gene therapy injection of fertilized egg –not applicable to humans! figure 11-38

Gene therapy• Injection of fertilized egg

– not applicable to humans!

Figure 11-38

Gene therapy

Figure 11-39

Gene therapy• Germ-line gene therapy

– Transgenes are inserted ectopically• insertional mutations

• rarely replace mutant gene, so defective allele still present and can segregate away from transgene

– For effective germ-line gene therapy, need efficient targeted gene replacement i.e. gene “knock-in” by double crossover

Gene therapy• Somatic gene therapy

– corrects disease phenotype in affected somatic cells– two methods to introduce cloned gene into cells:

• 1a) Viral – defective retroviruses – for replicating cells only– integrate into genome – potential insertional mutations leading to eg cancer

• 1b) Viral – defective adenoviruses and parvoviruses– remain extrachromosomal– infect non-dividing cells eg lungs, nerves, muscles, liver– problem with inflammatory response

Oncogene 24:7802, 2005

Gene therapy

Gene therapy• Methods of gene transfer

– Viral methods:• Retroviruses – diploid RNA genome; integrates

– Replace viral genes with selected DNA

– Produce virus in packaging cell line (with replication defective helper virus)

– Helper virus env gene can be modified to alter host range and to target virus to specific cells

– Advantages: moderate efficiency, long-term expression

– Disadvantages: chronic overexpression; insertional mutagenesis; only infect replicating cells

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Viral approach

Endosome

Reverse transcriptase

Integratedgene

By recombination, the viral DNA, carryingthe gene of interest, is integrated into achromosome of the target cell.

Fig. 19.20bb (TE Art)Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Target cell

The viral coat is disassembled in theendosome, and the RNA is releasedinto the cytosol.

Retrovirus is taken into the target cellvia endocytosis.

The RNA is reverse transcribed intoDNA, which travels into the nucleus.

Icosahedral capsidRNA

Retrovirus-RNA genome containsgene of interest.

Envelope with spike proteins

Fig. 19.20ba (TE Art)

Gene therapy


– Viral methods:• Adenovirus – DNA virus; episomal

– Replace E1 (replication) gene with selected DNA

– Package in cell line with E1 gene (only)

– Virus enters cell interaction of viral proteins with receptors

– Advantages: high efficiency, infects replicating and nonreplicating cells; no insertional mutagenesis

– Disadvantages: immunogenic, transient expression

Oncogene 24:7802, 2005

Gene therapy

Oncogene 24:7775, 2005

Cancer Gene Therapy 1 – 15, 2006

Gene therapy


– Viral methods:• Adenovirus-associated virus (AAV) – nonpathogenic

parvovirus; DNA virus; integrates – Replace most of viral genome with selected DNA– Advantage: high efficiency, long-term expression, infects

replicating and nonreplicating cells, less immune response

• Herpesvirus – infect specific cell types– Eg HSV (neurons); HV7 (T-cells)

• Vaccinia and poxviruses – don’t integrate; – No significant immune response; transient expression

• Reoviruses – ds RNA– Oncolytic; preferentially infects/kills tumour cells not normal

• Alphaviruses – ss RNA; infect many cell types– Selected DNA replaces structural genes


– Physical methods:• DNA injection – directly into cells eg smooth muscle

• “Gene gun” – microparticles coated with DNA

• Electroporation – naked DNA injected into tumours; followed by electric pulses to induce pore formation

• Lipofection – encapsulate DNA in liposomes– DNA complexed with cationic lipid vesicles, taken up by cells via

endocytosis

• Advantages: DNA can be any size; non-infectious and can’t replicate; no inflammatory response

• Disadvantages: low efficiency; non-specific


Nonviral approach

Endosome

Integratedgene

By recombination, the DNA carryingthe gene of interest is integrated intoa chromosome of the target cell.

