Apoptóza a genová terapie

Mgr. Jan Bouchal, PhD.

Laboratoř molekulární patologie LF UP Olomouc

http://www.researcherid.com/rid/A-3859-2008

Funkce nádorového supresoru p53

senescence

transdifferentiate

Cell options

keep working

fuse

hypertrophy

enlarge &

divide

apoptosis

stress responses

de-differentiate

shrink

RETREAT OR ADVANCE

Apoptóza neboli programovaná buněčná smrt

• Charakterizována kondenzací chromatinu,

puchýřkovatěním cytoplazmatické membrány

(budding) a tvorbou apoptotických tělísek

• Apoptotická tělíska jsou fagocytována a nedochází k

zánětlivé reakci

http://en.wikipedia.org/wiki/Apoptosis

http://www.celldeath.de/mainfram.htm

Incomplete differentiation in two toes (syndactyly) due to lack of apoptosis

THE APOPTOTIC PATHWAY

Triggers Modulators Effectors Substrates DEATH

. FADD

. TRADD

. FLIP

. Bcl-2 family

. Cytochrome c

. p53

. Mdm2

. Caspases . Many cellular

proteins

. DNA

. Growth factor

Deprivation

. Hypoxia

. Loss of adhesion

. Death receptors

. Radiation

. Chemotherapy

Caspázy (cysteinyl aspartate specific proteinases; neaktivní prekurzory -procaspázy)

Receptory smrti

Molekuly v mezimemránovém prostoru mitochondrií (nejasné teorie – jednou z nich je tvorba pórů)

Receptorová a

mitochondriální

dráha apoptózy

Caspázy (cysteinyl aspartate specific proteinases; neaktivní prekurzory -procaspázy)

www.sabiosciences.com

Review in Cell Proliferation, 45, 487–498 (Quyang et al. 2012)

Metody detekce apoptózy („alespoň dvě“) Světelná, fluorescenční a elektronová mikroskopie (morfologie)

• kondenzace chromatinu, puchýřkovatění membrány (budding), apoptotická tělíska

• další znaky (viz níže)

Substráty caspáz

• Imunohistochemická detekce štěpení PARP, laminu B, keratinu 18, transglutaminázy

Mitochondriální funkce

• změny membránového potenciálu mitochondriální membrány (rhodamin 123, Mitotracker)

• uvolňování cytochromu c

Změny na cytoplazmatické membráně

• expozice fosfatidylserinu a vazba Annexinu V

• permeabilita membrány a vazba barviv na DNA (propidium iodid, Hoechst, DAPI) – odlišení od nekrózy

Změny DNA

• Apoptotický DNA žebřík

• značení štěpených konců DNA (TUNEL, ISNTA)

Metody detekce apoptózy

Světelná, fluorescenční a elektronová mikroskopie

(morfologie)

kondenzace chromatinu, puchýřkovatění membrány

(budding), apoptotická tělíska

Metody detekce apoptózy

Substráty kaspáz

• detekce štěpení keratinu 18 (protilátka M30 proti štěpenému fragmentu), laminu B (inverzní průkaz)

Metody detekce

apoptózy

Změny na cytoplazmatické

membráně

- expozice fosfatidylserinu a

vazba Annexinu V

Změny DNA

Apoptotický DNA žebřík

Další typy

buněčné

smrti

Normální buňka Nekrotická

Apoptotická Autofagická

Nekróza

1. Pasivní buněčná smrt v důsledku

fyzikálního nebo chemického

poškození

2. Charakteristická vakuolizace,

permeabilizace cytoplazmatické

membrány a vyvolání místní

zánětlivé odpovědi (významné

během mikrobiálních infekcí)

1. Dochází ke strávení části vlastního buněčného materiálu (auto – fagie)

2. Nejasný význam

- buď další forma programované buněčné smrti

- nebo strategie pro přežití v období nedostatku energie

Uberall 2010, Vesmír 90: 41-44.

