ADENO-ASSOCIATED
VIRUSES
Current clinical trials of viral vector mediated
gene therapy
By: Michael Chang, Jerry Boun, Faraz Razzagh, and Jessie Lu
PHM142 Fall 2014
Coordinator: Dr. Jeffrey Henderson
Instructor: Dr. David Hampson
HISTORY ON AAV AND USES

Adenosine-Associated Virus

4.7 kb single stranded linear DNA

Non-pathogenic virus

Specific for AAVS1 on Human Chromosome 19

Two opening reading frames Capsid (cap) and Replication (rep)
sites

ITR (Internal Terminal Repeats) results in stem loops
PATHWAY
BREAKDOWN OF GENE THERAPY
1.
Virus attaches to cell membrane, specifically heparan receptor
2.
Receptor mediated endocytosis
3.
Endosomal trafficking (providing direction)
4.
Lyse of endosome
5.
Viral uncoating
6.
Viral entry into nucleus
7.
Use of host machinery; DNA replication
8.
Integration of viral DNA into human chromosome 19
9.
Use of helper cells (optional) allows for viral release
RESEARCH USES OF AAV

Helper-mediated AAV – can purify large titres of virus

Disadvantage – combination of helper virus and AAV can result
in immune response

AAV only – can purify large titres of virus

Disadvantage – expensive and very labour intensive to purify.
CLINICAL TRIAL #1: HEMOPHILIA

Lack of blood clotting factors, leads to excessive bleeding

Two types: A and B

Hemophilia B: genetic deficiency in FIX (Factor IX protein)

Can treat with FIX injections, but only lasts 2-3 weeks

AAV gene therapy proven successful
NEUROMUSCULAR DISEASES

Neuromuscular disorders: Amyotrophic Lateral Sclerosis (ALS), LimbGirdle Myasthenia and Emery-Dreifuss Muscular Dystrophy (EDMD),
Spinal Muscular Atrophy, Huntington’s and Parkinson’s disease.

Caused directly by problems with the muscle itself

Caused indirectly by the inability for the nerve and muscle to
communicate at the neuromuscular junction (NMJ)
CLINICAL TRIAL #2: LIMB-GIRDLE

Various forms of Limb-girdle myasthenia are caused by different mutations

It is commonly seen for a defective DOK7 to be a cause of this.

The DOK7 protein acts to work the NMJ.

General symptoms: muscle atrophy, difficulty breathing and weakening of
heart muscle

AAVs associated with a normal human DOK7 gene are injected in diseased
mice

The recipient cells begin producing normal DOK7 protein and the mice
eventually gained weight, strength, improved in motor tasks and life
expectancy increased.

EDME is also the result of defective NMJ’s and can be treated in the same way
CLINICAL TRIAL #3: SPINAL MUSCULAR
ATROPHY

Progressive degeneration of spinal cord motor neurons

Results in


muscular atrophy

paralysis

shortened life span
Genetic

Humans have two genes that code for SMN proteins, SMN1 and SMN2

The mutation is in SMN1 gene, but SMN2 cannot compensate for the loss
INSULIN GROWTH FACTOR-1 (IGF-1)

Pleiotropic hormone that modulates multiple fundamental cellular processes

Necessary for normal brain development and function

Previous injections of IGF-1 only produced modest results

Use of AAV factor showed significant improvement

Administered intravenously

Local administration only partially alleviated disease
SMN REPLACEMENT

Another way to alleviate the symptoms of the disease

Alter SMN2 splicing

SMN1 gene replacement

scAAV9 inserted into the SMNΔ7 mouse model

Fully rescued mouse
CLINICAL TRIAL #4: ALS

Amyotrophic lateral sclerosis

A progressive neurodegenerative disease that is usually fatal within 2-5 years

Progressive loss of lower motor neurons in the cerebral cortex, brainstem and spinal
cord

Results in weakness, muscle wasting, spasticity and eventually respiratory failure.

Current treatments available for ALS is riluzole

riluzole has proven to have limited efficacy

slows loss of limb muscle strength

extends life by only a few months
VEGF AND ALS

Vascular endothelial growth factor (VEGF) is primarily associated with
angiogenesis

Treatment with VEGF-A gene or VEGF-A protein yield positive therapeutic
outcomes

Reduced expression of VEGF-A protein in mice shows similar neurodegenerative
effects as seen in ALS mice

VEGF-A shown to stimulate neurogenesis


responsible for nerve migration and axon guidance in mice

neuron survival and protection factor

increases both cell diameter and number of emerging neurites

enhanced survival of dopaminergic neurons in the midbrain
Animal models used for testing are those that express the SOD1 mutation


