Gene therapy delivery Flashcards
What is somatic cell gene therapy?
Somatic cell gene therapy involves the placement of a human gene into a living person’s somatic cells—cells that do not produce the eggs and sperm that in turn produce the next generation.
Somatic cell gene therapy would aim to cure a disease only in the patient, not in the patient’s descendants.
What is germ line gene therapy?
Germline gene therapy involves the modification of germ cells (gametes) that will pass the change on to the next generation.
With germ line therapy genes could be corrected in the egg or the sperm that is being used to conceive.
What are the challenges with gene delivery?
Large molecules (high MW) and polar.
Limited absorption and cellular uptake.
Metabolic liability - oligonucleotides degraded by body nucleases.
Endocytosis is the major uptake mechanism; possible entrapment in endosomes leading to degradation by lysosomal enzymes.
What would be the optimal gene delivery system?
Protect oligonucleotides from damage/metabolic degradation.
Facilitate their entry into the target cells & their nuclei.
Promote an efficient endosomal release.
Ensure transcription in cells (high expression).
Minimal immunogenicity.
What are the 2 main classes of vectors?
Viral vectors (Transduction):
Adenovirus (AV).
Adeno-associated virus (AAV).
Retrovirus (γ-retrovirus or lentivirus).
Herpes simplex virus (HSV).
Non-viral vectors (Transfection):
Naked nucleic acids. Lipid complexes (Liposomes or Lipoplexes). Polymer complexes (Polyplexes). Protein conjugates for targeting. Nanocapsules/nanoparticles.
How are viral vectors constructed?
Viral vectors are usually derived from parental wild type viruses whose viral genes (essential for replication and virulence) have been replaced with the heterologous genes intended for cell manipulation.
What is the adenovirus (AV)?
Non enveloped virus (only capsid protein coat).
Genome is comprised of a linear double stranded DNA.
Does not integrate its genome into the host genome (i.e. episomal).
Used in combination with chemotherapy.
Adenovirus vector engineered to express WT p53.
Enters tumour cells by endocytosis.
p53 activates cellular apoptosis.
Advantages:
One of the best and most widely used gene delivery systems.
Ease of purification and concentration (i.e. ease of production).
High efficiency rate of infection for various types of host cells (dividing or non-dividing).
Good for direct in vivo gene transfer.
Disadvantages:
Can elicit immune response.
What is the adeno-associated virus (AVV)?
Non enveloped icosahedral (diamond like) shape.
Genome is comprised of a small single stranded DNA.
Target non-proliferating cells.
For treatment of lipoprotein lipase deficiency (LPLD) that causes severe pancreatitis.
Adeno-associated viral vector (AAV1) engineered to express WT LPL gene.
Injected into muscles (IM).
Advantages:
Non-pathogenic & does not trigger patient immune responses.
Disadvantages:
Limited cloning capacity (difficult to work with).
What is the retrovirus?
First constructed human gene therapy vector.
Enveloped virus particle.
Genome is comprised of 2 copies of RNA.
Reverse transcriptase converts RNA to double stranded DNA (reverse transcription).
Viral DNA integrate to host genome using integrase (non-specific site).
Used for ex vivo gene therapy as it is unable to efficiently infect non-dividing cells.
For treatment of severe combined immunodeficiency due to adenosine deaminase deficiency (ADA-SCID).
Autologous CD34+ cells transduced with retrovirus vector encoding the cDNA sequence for human ADA.
Limitations:
Low vector titer.
Low transduction efficiency.
Particle instability and difficulty to concentrate.
Infect only proliferating cells (i.e. inability to transduce non-dividing post-mitotic cells).
What are lentiviruses?
A special group of retroviruses with ability to infect both proliferating and non-dividing cells.
Ensure long-term expression and efficient transfer without inflammatory responses.
What is the herpes simplex virus (HSV)?
Enveloped virus.
Genome is comprised of a double stranded DNA.
Naturally is a human pathogen.
Has the ability to remain latent in tissues & to be re-activated at the original site of infection.
Used for treatment of melanoma.
Active substance T-Vec – genetically engineered oncolytic herpes simplex virus.
Promotes anti-tumour immune response.
Advantages:
Large size (i.e. can accommodate large pieces of foreign DNA).
Continuous expression of genes from long-lived infection.
Episomal (i.e. little risk of insertional mutagenesis).
Disadvantages:
Low infection efficiency.
Large genome size (i.e. more difficult to manipulate).
Tropism for neural cells (i.e. limited action); however, there are endeavours to exploit this feature to target neurons.
Compare the pros & cons of viral vectors:
Adenovirus (AV):
+ Infects many cell types.
- Doesn’t integrate into host genome (episomal) & can be lost.
Adeno-Associated Virus (AAV):
+ Integrates into host genome & can’t be lost.
- Limited cloning capacity (difficult to work with).
Retrovirus:
+ Integrates into host genome & can’t be lost.
- Integrates into host genome & can cause cancer.
Herpes Simplex Virus (HSV):
+ Continuous expression of genes from long-lived infection.
+ Episomal (i.e. little risk of insertional mutagenesis).
- Tropism for neural cells.
What do we need in a good viral vector?
Toxicity.
Immunogenicity.
Specificity / tropism.
Propagation.
Integration.
Insert size.
What are non-viral vectors?
Founded in attempts to overcome limitations of viral vectors.
Genetic material delivery by the less efficient transfection.
Advantages:
Unlimited insert size.
Low toxicity.
Low immunogenicity.
Low cost & ease of production at large scale.
Disadvantages:
Low efficiency.
What are naked nucleic acids?
DNA (or RNA) delivered by direct injection e.g. into the muscle.
Low efficiency of transfer.
Low expression levels.
