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Newer cancer therapies gene therapy gene therapy Direct genetic modification of cells in patients 3 challenges in gene therapy delivery delivery delivery 1) Package the gene 2) Protect the gene 3) 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 replicationdeficient Retroviral vectors Advantages 1) long-term expression 2) low toxicity 3) high capacity 4) 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 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 Severe Combined Immunodeficiency (SCID) Gene therapy by injection of retrovirally transduced autologous CD34+ hematopoietic stem cells (HSCs). insertional mutagenesis near the protooncogene LMO2 promoter (Science, 302:415-419, October 17, 2003) 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); Non-viral Vectors Advantages non-toxic no immune response Non-viral Vectors liposomes (lipoplexes) Non-viral Vectors amino acid polymers: cationic polymers e.g. B-cyclodextrins Non-viral Vectors Gene gun naked DNA artificial human chromosomes Non-viral Vectors Receptor-mediated endocytosis Gene therapy in cancer gene therapy clinical trials cancer monogenic disease infectious disease 8% 10% 6% vascular disease others 64% 12% retrovirus adenovirus lipofection naked DNA pox virus AAV others clinical trials by vector 6% 2% 11% Based on http://www.wiley.co.uk /genetherapy/clinical/ 12% 7% 34% Conditionally replicating viruses Replication of a conditionally replicating virus, ONYX015, in a cancer cell from a patient with head and neck cancer during Phase II clinical testing. Tumour-suppressor gene delivery Nature Reviews Cancer (2001) Vol 1; 130-141 Delivery of agents that block oncogene expression Nature Reviews Cancer (2001) Vol 1; 130141 Conditionally replicating viruses 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 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