1. Newer cancer therapies gene therapy

2. gene therapy
Direct genetic modification of cells in patients

3. ‘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

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

5. METHODS OF VECTOR DELIVERY

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

7. 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

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

9. 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

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

11. 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

12. 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

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

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

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

16. 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

17. 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.

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

19. 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

20. 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 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)

21. 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: , 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 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

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

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

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

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

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

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

28. Non-viral Vectors
Receptor-mediated endocytosis

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

30. 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.

31. Tumour-suppressor gene delivery
Nature Reviews Cancer (2001)
Vol 1;

32. 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;

33. 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;

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

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