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Simply, what is gene therapy?

Gene therapy is the delivery of nucleic acid polymers to a patient's cells as a drug to treat disease, usually to replace a mutated gene


What is the aim of gene therapy?

To replace, modify, or knock out defective, disease causing genes genes


What are the 3 routes by which gene therapy can be carried out?

1 Gene Addition: missing/mutant protein is supplied by expression of normal gene

2 Gene Correction/Alteration

3 Gene Knockdown

(last two are more challenging, use Zinc Finger Endonucleases - ZFN's)


What are the 4 barriers to successful gene therapy?

  • Uptake of vector, transport and uncoating
  • Vector genome persistence
  • Transcriptional activity
  • Immune response


What are the 2 types of cells that gene therapy can be applied to?

(again this is very broad)

  • Somatic: for cells found in the body
  • Germ-line: cells found in sperm and eggs (hereditary)


What are the 2 types of gene delivery approaches (vectors)?

Viral or Non-Viral


With gene therapy, what is the difference between insertion and transduction?

Insertion: integration of the DNA into the genome

Transduction: virus-mediated DNA transfer


How might gene therapy effects be short-lived?

Because it's hard to rapidly integrate therapeutic DNA into the genome, and the rapidly dividing nature of cells requires multiple rounds to make sure it's effective


What are the possible adverse responses to gene therapy?

Toxic, Immune and/or Inflammatory responses

May cause a new disease once inside

May induce a tumor if integrated in a tumor suppressor gene

May inactive an essential gene

Viral vector may infect surrounding healthy tissues


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

Ex vivo: cells are removed and grown in tissue plate, then therapeutic gene is made customized for the person, then reinserted into the body


In vivo: new DNA is delivered directly to cells with a virus


What 4 types of viruses are used in gene therapy?



Adeno-associated viruses

Herpes simplex viruses


What are the advantages of using a retrovirus for GT?

The coding region of the provirus is easily replaceable by the therapeutic gene


They infect cells at high efficiency, integrating a copy of their genome into the host cell


How might retroviruses be utilized for gene therapy?

What problems might occur?

Create double-stranded DNA copies from their RNA genome, using reverse transcriptase

Integrates into the human genome via integrase, which inserts the gene anywhere bc it has no specific site

-therefore this may disrupt the code of a gene, causing insertional mutagenesis-


Infectivity of retroviruses mostly limited to dividing cells

Only allows small length of code



What is the general map of the typical retrovirus genes?



(LTR = long terminal repeats)


What is the significance of the pol gene in retroviruses?

pol encodes for reverse transcriptase, RNase, and integrase



What is the significance of the env gene in the retrovirus?

Encodes for envelope glycoproteins, which mediate virus entry


What are lentiviral vectors?

Subgroup of retroviruses that infect both dividing and nondividing cells


So effective against neurons, muscle and liver cells, etc.


Potentially may lead to a cure for HIV


What are the advantages of using adenoviruses for GT?

-Can use very large inserts of DNA

-Can infect a broad range of mammal cells, both dividing and non-dividing

-High transduction efficiency



What are the disadvantages of using adenoviruses for GT?

-Transient expression! Viral DNA does not integrate

-So viral proteins can be expressed in host following vector administration

-Can be toxic with high dose

-Highly immunogenic


What are the advantages of using adeno-associated viruses (AAV) for GT?

-All viral genes are removed

-Does not stimulate immune response

-Enters both dividing and non-dividing cells

-Stable expression


What are the disadvantages of using adeno-associated viruses (AAV) for GT?

-Small insertional size of DNA

-Labor-intensive production

-Status of genome not fully known


What are the advantages of using herpes simplex virus for GT?

Affects neurons

-Allows large size of DNA

-Could provide long-term CNS gene expression

-Patient can take high dose


What are the disadvantages of using herpes simplex virus for GT?

Only infects cells of the nervous system

-Still under development

-Transient expression, currently

-Low transduction efficiency


What is the main barrier to using non-viral vectors in GT?

Currently, the nuclear membrane is difficult to bypass in order to alter DNA


So gene transfer is inefficient, and gene expression is transient


What is the strategy for knock-down of gene expression?

Antisense therapy