Gene Therapies in Neurological Disorders

Question 1

How can the viral delivery of gene-based therapies be beneficial in neurological diseases?

Answer 1

→ Conventional pharmacological approaches for treatment of various neurodegenerative diseases have been predominantly disappointing
- This is despite have a relatively good understanding of disease pathogenesis
- The novel targets are not readily “druggable”

There has been significant progress in the designing of viral vectors that now diffusely deliver genes throughout the CNS in addition to new genomic engineering tools that can subsequently alter disease pathways

Question 2

Gene replacement allows for..

Answer 2

correction of a significant gene mutations which ultimately causes a disorder
- can be NB in correction of loss of function (as seen in SMN protein in spinal muscular atrophy)

Question 3

What do gene based therapies offer?

Answer 3

• Ability to directly target disease pathogenesis
• Capacity to achieve a “permanent correction”
• A single long-lasting intervention (one and done) that provides sustainable pharmacology and efficacy is very attractive for CNS diseases

Question 4

What are the essential components of gene therapy?

Answer 4

Vector, Promoter, Transgene

Question 5

Why is a vector so NB?

Answer 5

responsible for tissue targeting and delivering the transgene and promoter to target cells, which can either increase or decrease expression of a certain gene
→ can include adenovirus, adeno-associated virus, retrovirus and lentivirus

Question 6

Why is a promoter so NB?

Answer 6

responsible for selective gene expressions and driving transgene expression in intended tissue targets

Question 7

Why is a transgene so NB?

Answer 7

responsible for producing a functioning version of the protein of interest
→ the genetic material introduced to targeted tissues by the vector and its expression is then driven by the promoter

Question 8

How do these components in gene therapy work together?

Answer 8

the vector binds to a specific receptor, enters the nucleus where the transgene and promoter are released and form an episome, this generates RNA which is responsible for protein generation of the essential protein… depending on the pathogenesis of that specific condition

Question 9

Why is the AAV Capsid so important?

Answer 9

It is NB in determining several key features of effective AAV gene therapy
- Tissue tropism: - viral purification methods can influence
- Distribution: - receptor interactions, anterograde and retrograde axonal transport
- Susceptibility to targeting by neutralising antibodies
→ Administration of AAV vectors to CNS via intraparenchymal route demonstrates sustained transduction of neurons
→ AAV serotypes from natural AAV can vary in terms of transduction efficiency, biodistribution if alternative modes of administration are used (intrathecal or IV)
→ AAV2 most frequently used in clinical trials, provides long term expression in CNS neurons but has limited restriction in biodistribution
→ Capsid properties can be engineered to increase their efficacy, mainly in preclinical models - none used in clinical trials
→ Biodistribution and homogeneity of cellular transduction - greatest challenges with AAV delivery for NG diseases

Question 10

AAV uses a range of proteoglycans including

Answer 10

Heparin Sulphate, as a primary cell surface receptor

Question 11

Secondary receptors have also been identified for several AAVs…

Answer 11

like fibroblast growth factor receptor, laminin receptor

Question 12

Receptor interactions can influence…

Answer 12

the biodistribution and tropism of the AAV capsid

Question 13

What does the route of administration and dosing paradigm determine?

Answer 13

The expression levels and homogeneity across cell types and tissue regions of interest in the brain
Also depends on which disease

Question 14

What are the most commonly used routes of administration of AAV?

Answer 14

Intraparenchymal (IPa), as it bypasses the BBB and delivers genes directly to the brain region and neurons of interest

→ One and Done features is essential due to required surgical procedure
→ Well tolerated in clinical trials to date
→ Minimal biodistribution to peripheral organs
→ Reduced immunogenicity
→ Significantly lower vector doses required compared to other routes of administration

Question 15

Other routes of administration:

Answer 15

Intrathecal (IT) - access via spinal cord cerebrospinal fluid via the space under the arachnoid membrane

Intracerebroventricular (ICV) - into the CSF via the lateral cerebral ventricles

Intracisternal (IC) – into the CSF via the cerebromedullary cistern
Currently mainly assessed in preclinical models, a small number of clinical trials have shown to be well tolerated, dosing regimes can be altered by varying volume delivered, rate of infusion & vector conc.
Compared to IPa, dosing via these routes results in lower tissue levels of vector genome and a more heterogeneous distribution, required doses are generally higher

IV administration - huge potential to transfer vector genes to entire CNS non-invasively with more uniform distribution

→ Some concerns to be addressed
IV injection exposes the virus to potential antibody neutralisation in subjects who have been pre-exposed to natural AAV infections
Approx. 90% of adults have been exposed to AAVs, and a smaller fraction harbour neutralising antibodies against AAV capsids profound negative impact on AAV vector transduction
IV administration typically requires higher total doses to achieve efficient transduction than IPa, IT or ICV administration- scale of manufacturing capacity must be sufficient

Question 16

Spinal Muscle Atrophy (SMA)

Answer 16

a progressive, monogenic lower motor neuron disease and is one of the leading genetic cause of infant mortality

Question 17

SMA is primarily caused by..

