delivery delivery delivery Package the gene Protect the gene targeted delivery to the nucleus and release in an active form Vectors ‘Trojan horses’ that sneak the gene into the cell

Vectors Carrier molecules designed specifically to enter cells & deposit therapeutic genes Vectors can be viral or non-viral

METHODS OF VECTOR DELIVERY

Gene therapy targets Germ line gene therapy Somatic cell gene therapy Gene augmentation Gene replacement Specific inhibition of gene expression Targeted cell death

Gene augmentation most therapies simply add a useful gene into a selected cell type to compensate for the missing or flawed version. Useful in treating loss of function mutations such as Tumour Genes

Gene replacement This strategy replaces the mutant copy with a correctly functioning copy in situ. Useful for gain of function mutations such as oncogenes

Specific inhibition of gene expression Involves silencing of specific genes like activated oncogenes, by using molecules that degrade RNA transcripts. Strategies include Antisense therapy siRNA (small interfering RNA) Ribozymes etc

Antisense therapy short stretches of synthetic ssDNA that target the mRNA transcripts of abnormal proteins preventing its translation

siRNA therapy Small interfering RNAs short stretches (21-23nt) of synthetic dsRNA Has 3’ overhangs of 2 nt Incorporates into RISC (RNA induced silencing complex) Target mRNA cleaved in the middle

Ribozymes Catalytic RNAs that cleave target mRNAs in a sequence-specific manner e.g. hammerhead ribozymes are engineered to recognise specific sequences and made resistant to nucleases

Targeted cell death Tissue specific toxicity as a result of gene therapy. Useful in cancer therapy direct approach

Targeted cell death Indirect approach stimulating an immune response against selected cells or eliminating the blood supply.

Viral vector strategy Replication & virulence genes can be substituted with therapeutic genes

Retroviral vectors designed to enter cell and deposit genes Special vectors are constructed by deleting or altering native sequence in retroviral and lentiviral vectors, to prevent the generation of replication competent retroviruses (RCR) typically caused by homologous recombination

Minimal HIV vector plasmid (1) consisting of the CMV/HIV LTR hybrid promoter followed by the packaging signal ( Ψ), the rev-binding element RRE for cytoplasmic export of the RNA, the transgene expression cassette consisting of internal promoter(s) and transgene(s), and the 3' self-inactivating (SIN) LTR. All genes coding for enzymatic or structural HIV proteins have been removed. Together with the HIV vector plasmid (1), the HIV packaging plasmid (2), HIV rev (3), and an envelope expressing plasmid (4) are needed for HIV vector production.

Packaging retroviral vectors Gag, pol and env genes on physically separate fragments without Ψ sequence Recombinant viral proteins are infective but replication-deficient

Retroviral vectors Advantages long-term expression low toxicity high capacity low antivector immunity allowing repeat administration Problems Lack of cell specificity: Promiscuous: depositing genes into several cell types resulting in reduced target efficiency and unwanted physiological effects Random splicing into host DNA resulting in normal gene disruption and/or alteration in gene function

Severe Combined Immunodeficiency Gene therapy in X-SCID patients Rare condition caused by the lack or reduction in the immune system (‘bubble baby syndrome’) Patients cannot make T lymphocytes and their B lymphocytes fail to make essential antibodies for fighting infections. X-SCID caused by mutations in the X-linked gene IL2RG, which encodes the common gamma chain (gc) of the lymphocyte receptors for interleukin-2 (IL-2) and many other cytokines XSCID (baby in the bubble) patients cannot make T lymphocytes, and their B lymphocytes fail to make essential antibodies for fighting infections. Without T and B lymphocytes patients develop serious infections in infancy and may die unless they are given a bone marrow transplant X-SCID, or better known as the “baby in the bubble” disease, is caused by mutations in the IL2RG gene. The gene encodes the gamma subunit of the interleukin-2 receptor. The protein made by the IL2RG gene is a receptor for chemical signals. The chemical signals direct the growth and activation of immune system cells. The gene is now called the “common gamma chain” because the protein is built into receptors for several different types of signals.   The IL2RG gene is located on the long (q) arm of chromosome X at position 13.1. The gene spans approximately 4.2kb, and has seven introns and eight exons. Exons 5 and 7 have mutation hot spots (CDC online). X-SCID is the most common form of SCID and has been estimated to account for 46% (Buckley, 2004) to 70% of all SCID cases (Stephan et al., 1993; Fischer et al., 1997) (CDC online). Severe Combined Immunodeficiency (SCID)

