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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