Answer 17

the homozygous deletion of the SMN1 gene that encodes the survival motor neuron (SMN) protein though the underlying reason for motor neuron vulnerability is not fully understood

As patients with SMA lack a functional SMN1 gene, the severity of their symptoms and the age of disease onset is determined by copy numbers of the SMN2 gene

Question 18

The most common type of SMA..

Answer 18

SMA type 1, patients tend to have two copies of the SMN2 gene

Hypotonia and motor delay before 6 months of age and death or the need for mechanical ventilation by the age of 2 years

Question 19

Other forms of SMA:

Answer 19

Patients with three to four copies of SMN2 have milder symptoms that appear later (6–18 months of age for type 2, and ~3 years for type 3).

Fewer than 5% of patients have four to eight copies of SMN2 (type 4) and have the mildest form of the disease, which has an adult onset

Question 20

Landmark gene therapy clinical trial in infants with SMA type 1

Answer 20

• They received a single IV dose of AAV9 vecotr carrying the SMN1 gene
• All patients in this trial were alive and event-free at 20 months compared with a historic survival rate of 8%
• Several patients receiving a higher dose showed unprecedented improvement in symptoms, including the ability to walk unassisted, although they were treated at a younger age
• During the trial, four patients developed transient elevations in liver transaminase levels post dose, although no additional liver dysfunctions were reported
• Subsequent 2-year follow-up study in the high-dose cohort showed a reduced need for pulmonary and nutritional support and improved motor function, and AVXS-101 was approved by the FDA in 2019

Question 21

What makes PD so amenable to gene therpay?

Answer 21

The focal nature of SN pathology

Question 22

Gene therapy in PD can lead to approaches which can:

Answer 22

• Restore NT imbalance
• Enhance neuronal survival
• Target genes directly linked to the disease

Question 23

Gene therapy & targeting PD familial genes

Answer 23

10% of PD cases are due to genetic mutations, for which there are multiple possible mutations…

• ? Attenuating LRRK2 expression as a therapeutic option
• However LRRK2 is also produced in the lungs, kidneys and spleen, so global inhibition of this gene could lead to pathological changes in other tissues

Question 24

Gene therapy in production of NTF

Answer 24

NTF are promising candidates involved in promoting survival of DA midbrain neurons (GDNF & NRTN)

• Currently undergoing phase 1 trials to determine safety in AAV-GDNF IPa injections to the putamen
• Phase 1 trial delivering AAV2-NRTN was well tolerated but did not improve motor function, post mortem analysis demonstrated increases NRTN expression at injection site but no upregulation in the SN

Question 25

Why is HD an appealing target for gene therapy?

Answer 25

It is a monogenic disorder!

• Preclinical models showcased positive results upon injection of AAV-HTT miRNA into the striatum → reduced HTT expression, leading to improved motor and behavioural deficits without neurotoxicity
• Phase I/II first in human trials have began with intra-striatal admin of AMT-130, which is an AAV5 vector carrying artificial miRNA specifically tailored to silence the huntingtin gene

Question 26

Translation research criteria for developing a viral-based gene therapy:

Answer 26

• High unmet medical need
• Favourable benefit-to-risk profile
• Strong target validation
• Ability to safely achieve therapeutic tissue levels of vector at the target CNS location-regulate transgene expression
• Transduction of the relevant cell type or types
• Adequate therapeutic index for pharmacological effects and safety
• Biomarkers and clinical assessments that will allow for measurement of pharmacological activity and clinical outcomes

Question 27

Challenges associated with AAV based gene therapies

Answer 27

• Ability to detect expression of the transgene in living patients is limited – no biomarkers and challenges with getting tissue biopsies
• Capsid proteins of recombinant AAV particles are similar to capsids of natural viruses that we are frequently exposed to hence neutralising host antibodies are produced which dampen transduction efficiency
• This can also affect patient involvement in clinical trials, often titers for AAV are detected which would exclude patients from participating
• Exogenous AAV capsids are immunogenic and can activate cytotoxic T-cells which eliminate the transduced cells
• Therapeutic index- function of both transgene and mechanism of action of vector
• Potential toxic effects – overexpression of therapeutic transgene within targeted cells, off-targets