2/11 X-SCID patients developed leukemia Gene therapy by injection of retrovirally transduced autologous CD34+ hematopoietic stem cells (HSCs). insertional mutagenesis near the proto-oncogene LMO2 promoter (Science, 302:415-419, October 17, 2003) XSCID (baby in the bubble) patients cannot make T lymphocytes, and their B lymphocytes fail to make essential antibodies for fighting infections. Without T and B lymphocytes patients develop serious infections in infancy and may die unless they are given a bone marrow transplant X-SCID, or better known as the “baby in the bubble” disease, is caused by mutations in the IL2RG gene. The gene encodes the gamma subunit of the interleukin-2 receptor. The protein made by the IL2RG gene is a receptor for chemical signals. The chemical signals direct the growth and activation of immune system cells. The gene is now called the “common gamma chain” because the protein is built into receptors for several different types of signals.   The IL2RG gene is located on the long (q) arm of chromosome X at position 13.1. The gene spans approximately 4.2kb, and has seven introns and eight exons. Exons 5 and 7 have mutation hot spots (CDC online). X-SCID is the most common form of SCID and has been estimated to account for 46% (Buckley, 2004) to 70% of all SCID cases (Stephan et al., 1993; Fischer et al., 1997) (CDC online). 2/11 X-SCID patients developed leukemia

Adenoviral vectors do not insert into genome temporary lack of specificity strong immune response

Adeno-associated viral vectors Integrate into genome but small in size Nature Reviews Genetics 1; 91-99 (2000);

Advantages non-toxic no immune response Non-viral Vectors Advantages non-toxic no immune response

liposomes (lipoplexes) Non-viral Vectors liposomes (lipoplexes)

amino acid polymers: cationic polymers e.g. B-cyclodextrins Non-viral Vectors amino acid polymers: cationic polymers e.g. B-cyclodextrins

naked DNA artificial human chromosomes Non-viral Vectors Gene gun naked DNA artificial human chromosomes

Non-viral Vectors Receptor-mediated endocytosis

Gene therapy in cancer http://www.wiley.co.uk/genetherapy/clinical/ Based on http://www.wiley.co.uk/genetherapy/clinical/

Conditionally replicating viruses Replication of a conditionally replicating virus, ONYX-015, in a cancer cell from a patient with head and neck cancer during Phase II clinical testing. Figure 4 | Conditionally replicating viruses. a | Mechanism of action. The viruses infect both normal and tumour cells, but can only replicate in tumour cells. The progeny then go on to kill surrounding tumour cells. b | Replication of a conditionally replicating virus, ONYX-015, in a cancer cell from a patient with head and neck cancer during Phase II clinical testing. 109 infectious particles were injected over a 5-day period. After 8 days, biopsy was performed and analysed by electron microscopy. The inset on the left panel is magnified on the right. Clearly, this cell is doomed to die: presumably the new virus particles it produces will infect its neighbours.

Tumour-suppressor gene delivery Nature Reviews Cancer (2001) Vol 1; 130-141

Delivery of agents that block oncogene expression Figure 2 | Cancer gene therapy by delivery of tumour-suppressor genes or inhibition of oncogene expression. a | Tumour-suppressor gene (TSG) delivery. Vectors encoding the tumour suppressor of choice are assumed to infect normal cells and tumour cells. In tumour cells they induce either growth arrest or apoptosis, whereas in normal cells they are assumed not to have any detrimental effects. Some tumour suppressors might also exert unexpected bystander effects. For example, p53 blocks angiogenesis by downregulating the production of vascular endothelial growth factor (VEGF) and by upregulating two anti-angigogenic molecules, thrombospondin and insulin-like growth factor 1 binding protein (IGF1BP). b | Delivery of agents that block oncogene (Onc) expression. These include genes that encode antisense oligonucleotides, which block oncogene expression, and ribozymes, which cleave oncogene transcripts. Again, they are expected to have no detrimental effects on normal cells, which don’t express oncogenes. By contrast, they should cause cancer cells to arrest or undergo apoptosis. In some cases, they also sensitize radio- or chemo-resistant tumour cells to radiotherapy or chemotherapy. No bystander effects have been reported for antioncogenic gene-therapy agents. Nature Reviews Cancer (2001) Vol 1; 130-141

Conditionally replicating viruses Figure 3 | Suicide gene delivery. The vector delivers a gene that encodes a prodrug-converting enzyme, such as herpes simplex virus thymidine kinase (HSV-tk), to tumour and normal cells alike. Local delivery of either the prodrug (in this case, ganciclovir) or the vector to the tumour provides specificity. The prodrug is converted to the active, cytotoxic metabolite in the tumour cell, and diffusion to neighbouring cells confers a potent bystander effect. Nature Reviews Cancer (2001) Vol 1; 130-141

Current status Food and Drug Administration (FDA) has not yet approved any human gene therapy product for sale

References Optional reading Chapter 28 Mol & Cell Biol of Cancer by Knowles and Selby Optional reading Human gene therapy by Ioannou, Panos A (www.els.net) Nature Reviews Cancer (2001) vol 1 pp 130-141 by Francis McCormick