Module 5.1

What is gene therapy or delivery?

Gene therapy is an experimental technique that uses genes to treat or prevent disease.

What approaches to gene therapy are researchers currently testing?

Gene replacement

Gene silencing

Gene addition

Gene editing

What are some applications of gene delivery to tissue engineering??

• Helping to protect the engineered tissue

• Providing stimuli for the engineered tissue to grow and/or differentiate

Why do gene therapy?

• Can be more efficient

• less expensive

• long lasting

• therapeutic applications

What are some of the ethical considerations for using gene therapy?

• How can “good” & “bad” gene therapy uses be distinguished?

• high costs may limit access mainly to wealthy

What are some of the most expensive gene therapies?

• Lenmeldy

• Skysona

• zynteglo

How does gene therapy deliver new genetic material into cells?

It uses vectors—often modified viruses—that naturally enter cells. Their own genes are removed and replaced with the engineered therapeutic gene.

What are viral and non-viral methods of gene delivery?

Viral use:

• retroviruses

• lentiviruses

• adenoviruses

Non-viral delivery

• mechanical 

• electrical

• chemical

Compare viral vs. non-viral delivery

Viral delivery:

• cost: several fold higher

• time: > month

• transfection efficiency: > 90%

Non-viral delivery:

• cost: low

• time: 1 week

• transfection efficiency: 1-90%

What are the methods of non-viral delivery?

• Mechanical: microinjection

• electrical: in vivo/vitro electroporation

• chemical: calcium phosphate, protein, other polymers

What are the some common used gene delivery methods?

• electroporation

• liposomes

• calcium phosphate

• gold bullets

• human artificial chromosomes

What are some physical methods of delivery and there challenges?

• direct injection into nucleus - manually injecting is impractical

• electroporation - very delicate optimization of power/frequency

• ballistic particle delivery (gene gun) - bombard cell w/ nucleotides at high velocity. can physically damage cells

What are the properties of cationic polymers and its advantages?

Properties:

• contain positive charged groups

• formation of polyplexes w/ dna

Advantages:

• relatively inexpensive

• ability to incorporate ligands

How does the gene gun work?

• Gold or Tungsten particles are coated with DNA

• • The DNA-coated particles are placed on the end of a plastic bullet

• • The plastic bullet is placed in the gene gun

• • The target tissue is placed at the end of the barrel • An explosive charge /air pressure propels the particle bullet forward

• • The DNA-coated particles are released and strike the target tissue

What are some electroporation based technologies & treatments

• cell fusion

• biotechnology

• electrochemotherapy

• transdermal drug delivery

What are the 4 major problems w/ gene therapy?

1. short-lived of gene therapy - very hard to achieve any long-term benefits w/o integration & even w/ it.

2. immune response - reduces gene therapy effectiveness

3. problems w/ viral vectors - fears that viral vector may recover disease-causing ability

4. multigene disorders - common diseases are caused by combined effects of variations in many genes

How is gene therapy different for different diseases?

• Gene transplantation (to patient w/ gene deletion)

• Gene correction (to revert specific mutation in the gene of interest)

• Gene augmentation (to enhance expression of gene of interest)

• Targeted killing of specific cells by introducing killer gene

• Gene ablation - targeted inhibition of gene expression

What are the pros and cons of reto/lenti vectors 

pros: 

• stable gene transfer in vitro

cons: 

• retroviral vectors primarily transduce diving cells

• lentiviral vectors transduce dividing and non-dividing cell

• insertional mutagenesis/oncogneic potential

What are adenoviruses and problems with adenoviral vectors?

Adenoviruses are non-enveloped viruses containing a linear doube stranded DNA genome.

Problems:

• cannot integrate w/ the host cell genome

• expression from adenoviral vectors is transient (5-10 days)

What are the key features of adeno-associated virus (AAV)

• AAV can infect both dividing & quiescent (non-dividing) cells

• it does not stimulate inflammation in the host

• does not elicit antibodies against itself

• can enter non-diving cells

What are the desired features of viral vectors for in vivo gene therapy?

Efficiency

stability

low immunigenicity/toxicity

How would you treat huntingtons disease?

Using uniQure AMT-130, where it uses AAV vectors to deliver micro-RNAs directly to the brain fro non-selective knockdown of the huntington gene.

What are the challenges in gene therapy?

• Immume response (Gelsinger death)

• Disrupting important genes in target cells

• Commercial viability (developing a new therapy is expensive