This mutation is associated with a relatively large fraction of familial ALS cases
VEGF-A overexpressed in mice resulted in significant delays in motor neuron loss
GENE THERAPY

AAV delivery vector found to target ependymal cells in both the brain and
spinal cord

AAV-4 VEGF-A administered by ICV injection (Mice study)


These had increased survival and improved motor function

Use of AAV shows sustained expression of VEGF-A thus allowing for a single delivery
as opposed to continuous infusions
Current clinical trial (Humans)

includes 18 ALS patients

use of an infusion pump not AAV

trying to determine the safety and tolerability of VEGF-A

determine if life is pro-longed in the 18 ALS patients
SUMMARY

AAV are non-pathogenic and can transduce genome into chromosome 19

4.7 kb single stranded linear DNA with inverted terminal repeats forming stem loop structures

AAV binds to heparan sulfate receptor, enters via endocytosis and utilizes host machinery to copy viral DNA and proteins

AAV can be more effective with helper virus (herpes simplex virus or adenovirus), but can also promote immune response
Clinical Trial 1:

Hemophilia X-linked recessive disorder – results in no blood clotting

Clotting Factor 9 plasma injections required every 2-3 weeks; gene therapy every 5-6 months.

Significant increase in clotting factor 9 (less than 1% to 2-5% FIX)
Neuromuscular disorders: Caused indirectly by the inability for the nerve and muscle to communicate at the neuromuscular junction (NMJ)
Clinical Trial 2:

Limb-girdle myasthenia is caused by a defective DOK7 gene

Results in the inability to produce the DOK7 protein which acts to work the NMJ.

The recipient cells of AAV began producing normal DOK7 protein and the mice eventually gained weight, strength and improved in motor tasks
Clinical Trial 3:

Spinal motor atrophy: Progressive degeneration of spinal cord motor neurons

Two methods of treatment:

Insulin growth factor -1(IGF-1), which is necessary for normal brain development and function, can be inserted

Replace the gene itself, by inserting the SMN1 gene without a mutation back into the cell
Clinical Trial 4:

AAV VEGF-A administration is well sustained in ALS mice

AAV VEGF-A administration improves motor function survival rate in ALS mice
REFERENCES

Arimura, S., S. Takeda, Y. Yamanashi, D. Beeson, N. Yoshida, M. Motomura, T. Yoshimura, Y. Kasahara, T. Chiyo, T. Tezuka, and T. Okada. "DOK7
gene therapy benefits mouse models of diseases characterized by defects in the neuromuscular junction." Science 345.6203 (2014): 1505-1508.
Print.

McDonald, C. M. (2002). Physical activity, health impairments, and disability in neuromuscular disease. American journal of physical medicine &
rehabilitation,81(11), S108-S120.

Wallace, D. C. (1989). Mitochondrial DNA mutations and neuromuscular disease. Trends in Genetics, 5, 9-13.

Nathwani, A. C., Zhou, J., Mingozzi, F., High, K. A., Gray, J. T., Reiss, U. M., et al. (2011). Adenovirus-Associated Virus Vector–Mediated Gene
Transfer in Hemophilia B. New England Journal of Medicine, 365(25), 2357-2365.

Deyle, D. R., & Russell, D. W. (2009). Adeno-associated virus vector integration. Curr Opin Mol Ther, 11(4), 442-447.

Hacein-Bey-Abina, S., Fischer, A., Latour, S., Rieux-Laucat, F., Martinache, C., Berry, C. C., et al. (2010). Efficacy Of Gene Therapy For X-Linked
Severe Combined Immunodeficiency. New England Journal of Medicine, 363(4), 355-364.

High, K. A. (2012). The gene therapy journey for hemophilia: are we there yet?. Blood, 120(23), 4482-4487.

Gene Therapy II. (n.d.). Gene Therapy II. Retrieved October 12, 2014, from
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/G/GeneTherapy2.html

Orion, P.k., O’Conner, M.O., and Boulis, N.M. (2014). Gene and Protein Therapies Utilizing VEGF for ALS . Pharmacology & Therapeutics. 141, 261271.

Robbins, K.L., Glascock, J.J., Osman, E.Y., Miller M.R. and Lorson, C.L. (2014). Defining the therapeutic window in a severe animal model of
spinal muscular atrophy. Hum.Mol.Genet. 23 (17), 4559-4568.

Tsai, L.K., Chen C.L., Ting, C.H., Chao, S.L., Hwu, W.L, Dodge, J.C., Passini M.A. and Cheng, S.H. (2014) Systemic administration of a recombinant
AAV1 vector encoding IGF-1 improves disease manifestations in SMA mice. Molecular Therapy. 22 (8), 1450-1459.