Download Viral vectors

Current clinical trials of viral vector mediated
gene therapy – adeno-associated viruses
https://www.scientificamerican.com/article/experts-gene-therapy/
PHM142 Fall 2016
Coordinator: Dr. Jeffrey Henderson
Instructor: Dr. David Hampson
By
Visar Berisha, Jerry Chen, Andew Greig, and Mehrdad Azimi
What is gene therapy?
• Gene therapy involves altering the genes
inside your body's cells in an effort to
treat or stop disease.
Basic Mechanism
• Insert gene into a vector (Virus,
Bacterium or Plasmid)
• Once you inject vector into target tissue,
it enters the cell and inserts its genome
into the host cell’s genome
http://www.yourgenome.org/facts/what-is-gene-therapy
Gene Therapy Techniques
• Augmentation
• Used to treat disease caused by a mutation that stops a gene from producing a
functioning product, such as a protein.
• Adds DNA containing a functional version of the lost gene back into cell.
• New Gene produces a functioning product at sufficient levels to replace the protein
that was originally missing.
• Inhibition
• Aim is to introduce a gene whose product either
• inhibits the expression of another gene
• Interferes with the activity of the product of another gene
What are adeno-associated viruses (AAVs)?
• Single-stranded DNA viruses
• Icosahedral capsid, non-enveloped
• Replication defective
• Replication is dependent on a
helper virus (e.g. adenoviruses)
• 12 serotypes
Transmission and Immune Response
• Transmission is via respiratory droplets or the fecal-oral route
• Adeno-associated viruses are not known to cause disease
• Humans have a very limited immune response to AAVs
• Most adults are seropositive (85-90%), but many have no antibodies
specific to AAVs
Genome
• Adeno-associated viruses have a 4.7 kilobase genome, which
contains 2 main genes (in 2 open reading frames)
• Rep gene
• contains 4 proteins which are involved in viral replication
• Rep78, Rep68, Rep52, Rep40
• Cap gene
• contains 3 capsid proteins
• VP1, VP2, VP3
Mechanism – to lyse or not to lyse
• AAVs bind to receptors on cell
surface for endocytosis
• Receptor is dependant on serotype
• Can be either lytic or lysogenic
infection
• Lysogenic – viral DNA is
incorporated into the host DNA on
chromosome 19
• Virus is dormant until cell is infected
with a helper virus
AAVs and Gene Therapy
• AAV vectors are based off serotype 2
• Target to muscles, liver, brain and lungs
• Several weeks before optimal expression is reached
• DNA is spliced into chromosome 19
Why Adeno-Associated
Viruses?
- Non-pathogenic
- Mild immune response
- Implant genes on Chromosome 19
- Reliable and predictable = no risk of mutagenesis
- Integrate into host genome (chrom. 19)
long-term strong transgene expression
- Long-term correction possible
- Ability to transduce variety of cells
- dividing and non-dividing cells
Limitations
- Small genome insert capacity
- 4 kb
- Adenoviruses and Retroviruses:
- ~ 8kb
Current Clinical Trials of
Viral Vector-Mediated
Gene Therapy – Adenoassociated Viruses
STUDY 1:
Recombinant Human Endostatin Adenovirus Combined With Chemotherapy for Advanced Head
and Neck Malignant Tumors
Chengdu Shi Endor Biological Engineering Technology Co., Ltd.
Collaborator: West China Hospital
Estimated Study Completion Date: October 2016
Phase II
Study Type:
Interventional
Study Design:
Allocation: Randomized
Endpoint Classification: Safety/Efficacy Study
Intervention Model: Parallel Assignment
Masking: Double Blind (Subject, Investigator)
Primary Purpose: Treatment
Primary Outcome Measure:
Change in Objective Response Rate (ORR) of Target Lesion determined by tumor
assessments based on radiological tests
Recombinant Human Endostatin Adenovirus Combined With Chemotherapy for Advanced Head
and Neck Malignant Tumors
Arms
Assigned Interventions
Experimental: Combination therapy A
Recombinant human endostatin adenovirus (EDS01), 5.0 × 1011 VP
intratumorally on days 0 and 7; paclitaxel, 160 mg/m2 intravenously
on day 1; cisplatin, 25 mg/m2 intravenously on days 1 to 3.
Drug: recombinant human endostatin adenovirus
Specification: 1mL/division, 1.0×1012 virus particle (VP).
Method of administration: Intratumor injection, once a week for 2
weeks, every 3 weeks for one cycle.
Drug: Cisplatin injection
Specification: 2ml: 10mg. Usage: 25mg/m2, days 1 to 3
Drug: Paclitaxel injection
Specification: 5ml: 30mg. Usage: 160mg/m2 intravenously on day 1
Experimental: Combination therapy B
Recombinant human endostatin adenovirus (EDS01), 1.0 × 1012 VP
intratumorally on days 0 and 7; paclitaxel, 160 mg/m2 intravenously
on day 1; cisplatin, 25 mg/m2 intravenously on days 1 to 3.
Drug: recombinant human endostatin adenovirus
Specification: 1mL/division, 1.0×1012 virus particle (VP).
Method of administration: Intratumor injection, once a week for 2
weeks, every 3 weeks for one cycle.
