Gene Therapy

Potential types of human genetic engineering

germ line gene therapy

human cloning using transformed cells (SCNT)

somatic cell therapy

Germ line gene therapy

sperm or egg cells are manipulated

used to correct damaged genes or for any other purpose

Human cloning using transformed cells (SCNT)

transforming cells in culture, then transferring the nucleus to an enucleated egg cell

used to correct gens ro make a variety of other changes

Somatic cell therapy

involves introducing DNA into cells either before or after birth

almost always used for treatment of disease

began in 1990: stormy process since then (Jesse Gelsinger)

successes slow in coming

lots of current or completed trials - ovarian cancer, breast cancer, brain tumors, arthritis, alzheimer’s, HIV-infected infants

Somatic cell therapy: 2 methods for inserting DNA into cells after birth

Ex vivo - cells are removed, cultured, transformed, then reinserted 

In vivo - DNA is introduced directly into the individual 

Somatic cell therapy: Approach (deal target)

the ideal target for gene therapy is a single recessive gene disorder in which a protein is not made

Adding in a functional gene can sometimes produce a protein to overcome the effects of a mutation

Various approaches to killing cancer cells (different targets)

Dominant mutations

broken genes that make damaging proteins (like alzheimers and huntingtons) are very difficult to control with gene therapy

gene silencing can be used - short interfering siRNA can be used to eliminate unwanted/altered RNA which allows normal RNA to function

gene editing may be useful in some of these cases

Ex vivo approach

straightforward

works well for tissue that can be replaced

In vivo approach

involve viral vectors (carriers) to deliver the DNA to the target cells 

Adenoviral vectors

DNA viruses

normally infect lungs; potential candidate for CF

triggers immune response; rapid elimination of the vector

short-term relief, but no integration - good because random integration can disrupt other genes

inhaler delivery

Adeno-associated virus (vector)

less hazardous than adenovirus; no immune response

Can integrate into chromosome 19, but rare

integration provides long term expression

requires another virus to be packaged, so should not spread beyond target cells

small genome, so limited capacity

Retroviral vectors

RNA viruses that use reverse transcriptase 

DNA is integrated 

used more often ex vivo 

LTRs contain strong promoters 

can make disabled vectors for therapy 

integrate randomly into the genome - long term expression, random = trouble 

Lentiviruses

type of retrovirus, similar to HIV

can avoid detection by the immune system

only retrovirus that can infect dividing or non-dividing cells

recent packaging systems developed that allowed for the development of viruses that will not spread

popular choice

Non-viral systems

liposomes

nanoparticles

CAR-T cells

Liposomes

used to target particular cells by including ligands in the membrane

CAR-T cells

chimeric antigen receptor 

an artificial receptor 

combines antigen-binding site of an antibody with signaling domains of T cell receptors 

activates T cells against tumor-specific antigens 

can be engineered to cause specific actions by the T cell

How are CAR-T cells created?

ex vivo delivery

CAR-T cell therapy process

1. remove blood from patient to get T cells

2. make CAR-T cells in the lab

3. grow millions of CAR-T cells

4. infuse CAR=T cells into patient

5. CAR-T cells bind to cancer cells and kill them

CAR-T cells: pros

personalized - receptor to patient’s own tumor antigens and immune antigens 

some very high cure rates (tested in patients with refractory or secondary tumors)

CAR-T cells: cons

lethal side effects - cytokine release syndrome (like in shock)

extremely expensive - $400-500K + hospitalization

tumor antigen escape

CAR-T cells: initial efforts and new efforts (are directed towards)

initial efforts against CD19 (B cell leukemias)

new efforts directed at solid tumors

CAR-T cells: 2 versions to control them

1. T cells are inactive until drug administered 

2. administering drug causes apoptosis of the T cells 

CAR-T cells: How do CAR-T cells help kill tumor cells but not normal cells with the same surface markers?

CAR gene in the T cells isn’t turned on until the T cell is in the right environment

cells in the target environment bear markers that bind to synNotch receptor which activates CAR transcription

Universal CAR-T cells

use antibodies as adapters to recognize different tumor Ag’s 

Advantages: cheaper, more flexible, can multi-task