GENE THERAPY

what needs to be specific about an ideal viral vector

-ability to integrate at a site specific location in the host chromosome or to be maintained as a stable episome -ability to target specific cell type

how do you chose an ideal virus vector to deliver gene product to normal tissue such as neurons?

-adenovirus vector because it infects non dividing cells such as neurons -retrovirus is not able to infect non dividing cells such as neurons

targeting cells ex-vivo

-cells removed from body -purified/enriched cell populations obtained -cells transfected with vector -transfected cells selected and returned to body

packaging cell line of recombinant adenoviruses

-complements the deficiency of the viral E1 gene -permits production of fully infectious but replication in complement virus

gene therapy approach for cancer that improves immune response to cancer

-delivery of cytokine genes into cancer cells to induce a local inflammatory reaction -enhance the recognition and reaction of T lymphocytes towards tumor cells by insertion of T cell receptor genes

properties of retroviral vector gene therapy

-directs stable integration of the therapeutic gene into the host genome -minimal risk since many retroviruses are non-pathogenic -murine leukemia virus (MuLV) commonly used & can incorporate large genes up to 7-7.5 kb -lentivurses are nonpathogenic to humans, but have the ability to transduce postmitotic cells/tissue, *including neurons, retinal, muscle, hematopoietic cells*

properties of an ideal vector

-easy and reproducible production in high concentrations -long term gene expression through insertion into genome or stable episomal persistence -gene expression that can be regulated (potential transcriptional unit whose promoter region can be activated/inhibited by small molecule drugs) -tissue specific expression -low immunogenicity/toxicity (should be nontoxic and not provoke undesired immune response) -high viral titre

disorders treated by gene therapy

-inherited single gene (monogenetic) disorders -acquired disorders (cancer)

properties of adenoviral vectors

-most widely used for in vivo -can hold large amounts of DNA (up to 35 kb) -human viruses which infect a wide variety of cell types at high efficiency -can infect dividing and non dividing cells -can be prepared in high titers (about 1000-fold higher than retro) -do not incorporate into the genome, must be re-administered, especially in actively dividing cells

non-viral vectors

-naked DNA injection -use of liposomes -receptor mediated endocytosis -low transfection efficiency (bad) -simple large scale production and low host immunogenicity (good)

adeno-associated viral (AAV) vectors

-nonpathogenic viruses that infect >80% of the human population and that DO NOT elicit an immune response -can be produced at large titers -only known viruses that show preferential integration into specific region of the genome at a specific site -since they are non-pathogenic, the insertion of the virus into the short arm of chromosome 19 indicates that this is a safe region of the genome -engineered AAV vectors have no retained the site specificity for insertion into the genome

ways to correct faulty genes

-nonspecific insertion into genome (gene augmentation therapy) -selective reverse mutation by homologous recombination or gene editing (CRISPR) -delivery of suicidal genes (direct killing) -delivery of immune enhancing genes (assisted killing) -regulation of gene expression (targeted inhibition)

approaches to replacing a gene w a healthy copy

-normal gene inserted into nonspecific location within the genome to replace nonfunctional gene -abnormal gene swapped for normal gene through homologous recombination -abnormal gene repaired through selective reverse mutation (gene editing-CRSPR)

limitations of retroviral vectors

-only infect actively dividing cells (except lentiviruses) -gene integration occurs randomly into the host chromosome -possibility exists for insertion mutagenesis (cancer) -hard to produce high viral titers -many cells express a MuLV receptor which limits the target specificity of viral infusion by in vivo approaches -developing a cost-effective way to manufacture

gene therapy approaches in cancer

-replace missing or altered genes with healthy genes -improve immune response to cancer -introduce "suicide genes" into cancer cells -delivery of genes into cancer cells which make them more sensitive to treatments -delivery of antisense DNA and ribozymes

inherited single gene (monogenetic) disorders

-severe combined immunodeficiency (SCID) (hematopoietic cells) -cystic fibrosis (airway epithelium) -duchenne muscular dystrophy (muscle) -familial hypercholesterolemia (liver)

