Gene Delivery

Gene Delivery
Ex vivo
Transplantation
of recombinant
cells with virus
Direct administration
of virus
In vivo

“New” Gene Therapy
Mucosal gene therapy using a pseudotyped lentivirus vector encoding murine interleukin-10 (mIL-10) suppresses the development and relapse of experimental murine colitis
H. Matsumoto et al. (2014)
BMC Gastroenterology 14:68

Gene Therapy Trials

Gene Therapy
Ex vivo
Transplantation
of transduced cells
myoblasts
hepatocytes
blood
bone marrow
fibroblasts
skin
stem cells

Ex vivo
1990

Cells? Which cells?
Focus on the patient!
Then focus on the disease (cells, tissues…)

Ex vivo Gene Therapy
Lentiviral Hematopoietic Stem Cell Gene Therapy Benefits Metachromatic Leukodystrophy
Alessandra Biffi et al. Luigi Naldini’s laboratory (Italy); Science 2013
Metachromatic leukodystrophy (MLD) is a neurodegenerative lysosomal storage disease caused by arylsulfatase A (ARSA) deficiency. The disease primarily affects children and invariably leads to premature death. In previous work with a mouse model of MLD, we used a lentiviral vector (LV) to introduce a functional
ARSA gene into hematopoietic stem cells (HSCs) ex vivo and
showed that reinfusion of the engineered HSCs prevented and corrected disease manifestations in the animals. To determine whether this gene therapy strategy is safe and can offer therapeutic benefit to patients with early-onset MLD, we designed a phase I/II trial. There was high-level stable engraftment of the transduced HSCs in the bone marrow and peripheral blood of all patients. Findings were associated with a clear therapeutic benefit.

Which vector to use? (rocket)

blood
Retrovirus
AAV
Adenovirus
Herpes
Target cell
DNA/RNA
oligo
DNA-poly(k)
conjugate +/- ligand
DNA
DNA liposomes
secretion
transport
genome
lysosome
transfection
proteins
Receptor-mediated endocytosis
cytoplasm
nucleus
mRNA

Virus Classification (classes)

Viral Replication

Viral Proteins

Ψ is essential for viral replication

Pseudotyped virus

Viral vectors
Genome RNAs DNAd DNAs RNAs DNAd
Size 9 30 4.7 9 150
Capacity >7 8-30 4 >7 130
Target cells Div Div/no Div/no Div/no Div/no
Integration yes no yes/no yes no
Retrov. / Adenov. / AAV / Lentiv. / Herpes

Viral Entry into Nucleus

RELEVANT QUESTIONS WHEN CHOSING A VECTOR
What disease am I going to target?
How long do I need to express the transgene for? Is it likely that re-administrations are required?
Which cells do I want to target?
What medical conditionings do patients have?
Choice of promoter? Viral? Mammalian?
Is regulation of expression required?
Vector tropism?

IDEAL VECTOR CANDIDATE
(does not yet exist)
High titer or concentrations (>108 particles/ml)
Method of production is convenient and reproducible
Precise introduction of the transgene
The transgene is responsive to its regulatory elements
Ability to target specific cells (pseudotyped)
Does not elicit host immune response
Persistence as required

DNA of interest
Transfection
Packaging cell
Viral particles
- Size limitation
- Viral titer
- Replication ability
genome
cytoplasm
nucleus
Production of Viral particles

RETROVIRUS
Single stranded RNA molecule
Only infects dividing cells
eco, amphotrophic
Mouse: cationic amino acid transporter
Integrates into host genome
Pseudotyped

RETROVIRUS

RETROVIRUS
Single stranded RNA molecule
Long terminal repeats LTR with promoter/enhancer sequences
Long-term persistence of DNA

LIMITATIONS OF RETROVIRUS
Retroviruses are inactivated by human sera
Transgene expression from LTR is often inactivated
Potential insertional mutagenesis
Oncogene activation

LENTIVIRUS
Based on HIV genome
Infect dividing / non-dividing cells
CD4/CCR5 receptor (co-recep)
Integrates into host genome
Sustained transgene expression

ADVANTAGES OF LENTIVIRUS
Targeting of stem cells
Gene expression is sustained, and often sustained through cellular differentiation
Promising in preclinical studies:
Hematopoietic cells
inhibition of genes (interference)

LIMITATIONS OF LENTIVIRUS
Gene expression is often not as high as with adenovirus
Same as retrovirus (except it can target non-dividing cells)
Potential use in gene therapy provided safety is proven

ADENOVIRUS
There are at least 10 proteins in the Adenovirus capsid
Double stranded DNA molecule
Infects dividing and non-dividing cells
Human CD46 receptor
Does not integrate into host genome (episomal)
Very high titer

Adenovirus
Large capacity as a vector
Very broad cell tropism
Infects dividing / non-dividing cells
Very high expression
Very antigenic
Expression is typically transient
Gutless
oncolytic
replication selective
Serotypes

AAV
Small size virus (< 5kbp)
S/s DNA genome
Adenovirus-dependent for efficient replication
Infects dividing / non-dividing cells
Heparin sulfate receptor
Integrates into host genome ??
Episomal vs integrated

Adeno-Associated (AAV)
• not very antigenic
• high expression
• long term (>1 year)
• AAV vectors are virtually empty of viral genes
• most promising viral vector

AAV
Lag phase (6 weeks) for max delivery
Neutralizing Abs to capsid do not prevent long-term delivery of therapeutic product
Small size of load (unsuitable for large genes)
Difficult to produce
Multiple administrations ?
Serotypes

HERPES
Large size DNA genome (150 kbp)
Human neurotropic virus
Suitable for targeting the CNS
Infects dividing / non-dividing cells
Very large payload
Does not integrate into host genome, but replicates as episome
Cytotoxic / inflammation

HYBRID VECTORS
AAV / adenovirus
Retrovirus / adenovirus
Retrovirus / Herpes

ALTERNATIVE VIRUS
Simbis
Poxvirus
Vaccinia
Baculovirus
Sendai
Foamy virus
SV40…..

KEY ISSUES
Delivery
Immune response
Logistics
Tropism
Persistence

IMMUNITY OF VIRAL VECTORS
Delivery
Immune response
Logistics
Tropism
Persistence