6. Gene therapies

Gene Therapy- The insertion, alteration, or removal of genes within an individual's cells and tissues in order to treat disease

Classifying Gene Therapy

How you introduce into patient’s gene

Terminology

Transformation = transfer of naked DNA into bacteria.

Transfection = transfer of naked DNA into cells.

Infection = infect with a wild type virus.

Transduction = A viral vector with foreign DNA introduced into another cell

Titer = way of expressing concentration of viral particles (Infectious Units per ml, IU/ml)

Genetic background of diseases

How disease can occur

  • Environmental factors: infectious diseases
  • Genetic factors: inherited disorders
  • Monogenic (caused by one gene)
  • Polygenic (requires many genes to manifest) ← uncommon
  • Environmental factors + genetic factors: cancers, chronic diseases

Ex vivo or In vivo

Ex vivo: Gene of interest transferred into patient’s cells. Ex vivo means "out of the living" in Latin.

In vivo: Viral vectors produced in the lab and injected into the target organ. In vivo means "within the living" in Latin.

Ex vivo: Allogenic and autologous

Allogeneic: from a donor

Autologous: from the patient itself

Autologues-

Advantages:

•Patient-specific

•No rejection by the immune system

•No risk for graft-vs-host disease

•Repeated doses possible

Challenges:

•High costs for manufacturing and quality testing

•Starting material variability (donor variability)

Allogenic-

Advantages:

•Larger-scale manufacturing possible

•Donor can be screened for desirable characteristics

•More reproducible manufacturing (less donor variability)

•Banking possible (immediate availability)

Challenges:

•Risk for graft-vs-host disease needs to be minimized through additional steps (cost increase)

•Risk of rapid rejection as cells are still recognized as foreign cells

Plasmids

  • Circular double stranded DNA molecule, can be propagated in bacteria
  • Naked DNA (i.e. no surrounding membrane, outside the nucleiod)
  • Can replicate independently of chromosomal DNA in the cell
  • Excellent gene carriers

Plasmid production

Bacterial transformation is a process of horizontal gene transfer (transmission of DNA between different genomes) by which some bacteria take up foreign genetic material (naked DNA) from the environment.

Plasmid mediated gene delivery

• Low gene transfer efficiency

• Only transient expression (non-integrating)

• Difficult with primary cells

→ This why people have moved onto Viral vector

Delivery Vehicle: Viral

Two main classes

  • Integrating
    • Retrovirus
    • Lentivirus
  • Non-integrating
    • Adenovirus
    • Adeno-associated virus

Adenovirus

  • Class of virus with double-stranded DNA genomes
  • infect dividing and non dividing cells
  • DNA is not integrated into host cell genome

Pros:

  • High efficiency of transduction
  • High levels of transgene expression
  • Rarely integrates into hest cell genome
  • Infect both dividing and non-dividing cells
  • Easily produced in high amounts

Cons:

  • Causes common cold symptoms in human
  • Difficult to achieve persistent gene expression - since non integrating

Adeno-associated virus (AAV)

  • Class of small, single-stranded DNA virus
  • transduce both dividing and non-dividing
  • DNA is not integrated into host cell genome (it can integrate but at low frequencies)

Pros:

  • Not pathogenic to humans
  • Low immunogencity
  • Low cytotoxicity
  • Smaller than Adenovirus (1/3 of the diameter), have a relatively small packing capability

Cons:

  • Difficult to achieve persistent gene expression - since non integrating

Retrovirus

  • Class of virus that can create double-stranded DNA copies of their RNA genomes.
  • These copies can be integrated into the chromosomes of host cells.
  • The vector genome integrates into the host cell genome, whereby persistent transgene expression is expected.
  • Most retrovirus can only infect dividing cells.

Pros:

  • Persistent transgene expression is expected
  • Non-dividing cells resistant

Cons:

  • Due to the integration, there is a potential risk of insertional mutagenesis

Lentivirus (e.g.HIV)

  • Sub-species of Retrovirus
  • Unique ability among retroviruses of being able to infect non-dividing cells
  • Successful transduction of host cells by lentiviral vectors does not require active cell division because of nuclear transport mechanisms (not require dividing cells due to that transport)

Pros:

  • Persistent transgene expression is expected
  • Less likely to activate adjacent oncogenes
  • Tends to integrate more within active genes, but less frequently upstream of transcriptionally active promoters.

Cons:

  • Due to the integration, there is a potential risk of insertional mutagenesis, although less than with gammaretroviral vectors, because it is more random

Clinical example: ADA-SCID

• A rare disorder caused by the retrovirus inserted near a proto-oncogene promoter.

• Caused by a mutation that results in the absence of protein called adenosine deaminase (ADA), which is required for the production of lymphocytes.

• Children born with ADA-SCID do not develop a healthy immune system so cannot fight off

everyday infections, which results in severe and life-threatening illness.

CAR-T cell therapy- CAR-T cell therapy involves re-engineering a patient’s own T cells to recognize and destroy cancer. T cells are currently being explored as carriers for chimeric antigen receptors (CARs)

  • A specific cell type (e.g. T, γδ Τ) is isolated from the patient’s own blood.
  • These T cells are genetically altered to express a TCR (T cell receptor) that binds to a specific antigen on the patient’s tumour.
  • Expansion of these cells and re-infusion follow.
  • CAR-T cells attack cancer cells

Drawback: tight control by natural cytotoxicity receptors and KIRs, which therefore might be educated over time to avoid tumour recognition

Pros:

  • When using patient’s own cells, no risk of graft-versus-host disease
  • Potential for lasting immunity even after a single infusion

Cons:

  • High cost (personalized gene therapy)
  • Length of time required for T-cell processing and modification (a delay can be detrimental)
  • Adverse events including cytokine release syndrome (CRS)
  • Natural cytotoxicity receptors and KIRs tightly regulate the activity of γδ T cells, which therefore might be educated over time to avoid tumour recognition

Functional Challenges in CAR T cell therapy (solid tumor)

  • hard to get the T cells to traffic into the tumor cells
  • hard to make the functional for a long time