gene therapy cancer

genetic alterations in cancer

-DNA Repair: genetic instability -tumor suppressor genes: interstitial deletion, inactivating mutation, hypermethylation -oncogenes: gene amplification, gene overexpression, activating mutation

pathways to activate oncogenic lesions

-RAS -MYC -E2F

what was the first successful HGT treatment in 2005?

-X-SCID -but patients got leukemia bc activation of oncogene

desired features of gene therapy viral vector

-able to enter wanted human cell -can generate recombinant viruses -can grow recombinant viruses in cell culture -replication-defective form is safe & can accomodate sufficient length of foreign DNA -deliver genetic material in form of DNA -no components that elicit immune response

limitation of retroviral vectors

-cannot infect nondividing cells (brain, muscle, lung, liver) -can randomly integrate vector DNA into host chrom

has 2 genes between inverted terminal repeats that define beginning and end of virus and contain packaging sequence

-cap and rep genes -adeno-associated viral vector

mechanisms for controlling progress through cell cycle

-checkpoints -telomere length -chemical signals inside & outside of cell

genetic instability of cancer cells

-cooperative genetic material -mutagenic agents -defective repair systems

construction of eukaryotic virus vectors

-delete some or all virus genes -retain cis-acting replication -package sequence in vector genome -insert genes of interest in vector geome

v-onc transduction

-derived from c-oncs and were transduced by viruses -fusion proteins w additional sequences derived from virus genes (gag sequence at amino terminus of protein is most common) -these sequences alter function or cellular localization of protein -these abnormal attributes cause transformation

basic biological features of neoplasms

-differentiation -abnormal proliferation -angiogenesis -invasion -apoptosis -senescence

self-maintained replication of cancer cells

-down regulation of apoptosis -lack of response to inhibitory factors -self-sustained proliferation

longer survival of cancer cells

-down regulation of apoptosis -telomerase reactivation

viruses that contribute to human cancer

-epstein-barr (HHV4) -Hep B -Hep C -Kaposi's sarcoma-associated herpesvirus (HHV8) -human papillomavirus -HTLV I & II

p53

-forms complex w SV40 T-antigen (& other DNA virus oncoproteins such as adeno- and papilloma-) -gene that encodes this is altered or mutated -central role in controlling cell cycle -is a TF to activate cellular gene expression so cell cycle can arrest at G1 -tviruses need ongoing cell DNA replication to proliferate, so thats why they target this gene

properties of transformed cells in vitro

-genetic changes (polyploidy) -immortalization -metabolic changes (rapid growth) -independent anchorage (loss of adhesion) -less serum needed in medium to grow -loss of cell cycle control -change in membrane structure and function -tumor-associated carbohydrate antigens

mutations in cell DNA from transformed cells

-loss of function: affects tumor-suppressor genes -acquire functions that disrupt normal behavior: oncogenes activated

transforming genes of DNA tumor viruses

-no cellular counterparts -functions of oncoproteins are less diverse compared to retroviruses -mostly nuclear proteins involved in control of DNA replication -interact w cell proteins which normally have negative role in cell proliferation, to achieve wanted effects

immortalization

ability to be passed indefinitely in cell culture, so the reactivation of telomerase is a key event/prevents cell aging

inherited case of cancer

all cells carry one mutation -second mutation nu probability

gene therapy

introduce DNA sequences into cells of patient to achieve clinical benefit (e.g. inherited single gene defects) -object is to destroy cancer cells

oncolytic virus

kills cancer cells

-infect nondividing and dividing cells -more complicated than retroviruses bc contain 6 more proteins (tat, rev, vpr, vpu, nef, vif) -no potent antibody response -low cellular immune response so good for in vivo gene delivery w sustained expression

lentiviral vectors

E2F

needed for transcription of adenovirus genes & involved in transcription of cellular genes

tumor-associated viruses

oncoviruses

transduction

process where genetic material (DNA) is inserted into a cell

adeno-associated virus vectors

pros- all viral genes are removed, safe(?), transduce nondividing cells, stable cons- small genome size limits size of foreign DNA, genome status not fully elucidated

ex vivo gene therapy

remove cell, genetically modify (reverse transcription for recombinant DNA), transplant back into patient

ideal oncolytic virus

-non human pathogen -not cause toxicity or severe side effects -can be genetically manipulated to help introduce therapeutic or suicide genes or genes that monitor viral spread -rapid life cycle (replicate, lyse, and spreads quickly) -can be administered systemically (IV) -can eradicate tumor and abolish antitumor immunity -does not enter target cell nucleus & cannot recombine w host cell genome -naturally replicates in tumor tissues only -susceptible to antiviral drug -characterized well by viral genome and protein function -well characterized mechanisms of oncolytic action and tumor specifitiy

adenovirus-based vectors

-nonenveloped -linear dsDNA -does not integrate into host genome -replicates as episomal element in nucleus -pros: higher titer, efficient transduction of nondividing cells in vitro and vivo cons: toxic, immunologic response, prior exposure, expression lasts short time post infection (5-10 days) *immune response causes cell-mediated and humoral (antibody producing) response

proto-oncogenes (c-oncogenes)

-normal cellular genes that control cell signaling and regulate cell division cycle