Drug: Cisplatin injection
Specification: 2ml: 10mg. Usage: 25mg/m2, days 1 to 3
Drug: Paclitaxel injection
Specification: 5ml: 30mg. Usage: 160mg/m2 intravenously on day 1
Experimental: Chemotherapy
Paclitaxel, 160 mg/m2 intravenously on day 1; cisplatin, 25 mg/m2
intravenously on days 1 to 3.
Drug: Cisplatin injection
Specification: 2ml: 10mg. Usage: 25mg/m2, days 1 to 3
Drug: Paclitaxel injection
Specification: 5ml: 30mg. Usage: 160mg/m2 intravenously on day 1
Recombinant Human Endostatin Adenovirus Combined With Chemotherapy for Advanced Head
and Neck Malignant Tumors
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Approximately 60% to 70% of patients have stage III or IV disease at the time of diagnosis.
-
The 5-year overall survival rate conventional treatment (surgery, chemotherapy and radiotherapy) is about 30%
-
Endostatin, an endogenous angiogenesis inhibitor and a C-terminal fragment of collagen XVIII, effectively inhibits tumor
angiogenesis by specific inhibition of neovascular endothelial cells
-
Previous studies have shown that the antitumor activity of recombinant human endostatin adenovirus is higher than that
of recombinant human endostatin protein.
-
Utilizes a recombinant adenovirus-recombined human endostatin gene (EDS01)
-
EDS01 uses recombined adenovirus type 5 as the vector for the human endostatin gene
-
Intratumor injection of EDS01 reportedly results in transportation of the human endostatin gene into tumor cells by
adenovirus infection, leading to the expression of endostatin protein
-
Expression of this protein inhibits neovascular endothelial cells, neovascularization, and tumor growth and metastasis
-
Both in vivo and in vitro experiments have shown that EDS01 significantly inhibits the growth of neovascular endothelial
cells and tumor growth in nude mouse xenograft models of laryngocarcinoma and nasopharyngeal carcinoma.
Source: ClinicalTrials.gov
STUDY 2:
A Gene Therapy Study for Homozygous Familial Hypercholesterolemia (HoFH)
University of Pennsylvania
Estimated Study Completion Date: January 2018
Phase I & II
Study Type:
Interventional
Study Design:
Endpoint Classification: Safety Study
Intervention Model: Single Group Assignment
Masking: Open Label
Primary Purpose: Treatment
Primary Outcome Measures:
-
Number of participants experiencing investigational product-related adverse events
Physical examinations; Clinical laboratory parameters; and adverse event reporting
Arms
Assigned Interventions
Experimental: AAV directed hLDLR gene therapy
- Single intravenous (IV) dose of human Low Density
Lipoprotein Receptor (LDLR) Gene Therapy
Genetic: AAV directed hLDLR gene therapy
- A novel adeno-associated viral (AAV) vector with human
low-density lipoprotein receptor (hLDLR) gene
A Gene Therapy Study for Homozygous Familial Hypercholesterolemia (HoFH)
-
Homozygous Familial Hypercholesterolemia (HoFH) is a rare genetic metabolic disorder characterized by absent or
severely reduced capacity to catabolize circulating LDL particles by the hepatic LDL receptor.
-
HoFH results from gene mutations approximately halving the number of functional LDL receptors in heterozygotes and a
greater lack in homozygotes.
-
As a consequence, HoFH subjects present abnormal total plasma cholesterol (LDL-C) levels, resulting in severe
atherosclerosis often leading to early onset of cardiovascular disease.
-
Early initiation of aggressive treatment for these patients is essential. Unfortunately, despite existing therapies
(PCSK9/CETP inhibition and ApoA1 mimetic), treated LDL-C levels remain well above acceptable levels.
-
Thus, the functional replacement of the defective LDLR via AAV-based liver-directed gene therapy may be a viable
approach to treat this disease and improve response to current lipid-lowering treatments.
Source: ClinicalTrials.gov
Summary
• Adeno-associated viruses (AAVs) can be used as vectors for gene therapy
• AAVs incorporate their genome into chromosome 19, which leads to long term expression
• AAVs do not induce a host immune response, and reliably integrate into the same spot, making them
good vectors
• However, their small genome means there is a limit to the size of gene that can be transfected
• Mechanism of adeno-associated viral vectors:
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Infection with viral particle
Endocytosis into the cell cytoplasm
Transport of viral capsid to nuclear pore
Nuclear transfer of viral genetic material
Transcription of viral-specific genes to induce viral progeny population
Release of viral progeny
• Numerous clinical trials utilizing viral vectors for cancer as well as genetic diseases.
• i.e. Neck and Head malignant tumors and Familial Hypocholesterolemia
• Aim is to target defective genes with gene therapy using a viral vector
References
YourGenome.org – What is gene therapy? http://www.yourgenome.org/facts/what-is-gene-therapy
VectorBio Labs - Introduction to AAV: http://vectorbiolabs.com/vbs/page.html?m=281
GeneTherapy.net - Adeno-Associated Viral Vectors: http://www.genetherapynet.com/viral-vector/adeno-associated-viruses.html
Daya S and Burns K. Gene Therapy Using Adeno-Associated Virus Vectors. Clin Microbio Rev. 2008 Oct; 21(4), 583-594.