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Newer cancer therapies gene therapy. gene therapy Direct genetic modification of cells in patients.

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Presentation on theme: "Newer cancer therapies gene therapy. gene therapy Direct genetic modification of cells in patients."— Presentation transcript:

1 Newer cancer therapies gene therapy

2 gene therapy Direct genetic modification of cells in patients

3 delivery 3 challenges in gene therapy deliverydelivery 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

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


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

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 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 Retroviral vectors

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 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

20 Severe Combined Immunodeficiency (SCID) 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. Gene therapy in X-SCID patients X-SCID caused by mutations in the X-linked gene IL2RG, which encodes the common gamma chain ( c) of the lymphocyte receptors for interleukin-2 (IL-2) and many other cytokines

21 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) 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 Nature Reviews Genetics 1; (2000); Integrate into genome but small in size

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

25 liposomes (lipoplexes)

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

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

28 Non-viral Vectors Receptor-mediated endocytosis

29 Gene therapy in cancer Based on /genetherapy/clinical/

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.

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

32 Delivery of agents that block oncogene expression Nature Reviews Cancer (2001) Vol 1;

33 Nature Reviews Cancer (2001) Vol 1; Conditionally replicating viruses

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

35 References 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

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