newer cancer therapies gene therapy. gene therapy direct genetic modification of cells in patients
TRANSCRIPT
Newer cancer therapiesNewer cancer therapies
gene therapygene therapy
gene therapygene therapyDirect genetic modification of cells in patients
deliverydelivery
3 challenges in gene therapy3 challenges in gene therapy
deliverydelivery deliverydelivery
1)1) Package the genePackage the gene2)2) Protect the geneProtect the gene3)3) targeted delivery to the nucleus and targeted delivery to the nucleus and
release in an active formrelease in an active form
Vectors ‘Trojan horses’ that sneak the gene into the cell
Carrier molecules designed Carrier molecules designed specifically to enter cells & deposit specifically to enter cells & deposit
therapeutic genestherapeutic genes
Vectors can be viral or non-viral Vectors can be viral or non-viral
VectorsVectors
METHODS OF VECTOR DELIVERYMETHODS OF VECTOR DELIVERY
Germ line gene therapyGerm line gene therapy
Somatic cell gene therapySomatic cell gene therapy
Gene augmentationGene augmentation
Gene replacementGene replacement
Specific inhibition of gene expressionSpecific inhibition of gene expression
Targeted cell death Targeted cell death
Gene therapy targetsGene therapy targets
Gene augmentationGene augmentationmost therapies simply add a useful gene into a selected cell type to 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 compensate for the missing or flawed version. Useful in treating loss of function mutations such as Tumour Genesfunction mutations such as Tumour Genes
Gene replacementGene replacement
This strategy replaces the mutant copy with a correctly This strategy replaces the mutant copy with a correctly functioning copy in situ. Useful for gain of function functioning copy in situ. Useful for gain of function mutations such as oncogenesmutations such as oncogenes
Specific inhibition of gene expressionSpecific inhibition of gene expressionInvolves silencing of specific genes like activated Involves silencing of specific genes like activated
oncogenes, by using molecules that degrade RNA oncogenes, by using molecules that degrade RNA transcripts. transcripts.
Strategies includeStrategies include
Antisense therapyAntisense therapy
siRNA (siRNA (small interfering RNA)small interfering RNA)
Ribozymes etcRibozymes etc
Antisense therapyAntisense therapy
short stretches of short stretches of synthetic ssDNA that synthetic ssDNA that target the mRNA target the mRNA transcripts of transcripts of abnormal proteins abnormal proteins preventing its preventing its translationtranslation
siRNA therapysiRNA therapy
Small interfering RNAsSmall interfering RNAs
short stretches (21-23nt) short stretches (21-23nt) of synthetic dsRNAof synthetic dsRNA
Has 3’ overhangs of 2 ntHas 3’ overhangs of 2 nt
Incorporates into RISC Incorporates into RISC (RNA induced (RNA induced silencing complex)silencing complex)
Target mRNA cleaved Target mRNA cleaved in the middlein the middle
RibozymesRibozymesCatalytic RNAs that cleave target mRNAs in a Catalytic RNAs that cleave target mRNAs in a
sequence-specific mannersequence-specific manner
e.g. hammerhead ribozymes are engineered to e.g. hammerhead ribozymes are engineered to recognise specific sequences and made resistant to recognise specific sequences and made resistant to nucleasesnucleases
Targeted cell deathTargeted cell death
Tissue specific toxicity as a result of gene Tissue specific toxicity as a result of gene therapy. Useful in cancer therapytherapy. Useful in cancer therapy
direct approachdirect approach
Targeted cell deathTargeted cell deathIndirect approachIndirect approach
stimulating an immune response against stimulating an immune response against selected cells or eliminating the blood supply.selected cells or eliminating the blood supply.
Viral vector strategyViral vector strategyReplication & virulence genes can be Replication & virulence genes can be
substituted with therapeutic genessubstituted with therapeutic genes
designed to enter cell and deposit designed to enter cell and deposit
genesgenes
Special vectors are constructed by Special vectors are constructed by
deleting or altering native sequence in deleting or altering native sequence in
retroviral and lentiviral vectors, to retroviral and lentiviral vectors, to
prevent the generation of replication prevent the generation of replication
competent retroviruses (RCR) typically competent retroviruses (RCR) typically
caused by homologous recombinationcaused by homologous recombination
Retroviral vectorsRetroviral vectors
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 vectorsRetroviral vectorsAdvantagesAdvantages1)1)long-term expression long-term expression 2)2)low toxicity low toxicity 3)3)high capacity high capacity 4)4)low antivector immunity allowing repeat low antivector immunity allowing repeat
administrationadministration
ProblemsProblemsLack of cell specificity:Lack of cell specificity:
Promiscuous: depositing genes into several Promiscuous: depositing genes into several cell types resulting in reduced target cell types resulting in reduced target efficiency and unwanted physiological effectsefficiency and unwanted physiological effects
Random splicing into host DNARandom splicing into host DNA resulting in resulting in normal gene disruption and/or alteration in normal gene disruption and/or alteration in gene functiongene function
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
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)
2/11 X-SCID patients developed 2/11 X-SCID patients developed leukemialeukemia
Adenoviral vectorsAdenoviral vectors
do not insert into genome
temporary
lack of specificity
strong immune response
Adeno-associated viral vectorsAdeno-associated viral vectors
Nature Reviews Genetics 1; 91-99 (2000);
Integrate into genome but small in size
AdvantagesAdvantages
non-toxicnon-toxic
no immune responseno immune response
Non-viral VectorsNon-viral Vectors
Non-viral VectorsNon-viral Vectorsliposomes (lipoplexes)liposomes (lipoplexes)
amino acid polymers: cationic polymers amino acid polymers: cationic polymers
e.g. B-cyclodextrinse.g. B-cyclodextrins
Non-viral VectorsNon-viral Vectors
naked DNA naked DNA
artificial human chromosomes artificial human chromosomes
Non-viral VectorsNon-viral Vectors Gene gun
Non-viral VectorsNon-viral VectorsReceptor-mediated endocytosis
Gene therapy in cancerGene therapy in cancergene therapy clinical trials
12%
6%
8%10%
64%
cancer
monogenic disease
infectious disease
vascular disease
others
clinical trials by vector
27%
12%
11%
2% 7%6%
34%
retrovirusadenoviruslipofectionnaked DNApox virusAAVothers
Based on
http://www.wiley.co.uk/genetherapy/clinical/
Conditionally replicating virusesConditionally replicating viruses
Replication of a conditionally replicatingvirus, ONYX-015, in a cancer cell from a patient with head and neck cancer during Phase II clinical testing.
Tumour-suppressor gene deliveryTumour-suppressor gene delivery
Nature Reviews Cancer (2001) Vol 1; 130-141
Delivery of agents that block Delivery of agents that block oncogene expressiononcogene expression
Nature Reviews Cancer (2001) Vol 1; 130-141
Nature Reviews Cancer (2001) Vol 1; 130-141
Conditionally replicating virusesConditionally replicating viruses
Current statusCurrent status
Food and Drug Administration (FDA) has not yet approved any human gene therapy
product for sale
ReferencesReferences
Chapter 28 Chapter 28
Mol & Cell Biol of Cancer by Mol & Cell Biol of Cancer by Knowles and Knowles and SelbySelby
Optional readingOptional reading
Human gene therapy by Ioannou, Panos A (www.els.net)
Nature Reviews Cancer (2001) vol 1 pp 130-141 by Francis McCormick