Autofagie

Hlavní znaky

Apoptóza

• Charakterizována kondenzací chromatinu, puchýřkovatěním cytoplazmatické membrány (budding) a tvorbou apoptotických tělísek, které jsou následně fagocytovány

• Receptorová (receptory smrti, caspáza-8) a mitochondriální dráha (rodina proteinů Bcl2, cytochrom C, caspáza-9)

Nekróza

• Charakteristická vakuolizace, permeabilizace cytoplazmatické membrány a vyvolání zánětlivé odpovědi (významné během mikrobiálních infekcí)

Autofagie

• Pravděpodobně strategie pro přežití v období nedostatku energie

Genová terapie

• Při zrodu genové terapie se uvažovalo především o léčbě vrozených monogenních chorob (např. ADA - Adenosine Deaminase Deficiency, cystic fibrosis, Huntington's chorea, muscular dystrophy )

• V současné době se většina úsilí orientuje na získané choroby, především zhoubné nádory

• Genová terapie v léčbě nádorů

– Je lehčí buňku zničit, než ji opravit (vyvolání apoptózy, …)

– Zpravidla stačí krátká exprese genu (problém umlčení cizorodých genů, zpravidla metylací)

– Vzhledem k povaze onemocnění jsou pacienti ochotni podstoupit nové postupy

Germ line gene therapy

In the case of germ line gene therapy, germ cells, i.e., sperm or eggs, are modified by

the introduction of functional genes, which are ordinarily integrated into their genomes.

This option is prohibited for application in human beings, at least for the present, for a

variety of technical and ethical reasons.

Somatic cell gene therapy

In somatic cell gene therapy, the gene is introduced only in somatic cells, especially of

those tissues in which expression of the concerned gene is critical for health.

Expression of the introduced gene relieves/ eliminates symptoms of the disorder.

Broad methods

•A normal gene may be inserted into a nonspecific location within the genome to

replace a nonfunctional gene. This approach is most common.

•An abnormal gene could be swapped for a normal gene through homologous

recombination.

•The abnormal gene could be repaired through selective reverse mutation, which

returns the gene to its normal function.

•The regulation (the degree to which a gene is turned on or off) of a particular gene

could be altered.

http://en.wikipedia.org/wiki/Gene_therapy

CGE, constitutive gene expression

SGE, specific gene expression

NGE, normal gene expression

Jia et al. 2012, Cancer Treatment Reviews 38: 868-876.

• Není možno zasáhnout všechny buňky

• Sebevražedné geny a by-stander efekt

Cross et Burmester: Gene Therapy for Cancer Treatment: Past, Present and Future. Clinical Medicine and Research 2006;4:218-227.

„…Currently gene therapy is also being used to

create recombinant cancer vaccines. …“ Cells engineered in vitro

to be more recognizable

to the immune system

by the addition of one or

more genes, which are

often cytokine genes

that produce pro-

inflammatory

immune stimulating

molecules, or highly

antigenic protein genes.

… delivery

of immunostimulatory

genes, mainly

cytokines, to the tumor

in vivo.

… directly alter the

patient’s immune

system in

order to sensitize it to

the cancer cells. A

tumor antigen, or other

stimulatory gene,

is then added to the

selected cell type.

From donated blood (usually leukapheresis) precursor cells – monocytes are extracted and in Step 1 of cell culture, transformed into dendritic cells. From tumour tissue specimens obtained from surgery, a tumour-specific antigen is produced with which dendritic cells are loaded. Additionally, dendritic cells are also loaded with a control antigen – key-hole limpet hemocyanin (KLH). The control antigen enables monitoring of the success of vaccination. After an activation step, Step 2, the ‘mature’ antigen-loaded dendritic cells are administered to the patient in the form of vaccination.