Fig. 19.20ab (TE Art)Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Liposome

DNA carrying thegene of interest

Target cell

The liposome is degraded within the endosomeand the DNA is released into the cytosol.

DNA-liposome complex is taken intothe target cell by endocytosis.

The DNA is imported into the cell nucleus.

Fig. 19.20aa (TE Art)

Gene therapy


– E.g. SCID (Severe Combined Immunodeficiency Disease) – usually fatal

• mutation in adenosine deaminase gene (ADA)

• results in accumulation of deoxyadenosine which is particularly toxic to T and B cells of immune system

• therapy cures symptoms

• problems with leukemia, perhaps due to insertion


Remove ADA-deficient lymphocytesfrom the SCID patient.

Culture the cells in a laboratory.

Infect the cells with a retrovirus thatcontains the normal ADA gene.

Reinfuse the ADA-gene-correctedlymphocytes back into the SCIDpatient.

Lymphocytes

Fig. 19.21 (TE Art)


– E.g. Cystic Fibrosis (CF) – death due to lung infections usually

• mutation in CFTR gene – encodes an epithelial cell ion transport protein

• defect leads to abnormal salt/water balance leading to accumulation of mucus in lungs

• need to treat cells in vivo, so use inhaled aerosol spray with either wt CF gene in a defective adenovirus or complexed with liposomes

Targeted cancer therapies

Eur J Cancer 41:2003, 2005

Acta Biochim Biophys Sinica 37:581, 2005

Gene therapy

Gene therapy• Strategies for gene therapy in cancer:

– Reversing oncogenic alterations• Antisense to inhibit oncogenes• Wt sense to replace tumour suppressor genes

– Transfer of genes to cause death of cell• Suicide genes• Proapoptotic genes

– Transfer of antiangiogenic genes– Enhance immune response

• Transfer of genes for passive and active immunity

– Increasing drug resistance of hematopoietic stem cells for protection during chemotherapy

Antisense gene therapy• Antisense oligonucleotides

– Sequences complementary to mRNA• Prevent protein translation, cause mRNA cleavage

– Sensitive to cytoplasmic degradation• Modify oligonucleotides to increase stability

– Phosphorothiolate oligomers (S replaces O in backbone)

• Expression vectors of RNAi constructs• Target genes involved in

– proliferation (MYC, HER2, PKC, IGF-IR, RAS, PKA, FOS, TERT, BCR-ABL, cyclinD1),

– anti-apoptosis (BCL2, MDM2, survivin, IGF1), – angiogenesis, metastasis (V integrin)

Gyn Oncol 99:736, 2005

Antisense gene therapy

Antisense gene therapy

Lancet Oncol. 2002 3:672

Ablation of oncogene function• Expression of gene encoding anti-ERBB2

intracellular single-chain antibody (sFv)– Downregulates cell surface ERBB2 levels– Induction of apoptosis in ERBB2 amp’n cells

• E1A represses ERBB2 promoter– Suppressed tumour growth, prolonged life in

tumour-bearing mice in model of breast cancer– Phase I trial for breast/ovarian cancer patients

(Ad5 E1A complexed with a cationic liposome)

Gene therapy• Tumour suppressor gene replacement

– Several TSGs induce apoptosis or cause cell cycle arrest in breast cancer cells

• Eg Rb, p53, p16, p27, p21, mda7, BRCA1, BRCA2, BARD1

– Clinical trials involving replacement of p53• Viral wt p53 sufficient to restore normal cell

proliferation and cell death in breast cancer cells• Associated with “bystander effect”, where surrounding

non-transduced cells also killed– Possibly via anti-angiogenesis, secretion of proapoptotic

factors, immune upregulation

– Registered clinical trials for Rb and mda7• Expression of Rb reduced tumorigenicity in nude mice

Gene therapy• Molecular chemotherapy/suicide gene therapy

– Toxin gene therapy• Transfection of genes encoding toxic molecules

– Gene-directed enzyme prodrug therapy (GDEPT)• Two-step: (1) deliver gene for enzyme to BrCa cell

