Gene therapy

For what abilities are particular viral vectors chosen?

-Ability to recognize and infect certain cells -to transfer the genes to cells -to alter DNA (permanently or temporarily)

What are some forms of transfection using non-viral mediated gene therapy?

-Chemicals, liposome mediated, electroporation and receptor mediated -direct transfer via (microinjection and particle bombardment) -bacterial gene transfer (involves live invasive bacteria [salmonella/shigella] those who transfer DNA through their pillus [agrobacteria tumefaciens]

How is mitochondrial DNA replacement therapy done?

-Oocytes from a patient's mito w mtDNA mutations (gray) & a healthy egg donor w normal mitochondria (purple) are retrieved. After successful exchange of the nuclear genome, the resulting oocyte contains normal mitochondria (purple) from the donor and the nuclear genome from the affected patient (blue) and can undergo IVF to produce an embryo

What are some challenges facing human gene therapy?

-human immune system may fight off the viral vector -appropriate vectors -gene therapy is still in its infancy -current therapies are experimental -short lived - not sustaining

Gene therapy is used for what disorders?

-if the disease is incurable/life threatening -depending on the genes involved - a copy of the target gene available -depending on what organ, tissue and cell types are affected ***can problem be corrected by using therapeutic DNA and can therapeutic gene be delivered to the affected tissue cells

How is gene therapy done?

1. normal (therapeutic) DNA is isolated 2. gene therapy -DNA packaged into a vector -vector unloads DNA into target cell -faulty or damaged cell restored to normal

What was the first usage of human gene therapy?

1990 - Ashanti DaSilva -ex vivo used for SCID with ADA gene

How can gene therapy be used to rid men of cancer?

1: Blood taken from patient 2: T cells infected with virus to carry key genes into them 3: DNA from genes helps cells develop receptors 4: Modified cells injected back into patient 5: Receptors target cancerous cells to be killed

What large vector is capable of carrying substantial amounts of genetic code and would exist autonomously alongside the standard 46, not chasing ant mutation?

47th chromosome - artificial human chromosome into target cells

What are the non-integrating viral vectors that are nonpathogenic single stranded DNA viruses, capable of infecting both dividing and non dividing cells (human parvoviruses) ?

Adeno-associated viruses

What are some non-integrating viral vectors used for gene therapy?

Adeno-associated viruses Adenoviruses Poxviruses Herpes viruses

What are the non-integrating viral vectors that are non-enveloped viruses containing a linear double stranded DNA genome (the virus causing the common cold)?

Adenoviruses

Germline gene therapy could potentially correct a genetic defect in a ...?

Affected individual and some or all of his/her descendants.

What are potential applications of gene therapy for the treatment of cancer?

Artificial killing of cells -insert a gene encoding toxin or a gene conferring sensitivity to a drug into tumor cells Stimulate natural killing of cancer cells -increase anti tumor activity of immune system cells -induce normal tissues to produce anti tumor substances (interluekin-2 and interferon) -protect surrounding normal tissues from effects of chemo/radio -selectively inhibit the expression of the oncogene -gene augmentation therapy, insert wild-type tumor suppressor gene

How is stem-cell based gene therapy used to cure sickle cell disease?

Combined RNA interference with globin gene transfer to create a therapeutic transgene therapeutic gene was engineered into a lentiviral vector and introduced into haematopoietic stem cells (HSC) to produce normal haemoglobin. two functions -produce normal haemoglobin -suppress the generation of haemoglobin S.

What type of gene therapy involves altering the regulation (the degree to which a gene is turned on/off) of a particular gene?

Gene alteration *using inhibitors which can bind to microRNA - will prevent the mRNA from being expressed

Which gene therapy involves inserting a normal gene into a NONSPECIFIC location within the genome to replace a nonfunctional gene?

Gene augmentation therapy

What type of gene therapy involves silencing a gene, or when a exogenous gene inhibits gain of function caused by mutation, or a pathogen?

