Gene Interventions – Part

AMB – L11
Pietro D SPANU
p.spanu@imperial.ac.uk
Intended Learning Objectives
• Reflect on why intervene in genetic makeup
• Define the scope of interventions
• Reflect on what is disease and what is therapy in humans and in a wider
biological context
• Describe the limitations and challenges of genetic basis of disease
• Explain the basis of Gene Therapy in humans
• Give examples of how genetic material may be delivered
• Differentiate between in vivo and ex vivo Gene Therapy
• Discuss the relative merits of (human) viral delivery

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Why intervene in genetic makeup?

Well-being

Health Economics

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The scope of interventions:
Environmental
Agriculture – crops and animals
Veterinary
Human health

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Gene Interventions in human health
Gene Therapy

Vaccines

Pathogens and Parasites (e.g. malaria)

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What is disease*?
https://www.menti.com/ald6247qvf
79

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Gene Therapy
(humans)
“In our view, gene therapy may
ameliorate some human genetic
diseases in the future”
Friedmann and Roblin (1972)
“Gene therapies are rapidly
becoming a critical component of
the therapeutic armamentarium…
in human diseases”
Dunbar et al (2018)
Promise:
Specificity
Long-term / Permanent
Overcoming intractability
Challenges:
Basis of disease/condition not fully understo
Insertional effects
Adverse reactions to delivery vectors
Immune suppression of engineered cells
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Gene Therapy (humans) –
Identifying basis of disease is essential

“Single gene” metabolic Complementation of defective

disorders (congenital) mutation

Acquired disorders (cancer) Killing affected cells and

replacement with healthy ones

Infection (HIV) Modification of viral receptors

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Gene Therapy (humans) – Delivering genetic
material

Naked DNA/RNA

Abiotic

Viruses

γ-retrovirus Lentivirus Adeno-Associated Virus

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Gene Therapy (humans) – Delivering genetic
material
Abiotic Delivery

Physical methods Biomaterials

Needles/injection Liposomes Inorganic particles
Biolistics Peptides Dendrimers
Electroporation Polymers: Extracellular Vesicles
Microneedles
Hydrodynamic pressure polyesters Microspheres
Magnetic fields Polyethyleneimine Hydrogels
Ultrasounds Polyaminoacids
“Natural” polymers

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Gene Therapy (humans) –
Delivering genetic material
Viruses
γ-retrovirus: Lentivirus: Adeno-Associated Virus:
• Packaging • High efficiency of • Do not cause disease
does not infection • Infect dividing and
require gag, • Long-term stable quiescent cells
pol, accessory expression • Integrate at specific site
genes • Low immunogenicity (AVVS1), but can be
• Can target non- modified to suppress
• Target dividing cells integration
dividing cells • Low immunogenicity
• Associated • Low cytotoxicity
with cancer • Integrate and may
cause mutation • Limited genome size
(4.8kb)
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Gene Therapy (humans) – ex vivo vs in vivo
therapies

Bulaklak and Gersbach, 2020

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Gene Therapy (humans) – A case study: Sickle Cell Disease
(SCD)

SCD cause:
e.g. mutation in haemoglobin (HbA to HbS)
single base A-T, codon 6, chromosome 11:
the “E6V” mutation

SCD Symptoms:
Formation of HbS polymers, distortion of
erythrocytes, capillary occlusion, haemolysis,
anemia, dactylitis, infarction of liver, spleen,
lymph nodes

SCD prevalence:
About 20M people worldwide
Prevention of death in early childhood leads to
increased numbers of adults with symptoms
(complex, life-limiting)

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Gene Therapy (humans) – A case study: Sickle Cell Disease
(SCD)

SCD non-Gene Therapy SCD Gene Therapy – ex vivo delivery

treatments: into autologous haematopoietic stem
Hydroxyurea cells:
L-glutamine γ-retrovirus
Blood transfusions Lentivirus
Monoclonal Abs (Crisanlizumab;
Voxelotor) Challenges:
Low expression
Allogenic Haematopoietic Stem Cell Limited genome capacity
transplant:
Activation of cis-loci
Transplant cells from healthy siblings
or matched donors Insertional mutagenesis

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Gene Therapy (humans) – A case study: Sickle Cell Disease
(SCD)

White et al (2022)
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Gene Therapy (humans) – A case study: Sickle Cell Disease
(SCD)

White et al
2022
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Gene Therapy (humans) – A case study: Sickle Cell Disease
(SCD)

Outlook:

Several clinical trials underway to

evaluate effectiveness, and safety

In vivo editing – may require less

complex, expensive therapeutical set
up

White et al
2022
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Gene Interventions (humans) – A case study: mRNA
Vaccines against COVID-19
• Transient gene expression to produce antigen
• Not considered transgenesis or Gene Therapy

COVID vaccines: Recombinant protein

• Inactivated virus
• Live attenuated virus
• Recombinant protein
• Adenovirus vector
• Influenza virus vector
• mRNA
• DNA

© Higgins et al, 2020

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Gene Interventions (humans) – A case study: mRNA
Vaccines against COVID-19

Delivery:
• Intramuscular
• Intracutaneous
• Subcutaneous

Transfected cells:
• Non-immune cells
• Immune cells at site
• Immune cells at periphery
(lymph nodes/spleen)

Fang et al 2022

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Gene Interventions (humans) – A case study: mRNA
Vaccines against COVID-19
How does the mRNA enter the cytoplasm (site
of translation)?

A proposed model:

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Gene Interventions (humans) – A case study: mRNA
Vaccines against COVID-19

Complexities involved in mRNA vaccines:

• Types of RNA vaccines • Carriers and delivery
• 5’ capping • Legal and commercial framework
• Base modification • Adverse effects
• Variable effectiveness
• 5’ and 3’ UTRs • Storage and delivery temperature
• polyA
• Which antigen?
• Codon optimisation

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The Future of Gene Therapy - Challenges
What do you think might be the difficulties, problems, challenges
posed by (human) Gene Therapy in the coming years?

Turn to the person sitting next to you and discuss this for 5
minutes.

Then summarise your thoughts in this Mentimeter poll

https://www.menti.com/alm9spjj1cgz
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