Lecture 8: Gene Therapy For Neurological Disease Flashcards
Gene Therapy For
Neurological Disease
Kevin Foust
Sepember 23
When should you use gene therapy?
Gene Therapy is not a solve-all solution, as previously believed.
Single-gene problems, such as Hemophilia, are much more likely to be cures using gene therapies; but multi-gene issues, like diabetes, are not issues that gene therapy can really solve, at this point.
Single gene vs. complex disorder
Define the targets—gene, cell, patient
What gene(s)? What cells must be affected to bring about cure? Are the patients young, old; do they have weak immune systems?
Blood Brain Barrier System
Keeps the environment of the brain constant.
Ensures the homeostasis of the CNS.
The BBB is formed primarily of Epithelial Cells that form tight junctions that keep things out, including disease and medications. Due to factors of size and charge, many molecules cannot get past the BBC
Cerebral Spinal Fluid
CSF is pressurized, so you have to be really careful with the volume of the injections; you don’t want to throw off the balance or cause hemorrhages.
ASGCT
The American Society of Gene & Cell Therapy
What is Gene Therapy?
Gene therapy is defined as a set of strategies that modify the expression of an individual’s genes or that correct abnormal genes.
Each strategy involves the administration of a specific DNA (or RNA).
Obstacles to CNS Gene Therapy
- Bones (skull, vertebral column)
Only certain patient populations can tolerate a procedure that drills through bone.
Blood Brain Barrier (BBB)
Complex anatomy
Widespread pathology
Multiple targets
Upper motor neurons
Upper motor neurons are motor neurons that originate in the motor region of the cerebral cortex or the brain stem and carry motor information down to the lower motor neurons.
Obstacles to CNS Gene Therapy:
Astrocytes
Once you have gotten beyond the BBB, astrocytes present another defense.
Astrocytes can also modulate the rate at which the brain’s vasculature lets things in and out.
Lower Motor Neurons
The final pathway in voluntary movement.
Lower motor neurons are motor neurons located in either the ventral horn of the spinal cord and anterior nerve roots (spinal lower motor neurons) or the cranial nerve nuclei of the brainstem and cranial nerves with motor function (cranial nerve lower motor neurons).
Lower motor neuron: A nerve cell that goes from the spinal cord to a muscle. The cell body of a lower motor neuron is in the spinal cord and its termination is in a skeletal muscle. The loss of lower motor neurons leads to weakness, twitching of muscle (fasciculation), and loss of muscle mass (muscle atrophy).
Which type of motor neurons die in diseases like ALS?
Lower Motor Neurons and upper…
Obstacles to CNS Gene Therapy:
Widespread pathology
Example of a disease with widespread pathology: Alzheimer’s
Can one possibly design a treatment that would go after literally everything in the brain and fix it?
Parkinson’s is different from Alzheimer’s.
It is very localized (Substantia Nigra), and sometimes it is treated with direct injections to the brain.
This is why Parkinson’s would be easier to treat with gene therapy methods than Alzheimer’s would.
How to Deliver a Gene Therapy to the CNS
Direct Injection
Cerebrospinal Fluid (CSF)
Retrograde Transport
Systemic Injection
Why Use Vectors?
Vectors can protect DNA from degradation and from causing an immune response, and vectors can enhance transfer into the target cell.
Low efficiency from naked
DNA/RNA transfer… basically meaning that if you throw just DNA at a cell, it will bounce right off. Cells don’t generally pick up nucleic acids.
In fact, the “rogue/unfamiliar” DNA of gene therapy can promote an immune response as though it was a disease.
Non-viral
Viral
Non-Viral Vectors
Scaleable, ease of manufacture:
“You can make a bucket of this stuff.” Viral vectors are more complicated.
Transient Expression: It’s active for a short time and then it goes away. This can be good or bad.
High payload capacity
Customizable
Liposomes
Polymers
Molecular Trojan Horses
Nuclear Entry –> RNAi
Molecular Trojan Horse
Antibody against human Insulin receptor
Attach protein, drug or DNA
Transient gene expression
All components FDA approved
Viral Vectors
Types
Adenovirus
Lentivirus
Adeno-Associated Virus
Adenovirus
The first virus to be used as a vector.
DNA Virus
3 stage replication cycle.
Based on the human Adenovirus 5, which causes Respiratory infections (like colds)
Payload size: Generations
Immunogenicity: Transient expression
Jessie Gelsinger died because of this therapy in early stages.
Lentivirus
RNA virus:
Complex biology
Based on many platforms, including HIV
Integrating
Payload ~9kb
Enveloped
Life-long expression, but harder to manufacture
Stable Expression
Low Titer
Why doesn’t DNA get through membranes?
Phosphates carry a negative charge. Both DNA and cell membranes have of these negative components. Negative and negative don’t go together, so they repel each other.
Thus a vector is needed to get DNA in the cell.
Adenovirus & Cancer
Cancer—
Adenovirus
HSV-TK + ganciclovir
Glioblastoma multiforme
Adenovirus vectors seem to always attract immune response, which is good when dealing with cancer
Adeno-Associated Virus (AAV)
VERY Small, non-pathogenic (unlike lenti. and ad. viruses) parvovirus
Gene therapy vector
Long-term transgene expression
Post-mitotic cells
Easy to convert to vector
Two types of recombinant genomes:
Single stranded—4.7kb
Self Complementary—2.5kb
Safe
Episomal
Long-term, stable expression
Efficacious
AAVs require helper viruses
Leber’s Congenital Amaurosis
genetic form of blindness
Three independent groups
Unilateral subretinal injections
AAV2-RPE65
AAV injections Treat bad Eye