cons of autologous ex vivo gene therapy

-stem cells are present in low abundance, and are difficult to isolate and culture in vitro -minimal number of available promoter-enhancer sequences that can drive long-term expression of various proteins

pros of autologous ex vivo gene therapy

-uses autologous cells -little chance of immune rejection since cells are taken from and infused back into same individual

targeting cells in vivo

-vector is injected directly into blood/placed directed into target tissue -adenoviral infection of tracheal epithelium/infection of tumor mass

limitations of recombinant adenovirus vectors

-vectors with E1 and E3 deletions elicit strong inflammatory/immune responses

the process of gene therapy

1) cells removed from body 2) in the lab, a virus is altered so that it cannot reproduce 3) gene inserted into virus 4) the altered virus is mixed with cells from the patient 5) cells from patient become genetically altered 6) altered cells returned to body 7) altered cells produce desired protein

choosing targets for gene therapy

1) is there any effective conventional treatment? 2) which genes are involved? 3) does the conditions result from mutations in one or more genes? (single gene is easier to treat) 4) will adding a normal copy of the gene fix the problem 5) are the tissues to which the new gene will be transferred accessible?

approaches to gene therapy

1) replacing missing/mutated genes that cause disease with healthy copies *most common approach* 2) introducing a new gene into the body to help fight a disease 3) changing the regulation of a gene

which of the follow is not an ideal vector for transferring genes to brain tumor cells? A) adenoviral vector B) retroviral vector except lentivirus C) vaccine vector D) none of the above

A) adenoviral vector

which of the follow is preferred vector for transferring genes to neurons? A) adenoviral vector B) retroviral vector except lentivirus C) vaccine vector D) none of the above

A) adenoviral vector

which of the follow is not an ideal vector for transferring genes to neurons? A) adenoviral vector B) retroviral vector except lentivirus C) vaccine vector D) none of the above

B) retroviral vector except lentivirus

which of the follow is preferred vector for transferring genes to brain tumor cells? A) adenoviral vector B) retroviral vector except lentivirus C) vaccine vector D) none of the above

B) retroviral vector except lentivirus

How does the CRISPR work?

CRISPR "spacer" sequences are transcribed into short RNA sequences ("CRISPR RNAs" or "crRNAs") capable of guiding the system to matching sequences of DNA. When the target DNA is found, Cas9 - one of the enzymes produced by the CRISPR system - binds to the DNA and cuts it, shutting the targeted gene off

gene therapy is to A) treat cancer using in vitro culture technology B) perform preclinical trial using animal model C) develop vaccine for enhancing immune response to drug treatment D) correct defective gene responsible for human disease

D) correct defective gene responsible for human disease

properties of an ideal vector for carrying a therapeutic gene are A) easy and reproducible production in high concentration B) high and long-term gene expression C) tissue specific expression D) high immunogenicity E) A-C

E) A-C

which region is used for insertion of recombinant DNA

E1

mutations in the cystic fibrosis gene (CFTR) cause

abnormally thick mucus

Which of the following vectors is not an ideal vector for transferring genes to brain tumor cells? A) adenoviral vector B) vaccine vector C) retroviral vector D) A & B

adenoviral vector

which of the following is a preferred vector for transferring genes to neurons? A) adenoviral vector B) retroviral vector C) vaccine vector D) none of the above

adenoviral vector

which of the following is a preferred vector for transferring genes to non-dividing cells such as neurons? A) adenoviral vector B) retroviral vector C) vaccine vector D) none of the above

adenoviral vector

3 approaches to therapeutic gene delivery

adenovirus, retrovirus, naked/plasma DNA

transfer of genes to somatic cells may be introduced to the cells when

all levels of development

targeting mRNA in gene therapy...antisense

antisense oligonucleotides hybridize with a specific target mRNA in the cell -may be introduced in a vector or as a synthetic oligonucleotide

adenovirus can infect

any kind of cell - dividing or nondividing - including neurons

brain tumor target cells and therapeutic genes

brain tumor cells ; HSV-tk

what type of disease is mostly treated by gene therapy treatments

cancer diseases (NOT genetic diseases)

