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