Gene Therapy (Dustin) Flashcards

1
Q

Simply, what is gene therapy?

A

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

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2
Q

What is the aim of gene therapy?

A

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

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3
Q

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

A

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)

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4
Q

What are the 4 barriers to successful gene therapy?

A
  • Uptake of vector, transport and uncoating
  • Vector genome persistence
  • Transcriptional activity
  • Immune response
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5
Q

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

(again this is very broad)

A
  • Somatic: for cells found in the body
  • Germ-line: cells found in sperm and eggs (hereditary)
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6
Q

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

A

Viral or Non-Viral

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7
Q

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

A

Insertion: integration of the DNA into the genome

Transduction: virus-mediated DNA transfer

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8
Q

How might gene therapy effects be short-lived?

A

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

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9
Q

What are the possible adverse responses to gene therapy?

A

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

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10
Q

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

A

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

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11
Q

What 4 types of viruses are used in gene therapy?

A

Retroviruses

Adenoviruses

Adeno-associated viruses

Herpes simplex viruses

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12
Q

What are the advantages of using a retrovirus for GT?

A

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

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13
Q

How might retroviruses be utilized for gene therapy?

What problems might occur?

A

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

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14
Q

What is the general map of the typical retrovirus genes?

A

LTR-gag-pol-env-LTR

(LTR = long terminal repeats)

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15
Q

What is the significance of the pol gene in retroviruses?

A

pol encodes for reverse transcriptase, RNase, and integrase

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16
Q

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

A

Encodes for envelope glycoproteins, which mediate virus entry

17
Q

What are lentiviral vectors?

A

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

18
Q

What are the advantages of using adenoviruses for GT?

A
  • Can use very large inserts of DNA
  • Can infect a broad range of mammal cells, both dividing and non-dividing
  • High transduction efficiency
19
Q

What are the disadvantages of using adenoviruses for GT?

A
  • 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
20
Q

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

A
  • All viral genes are removed
  • Does not stimulate immune response
  • Enters both dividing and non-dividing cells
  • Stable expression
21
Q

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

A
  • Small insertional size of DNA
  • Labor-intensive production
  • Status of genome not fully known
22
Q

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

A

Affects neurons

  • Allows large size of DNA
  • Could provide long-term CNS gene expression
  • Patient can take high dose
23
Q

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

A

Only infects cells of the nervous system

  • Still under development
  • Transient expression, currently
  • Low transduction efficiency
24
Q

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

A

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

So gene transfer is inefficient, and gene expression is transient

25
Q

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

A

Antisense therapy