best candidates for gene therapy

conditions/disorders that arise from mutations in a single gene

gene transfer to embryonic cells is primarily used for

creating recombinant proteins

adenosine deaminase (ADA) deficiency ->

defective immune cells -> body is open to infection by any germs that are around *treat by putting in normal, functioning copy f the ADA gene to solve the problem @ its source*

retroviruses infect ONLY

dividing cells (tumor cells not normal tissue)

retroviruses only infect ___ cels, which means that ___ are not infected by retroviruses

dividing; non-dividing (neurons)

after genetic modification, cells in non-autologous ex vivo gene therapy are ...

encapsulated in an artificial semi-permeable membrane, allowing for the diffusion of therapeutic protein but bypassing the immune response

involves transfer of a cloned gene into cells grown in culture. Those cells that have been transformed successfully are selected, expanded by cell culture in vitro, then introduced into the patient

ex vivo

used for cells that are accessible for initial removal and that can be induced to engraft and survive for a long time after replacement. Examples include cells of the hematopoietic system, skin cells, etc

ex vivo

cells in non-autologous ex vivo gene therapy may be

fibroblasts, keratinocytes, astrocytes, hepatocytes, or myoblasts (undifferentiated muscle cells).

viral vectors for gene delivery have what disabled and why

genes responsible for the pathogenicity of the viral vector ; exploits the natural ability of the virus to infect human cells (may elicit a high immune response thereby decreasing the efficiency of therapeutic gene expression)

what diseases are currently treated with gene therapy?

genetic disorders and others (cancer, etc) *cancer is #1 for gene therapy*

what does germ-line gene therapy target

germ cells (egg/sperm) which allows the inserted gene to be passed on to future generations *not practiced in human cells for ethical reasons*

what are different types of gene therapy?

germ line and somatic gene

what type of gene therapy has an impact on the next generation

germ-line

severe combined immune deficiency (SCID)

group of inherited disorders that cause severe abnormalities in the immune system by reducing the numbers/function of T/B lymphocytes

cancer cells are

highly proliferated and dividing cells

removal of E3 region could cause

immune response because its function is to suppress the immune response

the only option in tissues where the recipient cells cannot be cultured in vitro in sufficient numbers (e.g. brain cells) or where cultured cells cannot be re-implanted efficiently into patients

in vivo gene transfer

this approach is crucially dependent on the general efficiency of gene transfer and expression

in vivo gene transfer

non-autologous ex vivo gene therapy

involves isolating cells that grow well in culture and which can be transfected efficiently; these may originate from animals or humans (but NOT from the patient's own body)

ex vivo gene therapy is only possible if

it is relatively easy to take the effected cells from the patient's body (bone marrow stem cells, lymphocytes from the blood, skin and liver cells, lung epithelial cells, tumor cells) and propagate them in sufficient numbers outside the body

reason why adenoviruses don't have the possibility more mutagenesis

its a DNA virus and double stranded so one strand can replicate and make a lot of copies ... doesn't have to integrate into host cell's dna

insertion into host genome (viral vector)

must be specific in order to avoid insertion mutagensis

in the human body, what cells are non dividing?

neurons and mature cardiomyocytes

are changes to somatic cells heritable?

no

can we use retroviruses to deliver gene product to normal brain tissue such as neurons?

no

replication of episomes allows for

passage to daughter cells

why is using HSV TK-GCV system a good idea

phosphorylation of GCV kills tumor cells directly and indirectly by the bystander effect

how can we seek to manipulate genes in gene therapy?

replacing missing or mutated genes, introducing a new gene, changing the regulation of a gene

how do you chose an ideal virus vector to deliver gene products to brain tumors?

retroviral HSVTK-GCV system ONLY kills tumor cells NOT normal tissue such as neurons