Oncolytic gene therapy vectors are generally viruses that have been genetically engineered to

target and destroy cancer cells while remaining innocuous to the rest of the body. A number of

different viruses have been used for this purpose, including vaccinia, adenovirus, herpes simplex

virus type I, reovirus and Newcastle disease virus.

The most notable adenoviral therapy is the ONYX-015 viral therapy. ONYX-015 is an adenovirus

that has been engineered to lack the viral E1B protein. Without this protein, the virus is unable to

replicate in cells with a normal p53 pathway. Cancer cells often have deficiencies in the p53

pathway due to mutations and thus, allow ONYX-015 to replicate and lyse the cells. ONYX-

015 has been tested in phase I and II trials on squamous cell carcinoma of the head and neck that

resulted in tumor regression which correlated to the p53 status of the tumor.

Challenges in Gene Therapy

http://learn.genetics.utah.edu/units/genetherapy/gtchallenges/

From Research to Trials (15 years for ADA)

STEP 1: Learn about the disease

Is the disorder a good candidate for gene therapy? To find out, study the disease.

1) Get money for the project

2) Get approval for the project

3) Perform clinical research

4) Perform biological research

5) DECISION: Is the disorder a good candidate for gene therapy?

STEP 2: Design a gene therapy

1) Use your knowledge of the disorder to design a gene therapy

2) Test the therapy in appropriate models of the disease

3) DECISION: Does your therapy look promising?

http://learn.genetics.utah.edu/units/genetherapy/gtresearch/

STEP 3: Get money and approval for clinical trials

1) Get money for the trials

2) Get approval for the trials

STEP 4: Phase One clinical trial

1) Establish safety and dosage limits in a small group of people (20-80)

2) DECISION: Does your therapy still look promising?

STEP 5: Phase Two clinical trial

1)Test the efficacy and safety in a larger group of people (100-300)

2) DECISION: Is your therapy effective in a larger group of people?

STEP 6: Phase Three clinical trial

1) Test the therapy in a large group of people (1,000-3,000)

2) DECISION: Is your treatment successful?

STEP 7: Get FDA approval for general clinical use

1) Write proposals, fill out paperwork, answer questions and wait for approval

STEP 8: Phase Four clinical trial

1) Further test the efficacy and optimal use of the treatment in general use

www.wiley.co.uk/genetherapy/clinical/

Amariglio et al.: Donor-Derived Brain Tumor Following Neural Stem Cell

Transplantation in an Ataxia Telangiectasia Patient. Plos Medicine 2009; 6:e1000029.

A 13-y-old boy with AT, who is homozygous for the ATM gene mutation, presented to the

Sheba Medical Center in February 2005 with recurrent headaches. On examination he had

severe neurological deficits characteristic of AT, affecting mainly his motor functions and

making him wheelchair bound. The patient’s intelligence was normal and he is highly

motivated at school and socially active. His recent detailed immunologic and hematological

status is given in Table S2. Since the age of 7 y he has been treated for

hypogammaglobulinemia at the Sheba Medical Center with monthly intravenous

immunoglobulin.

In May 2001 at the age of 9y, in March 2002 at the age of 10y, and in July 2004 at the age

of 12 y, he was taken by his parents to be treated in Moscow with repeated transplantation

of fetal stem cells (see Text S1 for details as supplied to the parents by the patient’s

physicians in Moscow).

Four years after the first treatment he was diagnosed with a multifocal brain tumor. The

biopsied tumor was diagnosed as a glioneuronal neoplasm. Molecular and cytogenetic

studies showed that the tumor was of nonhost origin suggesting it was derived from the

transplanted neural stem cells. Microsatellite and HLA analysis demonstrated that the tumor

is derived from at least two donors.

Shrnutí

• Genová terapie v léčbě nádorů

– Je lehčí buňku zničit, než ji opravit a zpravidla stačí krátká exprese genu

– Vzhledem k povaze onemocnění jsou pacienti ochotni podstoupit nové postupy

• Genová terapie (by-stander efekt)

• Imunoterapie