Gene inhibition therapy *hungtington's disease

What type of gene therapy involves repairing an abnormal gene?

Gene repair therapy *zinc finger nuclease = ZFN - cleave where you have mutation - homologous recomb you introduce the normal gene --> repair

What is the gene therapy were you swap an abnormal gene for a normal gene through homologous recombination? What is this used in?

Gene replacement therapy -used in transgenic mice knockout experiments

What is the treatment of a disease by genetically modifying the cells of a patient (usually involving the insertion of normal gene DNA directly into the cell)?

Gene therapy

Which gene therapy has modification in gamete or fertilized egg (zygote or early embryo)?

Germline gene therapy

What is the non-integrating viral vector that is a linear double stranded DNA molecule in its genome a human neurotropic virus?

Herpes viruses (herpes simplex virus type 1)

What is the difference between in vivo gene therapy and ex vivo gene therapy?

In vivo - put the cloned gene DIRECTLY into patient's genes Ex vivo - remove cells, do gene transfer and now some cells have X, return genetically modified genes to patient

What uses an artificial lipid sphere with an aqueous core with a liposome?

In vivo liposome gene delivery

What is gene augmentation therapy usually used to treat?

Inherited disorders caused by loss of function of a gene - used to treat ADA - adenosine deaminase deficient patients suffering from severe combined immunodeficiency

What happens in injection of a fertilized egg vs germinal therapy vs somatic therapy?

Injection of fertilized egg - normal mouse Germinal therapy - injected into blastocyst stage and the mosaic soma is made Somatic therapy - injection of transgene - transgenic clones made

What is it when a viral coat protein binds to a specific receptor on the plasma membrane of cells, followed by endocytosis ?

Receptor mediated endocytosis

What are some common integrating viral vectors used for gene therapy?

Retroviruses - single stranded RNA viruses - moloney murine leukemia virus (Mo-MLV) Lentiviruses - specialized retroviruses that can infect both dividing and non-dividing cells (Human immunodeficiency virus)

What are the main differences between somatic and germline gene therapy?

Somatic -confined to a patient -changes not heritable -may have to be repeated in future generations Germline -changes are heritable -prevents the disease/trait from being passed on to future generations

Which gene therapy has modification introduced into non-reproductive cell implicated in disease?

Somatic gene therapy

What cells are collected from patients with ADA in gene augmentation therapy?

T lymphocytes

What is a strain of herpes virus (HSV-1) that primarily infects cell of the CNS an example of?

Targeted gene therapy (viral vector)

Why is gene therapy done?

To correct or prevent a pathological process -acquired and genetic diseases

What type of gene therapy is also called TRANSDUCTION and is when therapeutic DNA is delivered to the cell using a vector that is a virus?

Viral mediated gene therapy

The viral vectors are modified which means they ...

are unable to cause disease -increase their ability to deliver specific genes to a patient's cells

How has gene therapy been proven to prevent the onset of diabetic symptoms in mice?

by inserting a gene encoding for a cytokine (a protein that stimulates or inhibits the proliferation or function of immune cells) into their insulin-producing cells

What is the difference between integrated and non-integrated genes?

exogenous genes that integrate into chromosomes can be stably transmitted to all daughter cells, unlike episomal genes (extrachromosomal)

In "in vivo" liposome gene delivery, what carries therapeutic DNA and is capable of passing the DNA through the target cell's membrane when injected or sprayed into the tissue?

liposome

All of the integrated gene new cells will have the therapeutic DNA, where as in the extrachromosomal...?

only some will have the therapeutic DNA

What led to the death of Jesse Gelsinger in 1999 proving that human immune system may fight off the viral vector?

ornithine transcarbamoylase (OTC) deficiency, an inherited disorder: commonly causes death in affected newborn males from inability to properly process nitrogen in food proteins. Gelsinger's died from immune reaction to engineered adenovirus that was infused into his liver.

viral = ? nonviral = ?

transduction transfection