Which of the following vectors is not an ideal vector for transferring genes to neurons? A) adenoviral vector B) vaccine vector C) retroviral vector D) A & B

retroviral vector

which of the following is a preferred vector for transferring genes to brain tumor cells? A) adenoviral vector B) retroviral vector C) vaccine vector D) none of the above

retroviral vector

suicide gene therapy HSV TK-GCV system

retrovirus engineered to produce and deliver Herpes Simplex Virus Thymidine Kinase - phosphorylates the drug ganiclovir which then incorporates into the DNA of dividing tumor cells and terminates chain elongation

what kind of virus will be used for the treatment of brain tumors?

retrovirus will infect tumor cells (not normal cells)

limitations of viral vectors

small capacity for therapeutic DNA and safety concerts

what type of gene therapy has no impact on the next generation

somatic gene

in gene therapy in humans - which cells are targeted

somatic only!

episomes

special vectors which carry a mammalian origin of replication which allows them to be maintained in moderate copy number

genes

specific sequences of DNA bases

non-viral method vector

synthetic vectors not based on viral systems like naked plasmid DNA gene transfer through gene gun or transfer via liposome vesicle *low transfection efficiency*

viral vectors

target defined types of cells or tissues and infect stationary as well as dividing cells because most cells in an adult patient are in a post-mitotic state (aden, AAV, retro)

chemical modification of RNA is an example of what

targeting mRNA in gene therapy

when genes are altered via mutation...

the encoded proteins are unable to carry out their normal functions, resulting in genetic disorders

Requisite components of CRISPR systems include what two DNA-editing tools

the enzyme Cas9, which cuts DNA at specific loci, and guide RNA (gRNA), a small piece of predesigned RNA sequence about 20 bases long located within a longer RNA scaffold. The scaffold part binds to DNA and the predesigned sequence "guides" Cas9 to the right part of the genome, ensuring that the enzyme cuts at the right point in the genome.

When the patient is injected with the HSV-TK gene therapy construct and subsequently treated with ganciclovir

the tumor cells that have incorporated the HSV-TK gene will be selectively killed

human gene therapy is...

the use of genes as medicines or drugs to treat/prevent human diseases by correcting altered genes in patient's bodies to rid them of illness forever

what is gene therapy?

the use of genes as medicines/drugs to treat or prevent human daises by correcting altered genes

the objective of somatic gene therapy is

to cure hereditary or acquired genetic defects by insertion of normal genes to target cells & to restore normal functioning affected cells

germ-line gene therapy

transfer of genes to human embryonic cells; creation of transgenic humans

somatic cell gene therapy

transfer of genes to somatic cells in order to correct/replace function of affected genes

bystander effect

transport of triphosphorylated ganiclovir between cells helps kill even non-transfected tumor cells

what does the E3 region code for

viral proteins that suppress the host's immune responses to viral infection

what are the advantages and disadvantages of viral vs. non viral methods?

virus - specifically insert nonviral - low transfection efficiency but simple large scale production & low host immunogenicity

why type of vectors will be used for gene delivery

virus and non-virus

what happens when you replace E1 gene with foreign DNA

virus replication rendered incompetent since the E1 gene is necessary for viral replication

can we use retroviruses to deliver gene product to brain tumor?

yes

autologous ex vivo gene therapy

• Cells derived from the patient's own body • Involves the isolation of totipotent hematopoietic stem cells • Effective in diseases which respond to bone marrow transplantation • Transfection of stem cells may lead to a permanent cure

goals of gene therapy research

• Development of injectable vectors • Targeting of vectors to specific cell populations • Safe and efficient gene transfer into appropriate regions of the genome • Regulation of gene expression by either administration of simple small molecule drugs or by the body's own physiological signals • Cost effective to manufacture • Curing the disease

What factors have kept gene therapy from becoming an effective treatment?

• Difficulty in delivering large sections of DNA to the correct site on the comparatively large human genome - *insertion mutagenesis* • Immune response • Problems with viral vectors: toxicity, immune and inflammatory responses, gene control and targeting issues, reversion of the virus to its original form. • Affect reproductive cells. • Short-lived nature of gene therapy • Multigene disorders