• (2) non-toxic prodrug given, activated into toxic metabolite by the enzyme expressed in tumour

– Eg. viral thymidine kinase (TK) converts ganciclovir, bacterial cytosine deaminase (CD) converts 5-fluorocytosine and human cytochrome P450 (CYP2B6) converts cyclophosphamide

• Bystander effect – transfer of toxic drug via gap junctions, transduction of tumour cells, induced immunity

• Clinical trial with CYP2B6 showed some partial response

Gene therapy• Tumour specificity for suicide genes

– Use tumour specific promoter• Eg ERBB2 promoter (phase I trial targeted expression

of CD but no tumour regression)

– Use 5’UTR that limits efficient translation to tumour cells expressing high levels of specific factor

• Eg increased expression of translation initiation factor eIF4E in breast cancer

Proapoptotic Gene therapy• BCL2 family

– BCL-XS is dominant-negative repressor of BCL2 and BCL-XL (anti-apoptotic members)

• Induces apoptosis in human breast tumours in nude mice– Proapoptotic BIK is down-regulated in Br cancers

• BIK induced apoptosis in BrCa cells and tumours– Tumour expression of BAX cytotoxicity in BrCa

• Death receptor/ligand pathways– Tumour necrosis factor (TNF)-, TNF-related apoptosis-

inducing ligands (TRAIL), Fas ligand (FasL), caspase 3• TRAIL in BrCa cells/xenografts in mice cell death

• Ad5 E1A– Induces apoptosis and differentiation, inhibits cell

cycle, sensitizes to chemo- and radio-therapeutics


Proapoptotic Gene therapy

Antiangiogenic gene therapy• Plasmids encoding angiostatin and endostatin

– Complexed to liposomes inhibit breast cancer in nude mice

– Best if used in combination eg with tamoxifen

Genetic immunotherapy• Genes to enhance immune response

– Passive immunotherapy• Pre-formed antibodies, antitumour cytokines, tumoricidal

effector cells

• Expression of genes encoding single chain variable region of antibody vs tumour

– Active immunotherapy• Stimulates patient’s immune response via tumour vaccines

and immunostimulatory cytokines and chemokines

Nature reviews immunology 5:928, 2005

Genetic immunotherapy

Genetic immunotherapy• Immune-stimulation

– Transduction of tumour cells with cytokine genes• After radiation, cells used as vaccine (tumour antigen

presented with high levels immunstimulatory factor

– Autologous/allogenic normal cells expressing (IL2) cytokine, transfected with tumour cell DNA

• Array of weakly antigenic tumour-specific proteins expressed in highly immunogenic form

• Prolonged survival of mice with BrCa metastasis

Genetic immunotherapy• Immune costimulatory receptors/ligands

– BrCa cells often downregulate MHC molecules, etc• Replace by gene therapy

– Cytokines• Combination of Ad vectors with granulocyte-macrophage

colony-stimulating factor (GM-CSF), interleukin-2 (IL2) and HSV-TK reduced tumour growth more than TK alone

Genetic immunotherapy• Genetic vaccines

– Tumour-associated antigens (TAAs)• Aberrantly expressed normal host genes

– Eg ERBB2, carcinoembryonic antigen (CEA), MAGE-1, MUC-1, TERT, Fos-related antigen (Fra-1) in breast cancers

• Oncogenic mutated genes and gene fusions

– Enhance effectiveness by co-expression of cytokines and costimulatory molecules eg GM-CSF

– Express TAAs and cytokines in dendritic cells (DC) = antigen presenting cells

• Use these genetically modified cells as vaccines– Eg DCs with nonfunctional ERBB2 regression of BrCa in

BALB-neuT mice and induced lysis of ERBB2-expressing cells

Nature reviews immunology 5:928, 2005

Gene therapy• Somatic gene therapy (Toronto Star, 16/01/05)

– Potential for “gene-doping” in athletes • expected by 2008 Olympic Games in Beijing!