Rb

-optic nerve tumors: retinoblastomas -forms complex w E2F -inactive E2F-Rb complex formation also arrests cell cycle at G1 -E2F release by replacement w E1A-Rb, T-antigen-Rb, or E7-Rb complexes causes cell and viral DNA replication

most important cellular proteins involved in cell transformation by DNA viruses

-p53 -Rb

ways viruses can be engineered to deliver and express certain genes

-produce specific proteins in cultured cells -vaccinate animals or humans against certain disease -gene therapy in animals or humans to replace defective genes -anticancer agents to kill tumor cells

chemical or physical DNA delivery for gene therapy

-pros: elicits minimum host immune rxn, large size of DNA -cons: low transduction efficiency, transient expression -ex: electroporation, calcium phosphates, liposomes, naked DNA

replication defective viruses used in gene therapy

-pros: high transduction efficiency, transient/permanent expression -cons: elicit host immune rxn, limited size transgene -ex: adenovirus, adeno-associated virus, retrovirus, herpes

cis-activation of c-oncogenes

-provirus that integrates into host cell genome close to c-onc sequence may indirectly activate gene expression -occurs if provirus is integrated upstream of c-onc gene, expressed via read-through transcript of virus genome -insertional activation also occurs when provirus integrates downstream c-onc sequence or upstream in an inverted form

common features of DNA viruses

-replicate in nucleus -DdDp and DdRp replicates and transcribes viral genome -generally induce entry of cell into S phase since cells are not dividing (in G0 phase) -must find and infect cells going through S phase, though -always risk of viral genome integration into host chromosomes since most replicate in nucleus and have DNA genome

hallmarks of cancer cells

-self-maintained replication -longer survival -genetic instability -can induce neoangiogenesis -can invade and metastasis

apoptosis

-signal arrives at death receptor on cell (stimulus: biochemical signal, UV radiation, x rays, toxin) -caspase enzyme carries out cell destruction -WBCs destroy cell fragments (phagocytosis)

sporadic case of cancer

-somatic mutation in one cell -second mutation u probability

trans-activation of c-oncogenes

-tax protein acts in trans to stimulate transcription from virus LTR -tax protein activates transcription of cellular genes by interacting w TFs

mechanisms of cell transformation by retroviruses

-transduction by v-oncogenes -cis-activation of c-oncogenes -trans-activation of c-oncogenes by viral reg. proteins

production of eukarytoic virus vectors

-transfect genome into cell lines that are engineered to express virus genes deleted in vector -transfect genome into cells coinfected w helper virus that expresses missing genes

conditions for cell transformation by viruses

-virus capable of noncytolitic interaction w cell (latent) -virus gene must be stably inherited by daughter cells so that all or most of its genome persists in transformed cell and usually integrated into host cell chromatin

all homology with vector backbone is removed in retroviral vectors because??

-you wanna prevent recombination that results in a replication competent retrovirus

How do oncolytic viurses cause tumor cells to die?

1) as consequence of infecting and replicating in cancer cells 2) by inducing apoptosis 3) by causing cancer cell to lyse while expulling progeny virus particles -stimulating host immune system

How many divisions does it take to cause mitosis to stop, when shortening telomeres?

50

spindle assembly checkpoing

Anaphase during mitosis

telomerase

an enzyme that elongates telomeres

acquired diseases

cancer cardiovascular neurological disorders infectious

oncoviruse

causes cancer

carcinogenesis (oncogenesis)

complex, multistep process where cellular transformation could be only the first, essential, step along the way

cell destruction by caspase enzymes

condenses chromatin collapses nucleus DNA repair turned off DNA replication stops DNA cutting enzymes turned on cytoskeleton disassbles blocks cell adhesion marks cell surface for phagocytosis

genetic diseases

cystic fibrosis blood disorders muscular dystrophy diabetes

in vivo gene therapy

direct transfer of genetic material into patient

cap gene

encodes viral capsid proteins

apoptosis checkpoint

end of G2

`the great majority of viruses of vertebrates are oncogenic: true or false?

false

transformed cells automatically result in cancer: true or false?

false

transformation is usually accompanied by productive infection: true or false?

false! it is accompanied by continued expression of a repertoire of virus genes!

infection of oncogenic viruses inevitably leads to cancer: true or false?

false! this is a rare and multifactorial event!

cell transformation and tumor formation are one in the same, so more events are NOT required to develop leukemia: true or false?

false-- additional events ARE required

this response prevents any subsequent infection of a second injection of antibodies to adenoviral proteins

humoral response

-can permanetly introduce foreign gene into tissue by integration -less immunogenic -packaged w G envelope protein of stomatitis virus to allow infection of many cells -based on Mo-MLV -gag, pol, env replaced w transgene of interest/expressed on plasmids in packaging cell lines -genes are non-essential & lack packaging sequence so not included in virion particle

retrovirus vectors

transformation

the change in the morphological, biochemical, or growth parameters of a cell culture; usually accomplished by immortalization

tax gene

transcriptional activator protein involved in trans-activation of c-oncogenes

neoplastic transformation

transformation that does NOT result in cells able to produce tumors

What is pertinent if therapy is to be permanent?

transgene to persist indefinitely in cells: chromosome integration, autonomous replication of its genome, partition during cell division, etc..

HIV has been disabled and developed as vector for in vivo gene delivery: true or false?

true

In cancer cells, telomerase activation occurs to restore telomere length: true or false?

true

True or false: retrovirus can only infect dividing cells

true

chromosomal abnormalilties that occur in HTLV-transformed cell population are also required to produce malignant tumor: true or false?

true

in a tumorigenic interaction w a host, many viruses lose their ability to produce progeny and transmit to other hosts instead: true or false?

true

many transforming retroviruses do not contain oncogenes- they activate c-oncs by insertional activation: true or false?

true

oncolytic viruses propogate more effectivly in tumor cells compared to normal host cells: true or false?

true

poxvirus can infect dividing cells & selectively inhibit tumor cells: true or false?

true

true or false: adeno-associated virus can infect dividing and nondividing cells, but needs helper virus to replicate inside cells

true

virus genomes in transformed cells are usually replication defective: true or false?

true

rep gene

viral replication and integration -viral DNA can integrate preferentially into human chrom 19 w rep gene