• already labeled as banned procedure in 2003 (by WADA)

– Instead of replacing defective genes, add new ones to enhance muscle strength or endurance, or increase the cardiovascular system’s capacity

• e.g. using genes identified for the treatment of muscle degeneration due to aging or disease like muscular dystrophy

• e.g. genes shown to enhance mouse muscle

– Unlike drugs, gene doping is one-time deal and hard to detect in blood or urine (e.g. need muscle biopsy)

Oncolytic viruses

• Viruses that replicate selectively in cancer cells vs normal cells– Cancer cells inactivate interferon pathway or

mutate tumour suppressor genes, enabling viral replication to proceed

• When they replicate, they lyse host cells• Commonly use adenovirus and herpes

simplex virus, mutated to replicate faster in cancer cells– Eg ONYX-015, a replicating adenovirus; >250

patients treated so far (2006)

Oncolytic viruses• Tumour-selective adenovirus –

– Delete viral proteins required to inactivate p53/Rb (eg E1B inhibits p53)

– Replicates only in cells deficient in p53 function already, killing them (oncolytic)

– Disadvantage: requires direct injection into tumour

Oncolytic virus infection


Eg p53-/-, Rb -/-

Oncogene 24:7775, 2005

Oncolytic virus infection

Oncolytic viruses• Problem: viruses are highly immunogenic

– Limits effectiveness to sites of injection, few doses

• Solution proposed: engineer viruses to induce immune response to tumour antigens or with therapeutic genes (eg cytokines, pro-drug activating enzymes, anti-angiogenic factors)– “arming” the viruses

• Potential side-effect: autoimmune disease– Many tumour antigens expressed in normal cells– Eg melanoma gene therapy with vaccinia virus

vitiligo in some patients due to identical antigens on normal melanocytes


Oncolytic viruses

Cancer Gene Therapy 1 – 15, 2006

Oncolytic viruses

Toxin geneGDEPT

(MRI or Heat-therapy)

Therapeutic cloning

• Cloning of mammals– e.g. Dolly the sheep

– difficult and low efficiency

– adult may age faster due to “old” nucleus to start with


The donor nucleus and the maternal proteinswithin the egg initiate development of theegg into an embryo.

The embryo is transferred into a surrogate ewe.

Allow pregnancy to proceed.

Surrogateewe

A lamb genetically identical to the donor sheep isthen born.

Fig. 19.9b (TE Art)


Mammarycell Unfertilized

egg

Nucleus

Mammary cell

The cells are fusedtogether.

Egg with nucleusremoved

Donor sheep's mammary cell is extracted and grownin a tissue culture flask. Another sheep's unfertilizedegg is extracted, and the nucleus is removed.

Fig. 19.9a (TE Art)

Therapeutic cloning• Source of tissues to replace damaged body parts

– e.g. brain cells for Alzheimer’s– e.g. pancreatic cells for diabetes– e.g. myoblasts for muscular dystrophy

• Disease models – somatic cells from patients e.g. with motor neuron disease– ES cells from blastocyst stimulated to differentiate into

specific cell type to study e.g. neurons

• Permitted in China, South Korea, Singapore– controlled in U.K., Australia– no legislation in U.S.A. (private funding), E.U.– banned in Canada


Blastocyst

Fetus

Inner cellmass

Embryonic stem cellsES cells(pluripotent)

Embryonic germ cellsEG cells(pluripotent)

Many types of adultstem cells(multipotent orunipotent)

Fertilized egg(totipotent)

Fig. 19.11 (TE Art)

Therapeutic cloning–Totipotent = potential to create entire organism–Pluripotent = potential to create any cell type–Multipotent = potential to create limited cell types–Unipotent = potential to create single cell type

gene therapy injection of fertilized egg –not applicable to humans! figure 11-38

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